GPR30 and estrogen receptor expression: new insights into hormone dependence of inflammatory breast cancer.

PubMed

Arias-Pulido, Hugo; Royce, Melanie; Gong, Yun; Joste, Nancy; Lomo, Lesley; Lee, Sang-Joon; Chaher, Nabila; Verschraegen, Claire; Lara, Juanita; Prossnitz, Eric R; Cristofanilli, Massimo

2010-08-01

GPR30 is a novel G protein-coupled estrogen receptor (ER) associated with metastases in breast cancer (BC) and poor survival in endometrial and ovarian tumors. The association of GPR30 expression with inflammatory breast cancer (IBC), an aggressive and commonly hormone-independent form of BC, has not been studied. GPR30, ER, progesterone receptor (PR), epidermal growth factor receptor (EGFR), and HER-2 expression were assessed by immunohistochemistry (and FISH for HER-2) in 88 primary IBCs. GPR30 expression was correlated with patient overall survival (OS), disease-free survival (DFS), pathologic variables, and other biomarkers. GPR30 expression was found in 69% of IBC cases. ER, PR, HER-2, and EGFR were found in 43, 35, 39, and 34% of IBC cases, respectively. GPR30 expression correlated inversely with ER expression (P = 0.02). Co-expression of ER and GPR30 was found in 24% of IBC samples; 19% expressed only ER and 46% expressed only GPR30. Univariate analysis showed no association between GPR30 expression and OS or DFS. However, co-expression of ER and GPR30 was associated with improved OS (P < 0.03) and marginally with DFS (P < 0.06); the absence of both ER and GPR30 was associated with worse OS and DFS (P = 0.03 for both). Multivariate analysis identified ER as an independent prognostic factor of OS (P = 0.008) and DFS (P = 0.02). The majority of IBC tumors are GPR30-positive, suggesting that estrogen signaling may be active in ER-negative IBC patients. These findings suggest potential new therapeutic targets for IBC such as novel endocrine agents or direct modulation of GPR30.

G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells.

PubMed

Albanito, Lidia; Madeo, Antonio; Lappano, Rosamaria; Vivacqua, Adele; Rago, Vittoria; Carpino, Amalia; Oprea, Tudor I; Prossnitz, Eric R; Musti, Anna Maria; Andò, Sebastiano; Maggiolini, Marcello

2007-02-15

Estrogens play a crucial role in the development of ovarian tumors; however, the signal transduction pathways involved in hormone action are still poorly defined. The orphan G protein-coupled receptor 30 (GPR30) mediates the nongenomic signaling of 17beta-estradiol (E2) in a variety of estrogen-sensitive cancer cells through activation of the epidermal growth factor receptor (EGFR) pathway. Whether estrogen receptor alpha (ERalpha) also contributes to GPR30/EGFR signaling is less understood. Here, we show that, in ERalpha-positive BG-1 ovarian cancer cells, both E2 and the GPR30-selective ligand G-1 induced c-fos expression and estrogen-responsive element (ERE)-independent activity of a c-fos reporter gene, whereas only E2 stimulated an ERE-responsive reporter gene, indicating that GPR30 signaling does not activate ERalpha-mediated transcription. Similarly, both ligands up-regulated cyclin D1, cyclin E, and cyclin A, whereas only E2 enhanced progesterone receptor expression. Moreover, both GPR30 and ERalpha expression are required for c-fos stimulation and extracellular signal-regulated kinase (ERK) activation in response to either E2 or G-1. Inhibition of the EGFR transduction pathway inhibited c-fos stimulation and ERK activation by either ligand, suggesting that in ovarian cancer cells GPR30/EGFR signaling relays on ERalpha expression. Interestingly, we show that both GPR30 and ERalpha expression along with active EGFR signaling are required for E2-stimulated and G-1-stimulated proliferation of ovarian cancer cells. Because G-1 was able to induce both c-fos expression and proliferation in the ERalpha-negative/GPR30-positive SKBR3 breast cancer cells, the requirement for ERalpha expression in GPR30/EGFR signaling may depend on the specific cellular context of different tumor types.

Post-synaptic Density-95 (PSD-95) Binding Capacity of G-protein-coupled Receptor 30 (GPR30), an Estrogen Receptor That Can Be Identified in Hippocampal Dendritic Spines*

PubMed Central

Akama, Keith T.; Thompson, Louisa I.; Milner, Teresa A.; McEwen, Bruce S.

2013-01-01

The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity. PMID:23300088

Post-synaptic density-95 (PSD-95) binding capacity of G-protein-coupled receptor 30 (GPR30), an estrogen receptor that can be identified in hippocampal dendritic spines.

PubMed

Akama, Keith T; Thompson, Louisa I; Milner, Teresa A; McEwen, Bruce S

2013-03-01

The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity.

Role of ERβ and GPR30 in the endocrine pancreas: A matter of estrogen dose.

PubMed

Ropero, Ana B; Pang, Yefei; Alonso-Magdalena, Paloma; Thomas, Peter; Nadal, Angel

2012-08-01

The endocrine pancreas has emerged as a target for estrogens. The functions of pancreatic α-, β- and δ-cells are modulated by the endogenous hormone, 17β-estradiol (E2). Low physiological concentrations (100pM-1nM) of E2 rapidly decrease the activity of the ATP-sensitive potassium channel (K(ATP)) and enhance glucose-induced insulin release in β-cells in an estrogen receptor β (ERβ)-dependent manner. In addition to the insulinotropic action of ERβ, the newly described estrogen receptor, GPR30, is involved in the insulinotropic effects of high doses of E2 (100nM-5μM). The specific GPR30 agonist G1 also increases insulin secretion in β-cells. Low glucose-induced calcium oscillations and glucagon secretion are suppressed by E2. The effects on glucagon secretion may be mediated by GPR30. Somatostatin release is also decreased by E2 and G1. In this review we summarize all the data published up to date on the rapid insulinotropic effects of estrogens in the endocrine pancreas and propose a model to integrate the estrogen actions mediated through both receptors. Copyright © 2012 Elsevier Inc. All rights reserved.

Proton-sensing G protein-coupled receptors as regulators of cell proliferation and migration during tumor growth and wound healing.

PubMed

Weiß, Katharina T; Fante, Matthias; Köhl, Gudrun; Schreml, Julia; Haubner, Frank; Kreutz, Marina; Haverkampf, Sonja; Berneburg, Mark; Schreml, Stephan

2017-02-01

Dysregulation of pH is a feature of both tumor growth and tissue repair. In tumors, microenvironmental changes, like in lactate metabolism, lead to altered intra- and extracellular pH (pH i , pH e ) and vice versa. In wounds, barrier disruption results in extensive variations in pH e on the wound surface. It is known that altered extracellular proton concentrations have a major impact on cell turnover and migration as well as on the metabolic activity of cells involved in tumor spread and wound closure. The proton-sensing G protein-coupled receptors (GPCRs) GPR4, GPR65 (TDAG8), GPR68 (OGR1) and GPR132 (G2A) are activated via a decrease in pH e and transduce this signal to molecular intracellular pathways. Based on the current knowledge, we speculate on the role of proton-sensing GPCRs in wound healing and on their potential as mechanistic linkers of tumor growth and tissue repair. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Involvement of estradiol-17beta and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rario.

PubMed

Pang, Yefei; Thomas, Peter

2009-03-01

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20beta-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17beta, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.

Obstructive sleep apnea and obesity are associated with reduced GPR 120 plasma levels in children.

PubMed

Gozal, David; Kheirandish-Gozal, Leila; Carreras, Alba; Khalyfa, Abdelnaby; Peris, Eduard

2014-05-01

Obstructive sleep apnea (OSA) is a common health problem, particularly in obese children, in whom a vicious cycle of obesity and OSA interdependencies promotes increased food intake. G protein-coupled receptor 120 (GPR 120) is a long-chain free fatty acid (FFA) receptor that plays an important role in energy homeostasis, and protects against insulin resistance and systemic inflammation. We hypothesized that GPR 120 levels would be reduced in children with OSA, particularly among obese children. Cross-sectional prospectively recruited cohort. Academic pediatric sleep program. Two hundred twenty-six children (mean age: 7.0 ± 2.1 y) underwent overnight polysomnographic evaluation and a fasting blood draw the morning after the sleep study. In addition to lipid profile, homeostasis model assessment of insulin resistance (HOMA-IR) and high-sensitivity C-reactive protein (hsCRP) assays, monocyte GPR 120 expression, and plasma GPR 120 levels were assessed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay kits. Obese children and those with OSA had significantly lower GPR 120 monocyte expression and plasma GPR 120 levels. Furthermore, when both obesity and OSA were present, GPR 120 levels were lowest. Linear associations emerged between GPR 120 plasma levels and body mass index (BMI) z score, as well as with apnea-hypopnea index (AHI), saturation of peripheral oxygen (SpO2) nadir, and respiratory arousal index (RAI), with RAI remaining statistically significant when controlling for age, ethnicity, sex, and BMI z score (P < 0.001). Similarly, HOMA-IR was significantly associated with GPR 120 levels, but neither low density lipoprotein nor high density lipoprotein cholesterol or hsCRP levels exhibited significant correlations. G protein-coupled receptor 120 (GPR 120) levels are reduced in pediatric OSA and obesity (particularly when both are present) and may play a role in modulating the degree of insulin resistance. The short- and long-term significance of reduced GPR 120 relative to food intake and glycemic deregulation remains undefined.

Development of novel ligands for peptide GPCRs.

PubMed

Moran, Brian M; McKillop, Aine M; O'Harte, Finbarr Pm

2016-12-01

Incretin based glucagon-like peptide-1 receptor (GLP-1R) agonists which target a G-protein coupled receptor (GPCR) are currently used in the treatment of type 2 diabetes. This review focuses on GPCRs from pancreatic β-cells, including GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucagon, somatostatin, pancreatic polypeptide (PP), cholecystokinin (CCK), peptide YY (PYY), oxyntomodulin (OXM) and ghrelin receptors. In addition, fatty acids GPCRs are thought to have an increasing role in regulating peptide secretions namely short fatty acids GPCR (GPR41, GPR43), medium chain fatty acid GPCR (GPR84), long chain fatty acid GPCR (GPR40, GPR120) and cannabinoid-like GPCR (GPR55, GPR119). Several pre-clinical and clinical trials are currently ongoing in peptide GPCR based therapies, including dual and triple agonist peptides which activate two or more GPCRs simultaneously. Copyright © 2016 Elsevier Ltd. All rights reserved.

GPR30 mediates anorectic estrogen-induced STAT3 signaling in the hypothalamus.

PubMed

Kwon, Obin; Kang, Eun Seok; Kim, Insook; Shin, Sora; Kim, Mijung; Kwon, Somin; Oh, So Ra; Ahn, Young Soo; Kim, Chul Hoon

2014-11-01

Estrogen plays an important role in the control of energy balance in the hypothalamus. Leptin-independent STAT3 activation (i.e., tyrosine(705)-phosphorylation of STAT3, pSTAT3) in the hypothalamus is hypothesized as the primary mechanism of the estrogen-induced anorexic response. However, the type of estrogen receptor that mediates this regulation is unknown. We investigated the role of the G protein-coupled receptor 30 (GPR30) in estradiol (E2)-induced STAT3 activation in the hypothalamus. Regulation of STAT3 activation by E2, G-1, a specific agonist of GPR30 and G-15, a specific antagonist of GPR30 was analyzed in vitro and in vivo. Effect of GPR30 activation on eating behavior was analyzed in vivo. E2 stimulated pSTAT3 in cells expressing GPR30, but not expressing estrogen receptor ERα and ERβ. G-1 induced pSTAT3, and G-15 inhibited E2-induced pSTAT3 in primary cultures of hypothalamic neurons. A cerebroventricular injection of G-1 increased pSTAT3 in the arcuate nucleus of mice, which was associated with a decrease in food intake and body weight gain. These results suggest that GPR30 is the estrogen receptor that mediates the anorectic effect of estrogen through the STAT3 pathway in the hypothalamus. Copyright © 2014 Elsevier Inc. All rights reserved.

GPR30 Regulates Glutamate Transporter GLT-1 Expression in Rat Primary Astrocytes*

PubMed Central

Lee, Eunsook; Sidoryk-Wêgrzynowicz, Marta; Wang, Ning; Webb, Anton; Son, Deok-Soo; Lee, Kyuwon; Aschner, Michael

2012-01-01

The G protein-coupled estrogen receptor GPR30 contributes to the neuroprotective effects of 17β-estradiol (E2); however, the mechanisms associated with this protection have yet to be elucidated. Given that E2 increases astrocytic expression of glutamate transporter-1 (GLT-1), which would prevent excitotoxic-induced neuronal death, we proposed that GPR30 mediates E2 action on GLT-1 expression. To investigate this hypothesis, we examined the effects of G1, a selective agonist of GPR30, and GPR30 siRNA on astrocytic GLT-1 expression, as well as glutamate uptake in rat primary astrocytes, and explored potential signaling pathways linking GPR30 to GLT-1. G1 increased GLT-1 protein and mRNA levels, subject to regulation by both MAPK and PI3K signaling. Inhibition of TGF-α receptor suppressed the G1-induced increase in GLT-1 expression. Silencing GPR30 reduced the expression of both GLT-1 and TGF-α and abrogated the G1-induced increase in GLT-1 expression. Moreover, the G1-induced increase in GLT-1 protein expression was abolished by a protein kinase A inhibitor and an NF-κB inhibitor. G1 also enhanced cAMP response element-binding protein (CREB), as well as both NF-κB p50 and NF-κB p65 binding to the GLT-1 promoter. Finally, to model dysfunction of glutamate transporters, manganese was used, and G1 was found to attenuate manganese-induced impairment in GLT-1 protein expression and glutamate uptake. Taken together, the present data demonstrate that activation of GPR30 increases GLT-1 expression via multiple pathways, suggesting that GPR30 is worthwhile as a potential target to be explored for developing therapeutics of excitotoxic neuronal injury. PMID:22645130

Mutation analysis and molecular modeling for the investigation of ligand-binding modes of GPR84.

PubMed

Nikaido, Yoshiaki; Koyama, Yuuta; Yoshikawa, Yasushi; Furuya, Toshio; Takeda, Shigeki

2015-05-01

GPR84 is a G protein-coupled receptor for medium-chain fatty acids. Capric acid and 3,3'-diindolylmethane are specific agonists for GPR84. We built a homology model of a GPR84-capric acid complex to investigate the ligand-binding mode using the crystal structure of human active-state β2-adrenergic receptor. We performed site-directed mutagenesis to subject ligand-binding sites to our model using GPR84-Giα fusion proteins and a [(35)S]GTPγS-binding assay. We compared the activity of the wild type and mutated forms of GPR84 by [(35)S]GTPγS binding to capric acid and diindolylmethane. The mutations L100D `Ballesteros-Weinstein numbering: 3.32), F101Y (3.33) and N104Q (3.36) in the transmembrane helix III and N357D (7.39) in the transmembrane helix VII resulted in reduced capric acid activity but maintained the diindolylmethane responses. Y186F (5.46) and Y186H (5.46) mutations had no characteristic effect on capric acid but with diindolylmethane they significantly affected the G protein activation efficiency. The L100D (3.32) mutant responded to decylamine, a fatty amine, instead of a natural agonist, the fatty acid capric acid, suggesting that we have identified a mutated G protein-coupled receptor-artificial ligand pairing. Our molecular model provides an explanation for these results and interactions between GPR84 and capric acid. Further, from the results of a double stimulation assay, we concluded that diindolylmethane was a positive allosteric modulator for GPR84. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules.

PubMed

Simon, Katharina; Hennen, Stephanie; Merten, Nicole; Blättermann, Stefanie; Gillard, Michel; Kostenis, Evi; Gomeza, Jesus

2016-01-08

Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gαi/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Zinc Supplementation, via GPR39, Upregulates PKCζ to Protect Intestinal Barrier Integrity in Caco-2 Cells Challenged by Salmonella enterica Serovar Typhimurium.

PubMed

Shao, Yu-Xin; Lei, Zhao; Wolf, Patricia G; Gao, Yan; Guo, Yu-Ming; Zhang, Bing-Kun

2017-07-01

Background: Zinc has been shown to improve intestinal barrier function against Salmonella enterica serovar Typhimurium ( S. typhimurium ) infection, but the mechanisms involved in this process remain undefined. Objective: We aimed to explore the roles of G protein-coupled receptor (GPR)39 and protein kinase Cζ (PKCζ) in the regulation by zinc of intestinal barrier function. Methods: A Transwell Caco-2 monolayer was pretreated with 0, 50, or 100 μM Zn and then incubated with S. typhimurium for 0-6 h. Afterward, cells silenced by the small interfering RNA for GPR39 or PKCζ were pretreated with 100 μM Zn and incubated with S. typhimurium for 3 h. Finally, transepithelial electrical resistance (TEER), permeability, tight junction (TJ) proteins, and signaling molecules GPR39 and PKCζ were measured. Results: Compared with controls, S. typhimurium decreased TEER by 62.3-96.2% at 4-6 h ( P < 0.001), increased ( P < 0.001) permeability at 6 h, and downregulated ( P < 0.05) TJ protein zonula occludens (ZO)-1 and occludin by 104-123%, as well as Toll-like receptor 2 and PKCζ by 35.1% and 75.2%, respectively. Compared with S. typhimurium- challenged cells, 50 and 100 μM Zn improved TEER by 26.3-60.9% at 4-6 h ( P < 0.001) and decreased ( P < 0.001) permeability and bacterial invasion at 6 h. A total of 100 μM Zn increased ZO-1, occludin, GPR39, and PKCζ 0.72- to 1.34-fold ( P < 0.05); however, 50 μM Zn did not affect ZO-1 or occludin ( P > 0.1). Silencing GPR39 decreased ( P < 0.05) zinc-activated PKCζ and blocked ( P < 0.05) the promotion of zinc on epithelial integrity. Furthermore, silencing PKCζ counteracted the protective effect of zinc on epithelial integrity but did not inhibit GPR39 ( P = 0.138). Conclusion: We demonstrated that zinc upregulates PKCζ by activating GPR39 to enhance the abundance of ZO-1, thereby improving epithelial integrity in S. typhimurium- infected Caco-2 cells. © 2017 American Society for Nutrition.

Neuro-psychopharmacological perspective of Orphan receptors of Rhodopsin (class A) family of G protein-coupled receptors.

PubMed

Khan, Muhammad Zahid; He, Ling

2017-04-01

In the central nervous system (CNS), G protein-coupled receptors (GPCRs) are the most fruitful targets for neuropsychopharmacological drug development. Rhodopsin (class A) is the most studied class of GPCR and includes orphan receptors for which the endogenous ligand is not known or is unclear. Characterization of orphan GPCRs has proven to be challenging, and the production pace of GPCR-based drugs has been incredibly slow. Determination of the functions of these receptors may provide unexpected insight into physiological and neuropathological processes. Advances in various methods and techniques to investigate orphan receptors including in situ hybridization and knockdown/knockout (KD/KO) showed extensive expression of these receptors in the mammalian brain and unmasked their physiological and neuropathological roles. Due to these rapid progress and development, orphan GPCRs are rising as a new and promising class of drug targets for neurodegenerative diseases and psychiatric disorders. This review presents a neuropsychopharmacological perspective of 26 orphan receptors of rhodopsin (class A) family, namely GPR3, GPR6, GPR12, GPR17, GPR26, GPR35, GPR39, GPR48, GPR49, GPR50, GPR52, GPR55, GPR61, GPR62, GPR63, GPR68, GPR75, GPR78, GPR83, GPR84, GPR85, GPR88, GPR153, GPR162, GPR171, and TAAR6. We discussed the expression of these receptors in mammalian brain and their physiological roles. Furthermore, we have briefly highlighted their roles in neurodegenerative diseases and psychiatric disorders including Alzheimer's disease, Parkinson's disease, neuroinflammation, inflammatory pain, bipolar and schizophrenic disorders, epilepsy, anxiety, and depression.

Fluoxetine, 17-β estradiol or folic acid combined with intra-lateral septal infusions of neuropeptide Y produced antidepressant-like actions in ovariectomized rats forced to swim.

PubMed

Molina-Hernández, Miguel; Téllez-Alcántara, N Patricia

2011-12-01

Folic acid is antidepressant, either alone or combined with several antidepressant drugs. However, the antidepressant-like actions of folic acid combined with intra-lateral septal (LSN) infusions of neuropeptide Y (NPY) in the forced swimming test (FST) have not been tested before. Thus, systemic injections of fluoxetine (20.0mg/kg, P<0.05; s.c.) or 17-β estradiol (10.0 μg/rat, P<0.05; s.c.) or oral administrations of folic acid (50.0 mg/kg, P<0.05; 75.0 mg/kg, P<0.05) or NPY intra-LSN (3.0 μg, P<0.05; 3.5 μg, P<0.05) reduced immobility of ovariectomized Wistar rats. Subthreshold doses of: folic acid (25.0 mg/kg) or 17-β estradiol (5.0 μg/rat, P<0.05) or fluoxetine (15.0 mg/kg, P<0.05; s.c.) combined with subthreshold doses of NPY (2.5 μg/rat, P<0.05; intra-LSN) and these combinations produced antidepressant-like actions; which were canceled by BIBP 3226 (a NPY-Y1 receptor antagonist). It is concluded that folic acid produced antidepressant-like effects probably through the participation of the NPY Y1 receptors found in the lateral septal nuclei. Copyright © 2011 Elsevier Inc. All rights reserved.

Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses

PubMed Central

Tobo, Ayaka; Tobo, Masayuki; Nakakura, Takashi; Ebara, Masashi; Tomura, Hideaki; Mogi, Chihiro; Im, Dong-Soon; Murata, Naoya; Kuwabara, Atsushi; Ito, Saki; Fukuda, Hayato; Arisawa, Mitsuhiro; Shuto, Satoshi; Nakaya, Michio; Kurose, Hitoshi; Sato, Koichi; Okajima, Fumikazu

2015-01-01

G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions. PMID:26070068

Characterization of Imidazopyridine Compounds as Negative Allosteric Modulators of Proton-Sensing GPR4 in Extracellular Acidification-Induced Responses.

PubMed

Tobo, Ayaka; Tobo, Masayuki; Nakakura, Takashi; Ebara, Masashi; Tomura, Hideaki; Mogi, Chihiro; Im, Dong-Soon; Murata, Naoya; Kuwabara, Atsushi; Ito, Saki; Fukuda, Hayato; Arisawa, Mitsuhiro; Shuto, Satoshi; Nakaya, Michio; Kurose, Hitoshi; Sato, Koichi; Okajima, Fumikazu

2015-01-01

G protein-coupled receptor 4 (GPR4), previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G protein-coupled receptor (GPCR) coupling to multiple intracellular signaling pathways, including the Gs protein/cAMP and G13 protein/Rho. In the present study, we characterized some imidazopyridine compounds as GPR4 modulators that modify GPR4 receptor function. In the cells that express proton-sensing GPCRs, including GPR4, OGR1, TDAG8, and G2A, extracellular acidification stimulates serum responsive element (SRE)-driven transcriptional activity, which has been shown to reflect Rho activity, with different proton sensitivities. Imidazopyridine compounds inhibited the moderately acidic pH-induced SRE activity only in GPR4-expressing cells. Acidic pH-stimulated cAMP accumulation, mRNA expression of inflammatory genes, and GPR4 internalization within GPR4-expressing cells were all inhibited by the GPR4 modulator. We further compared the inhibition property of the imidazopyridine compound with psychosine, which has been shown to selectively inhibit actions induced by proton-sensing GPCRs, including GPR4. In the GPR4 mutant, in which certain histidine residues were mutated to phenylalanine, proton sensitivity was significantly shifted to the right, and psychosine failed to further inhibit acidic pH-induced SRE activation. On the other hand, the imidazopyridine compound almost completely inhibited acidic pH-induced action in mutant GPR4. We conclude that some imidazopyridine compounds show specificity to GPR4 as negative allosteric modulators with a different action mode from psychosine, an antagonist susceptible to histidine residues, and are useful for characterizing GPR4-mediated acidic pH-induced biological actions.

GPR39 (zinc receptor) knockout mice exhibit depression-like behavior and CREB/BDNF down-regulation in the hippocampus.

PubMed

Młyniec, Katarzyna; Budziszewska, Bogusława; Holst, Birgitte; Ostachowicz, Beata; Nowak, Gabriel

2014-10-31

Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. In the present study, we investigated whether GPR39 knockout would cause depressive-like and/or anxiety-like behavior, as measured by the forced swim test, tail suspension test, and light/dark test. We also investigated whether lack of GPR39 would change levels of cAMP response element-binding protein (CREB),brain-derived neurotrophic factor (BDNF) and tropomyosin related kinase B (TrkB) protein in the hippocampus and frontal cortex of GPR39 knockout mice subjected to the forced swim test, as measured by Western-blot analysis. In this study, GPR39 knockout mice showed an increased immobility time in both the forced swim test and tail suspension test, indicating depressive-like behavior and displayed anxiety-like phenotype. GPR39 knockout mice had lower CREB and BDNF levels in the hippocampus, but not in the frontal cortex, which indicates region specificity for the impaired CREB/BDNF pathway (which is important in antidepressant response) in the absence of GPR39. There were no changes in TrkB protein in either structure. In the present study, we also investigated activity in the hypothalamus-pituitary-adrenal axis under both zinc- and GPR39-deficient conditions. Zinc-deficient mice had higher serum corticosterone levels and lower glucocorticoid receptor levels in the hippocampus and frontal cortex. There were no changes in the GPR39 knockout mice in comparison with the wild-type control mice, which does not support a role of GPR39 in hypothalamus-pituitary-adrenal axis regulation. The results of this study indicate the involvement of the GPR39 Zn(2+)-sensing receptor in the pathophysiology of depression with component of anxiety. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Aldosterone sensitizes connecting tubule glomerular feedback via the aldosterone receptor GPR30

PubMed Central

Ren, YiLin; D'Ambrosio, Martin A.; Garvin, Jeffrey L.; Leung, Pablo; Kutskill, Kristopher; Wang, Hong; Peterson, Edward L.

2014-01-01

Increasing Na delivery to epithelial Na channels (ENaC) in the connecting tubule (CNT) dilates the afferent arteriole (Af-Art), a process we call connecting tubule glomerular feedback (CTGF). We hypothesize that aldosterone sensitizes CTGF via a nongenomic mechanism that stimulates CNT ENaC via the aldosterone receptor GPR30. Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing luminal NaCl in the CNT. During the control period, the concentration of NaCl that elicited a half-maximal response (EC50) was 37.0 ± 2.0 mmol/l; addition of aldosterone 10−8 mol/l to the CNT lumen caused a left-shift (decrease) in EC50 to 19.3 ± 1.3 mmol/l (P = 0.001 vs. control; n = 6). Neither the transcription inhibitor actinomycin D nor the translation inhibitor cycloheximide prevented the effect of aldosterone (control EC50 = 34.7 ± 1.9 mmol/l; aldosterone+actinomycin D EC50 = 22.6 ± 1.6 mmol/l; P < 0.001 and control EC50 = 32.4 ± 4.3 mmol/l; aldosterone+cycloheximide EC50 = 17.4 ± 3.3 mmol/l; P < 0.001). The aldosterone antagonist eplerenone prevented the sensitization of CTGF by aldosterone (control EC50 = 33.2 ± 1.7 mmol/l; aldosterone+eplerenone EC50 = 33.5 ± 1.3 mmol/l; n = 7). The GPR30 receptor blocker G-36 blocked the sensitization of CTGF by aldosterone (aldosterone EC50 = 16.5 ± 1.9 mmol/l; aldosterone+G-36 EC50 = 29.0 ± 2.1 mmol/l; n = 7; P < 0.001). Finally, we found that the sensitization of CTGF by aldosterone was mediated, at least in part, by the sodium/hydrogen exchanger (NHE). We conclude that aldosterone in the CNT lumen sensitizes CTGF via a nongenomic effect involving GPR30 receptors and NHE. Sensitized CTGF induced by aldosterone may contribute to renal damage by increasing Af-Art dilation and glomerular capillary pressure (glomerular barotrauma). PMID:24966088

Estrogen stimulated migration and invasion of estrogen receptor-negative breast cancer cells involves an ezrin-dependent crosstalk between G protein-coupled receptor 30 and estrogen receptor beta signaling.

PubMed

Zhou, Kewen; Sun, Peng; Zhang, Yaxing; You, Xinchao; Li, Ping; Wang, Tinghuai

2016-07-01

Estrogen mediates important cellular activities in estrogen receptor negative (ER-) breast cancer cells via membrane associated G protein-coupled receptor 30 (GPR30). However, the biological role and mechanism of estrogen action on cell motility and invasion in this aggressive kind of tumors remains poorly understood. We showed here that treatment with 17β-estradiol (E2) in ER-negative cancer cells resulted in ezrin-dependent cytoskeleton rearrangement and elicited a stimulatory effect on cell migration and invasion. Mechanistically, E2 induced ezrin activation was mediated by distinct mechanisms in different cell contexts. In SK-BR-3 cells with a high GPR30/ERβ ratio, silencing of GPR30 was able to abolish E2 induced ERK1/2, AKT phosphorylation and ezrin activation, whereas in MDA-MB-231 cells with low GPR30/ERβ ratio, E2 stimulated ezrin activation was mediated by the ERβ/PI3K/AKT signaling pathway. Importantly, we showed that activation of GPR30 signaling significantly prevents ERβ activation induced ezrin phosphorylation, cell migration and invasion, indicating an antagonist effect between GPR30 and ERβ signaling in MDA-MB-231 cells. These findings highlight the important interplay between different estrogen receptors in estrogen induced cell motility and invasiveness in ER-negative breast cancer cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Increased Retinal Expression of the Pro-Angiogenic Receptor GPR91 via BMP6 in a Mouse Model of Juvenile Hemochromatosis

PubMed Central

Arjunan, Pachiappan; Gnanaprakasam, Jaya P.; Ananth, Sudha; Romej, Michelle A.; Rajalakshmi, Veeranan-Karmegam; Prasad, Puttur D.; Martin, Pamela M.; Gurusamy, Mariappan; Thangaraju, Muthusamy; Bhutia, Yangzom D.; Ganapathy, Vadivel

2016-01-01

Purpose Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone morphogenetic protein (BMP) coreceptor, underlie most of the juvenile type. Hjv−/− mice accumulate excess iron in retina and exhibit aberrant vascularization and angiomas. A succinate receptor, GPR91, is pro-angiogenic in retina. We hypothesized that Hjv−/− retinas have increased BMP signaling and increased GPR91 expression as the basis of angiomas. Methods Expression of GPR91 was examined by qPCR, immunofluorescence, and Western blot in wild-type and Hjv−/− mouse retinas and pRPE cells. Influence of excess iron and BMP6 on GPR91 expression was investigated in ARPE-19 cells, and wild-type and Hjv−/− pRPE cells. Succinate was used to activate GPR91 and determine the effects of GPR91 signaling on VEGF expression. Signaling of BMP6 was studied by the expression of Smad1/5/8 and pSmad4, and the BMP-target gene Id1. The interaction of pSmad4 with GPR91 promoter was studied by ChIP. Results Expression of GPR91 was higher in Hjv−/− retinas and RPE than in wild-type counterparts. Unexpectedly, BMP signaling was increased, not decreased, in Hjv−/− retinas and RPE. Bone morphogenetic protein 6 induced GPR91 in RPE, suggesting that increased BMP signaling in Hjv−/− retinas was likely responsible for GPR91 upregulation. Exposure of RPE to excess iron and succinate as well as BMP6 and succinate increased VEGF expression. Bone morphogenetic protein 6 promoted the interaction of pSmad4 with GPR91 promoter in RPE. Conclusions G-protein-coupled receptor 91 is a BMP6 target and Hjv deletion enhances BMP signaling in retina, thus underscoring a role for excess iron and hemochromatosis in abnormal retinal vascularization. PMID:27046124

Increased Retinal Expression of the Pro-Angiogenic Receptor GPR91 via BMP6 in a Mouse Model of Juvenile Hemochromatosis.

PubMed

Arjunan, Pachiappan; Gnanaprakasam, Jaya P; Ananth, Sudha; Romej, Michelle A; Rajalakshmi, Veeranan-Karmegam; Prasad, Puttur D; Martin, Pamela M; Gurusamy, Mariappan; Thangaraju, Muthusamy; Bhutia, Yangzom D; Ganapathy, Vadivel

2016-04-01

Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone morphogenetic protein (BMP) coreceptor, underlie most of the juvenile type. Hjv(-/-) mice accumulate excess iron in retina and exhibit aberrant vascularization and angiomas. A succinate receptor, GPR91, is pro-angiogenic in retina. We hypothesized that Hjv(-/-) retinas have increased BMP signaling and increased GPR91 expression as the basis of angiomas. Expression of GPR91 was examined by qPCR, immunofluorescence, and Western blot in wild-type and Hjv(-/-) mouse retinas and pRPE cells. Influence of excess iron and BMP6 on GPR91 expression was investigated in ARPE-19 cells, and wild-type and Hjv(-/-) pRPE cells. Succinate was used to activate GPR91 and determine the effects of GPR91 signaling on VEGF expression. Signaling of BMP6 was studied by the expression of Smad1/5/8 and pSmad4, and the BMP-target gene Id1. The interaction of pSmad4 with GPR91 promoter was studied by ChIP. Expression of GPR91 was higher in Hjv(-/-) retinas and RPE than in wild-type counterparts. Unexpectedly, BMP signaling was increased, not decreased, in Hjv(-/-) retinas and RPE. Bone morphogenetic protein 6 induced GPR91 in RPE, suggesting that increased BMP signaling in Hjv(-/-) retinas was likely responsible for GPR91 upregulation. Exposure of RPE to excess iron and succinate as well as BMP6 and succinate increased VEGF expression. Bone morphogenetic protein 6 promoted the interaction of pSmad4 with GPR91 promoter in RPE. G-protein-coupled receptor 91 is a BMP6 target and Hjv deletion enhances BMP signaling in retina, thus underscoring a role for excess iron and hemochromatosis in abnormal retinal vascularization.

Insulinotropic and antidiabetic effects of 17β-estradiol and the GPR30 agonist G-1 on human pancreatic islets.

PubMed

Kumar, Rajesh; Balhuizen, Alexander; Amisten, Stefan; Lundquist, Ingmar; Salehi, Albert

2011-07-01

We have recently shown that 17β-estradiol (E2) and the synthetic G protein-coupled receptor 30 (GPR30) ligand G-1 have antiapoptotic actions in mouse pancreatic islets, raising the prospect that they might exert beneficial effects also in human islets. The objective of the present study was to identify the expression of GPR30 in human islets and clarify the role of GPR30 in islet hormone secretion and β-cell survival. GPR30 expression was analyzed by confocal microscopy, Western blot, and quantitative PCR in islets from female and male donors. Hormone secretion, phosphatidylinositol hydrolysis, cAMP content, and caspase-3 activity in female islets were determined with conventional methods and apoptosis with the annexin-V method. Confocal microscopy revealed GPR30 expression in islet insulin, glucagon, and somatostatin cells. GPR30 mRNA and protein expression was markedly higher in female vs. male islets. An amplifying effect of G-1 or E2 on cAMP content and insulin secretion from isolated female islets was not influenced by the E2 genomic receptor (ERα and ERβ) antagonists ICI 182,780 and EM-652. Cytokine-induced (IL-1β plus TNFα plus interferon-γ) apoptosis in islets cultured for 24 h at 5 mmol/liter glucose was almost abolished by G-1 or E2 treatment and was not affected by the nuclear estrogen receptor antagonists. Concentration-response studies on female islets from healthy controls and type 2 diabetic subjects showed that both E2 and G-1 displayed important antidiabetic actions by improving glucose-stimulated insulin release while suppressing glucagon and somatostatin secretion. In view of these findings, we propose that small molecules activating GPR30 could be promising in the therapy of diabetes mellitus.

G protein-coupled receptor 30 (GPR30) forms a plasma membrane complex with membrane-associated guanylate kinases (MAGUKs) and protein kinase A-anchoring protein 5 (AKAP5) that constitutively inhibits cAMP production.

PubMed

Broselid, Stefan; Berg, Kelly A; Chavera, Teresa A; Kahn, Robin; Clarke, William P; Olde, Björn; Leeb-Lundberg, L M Fredrik

2014-08-08

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Involvement of estrogen receptor variant ER-alpha36, not GPR30, in nongenomic estrogen signaling.

PubMed

Kang, Lianguo; Zhang, Xintian; Xie, Yan; Tu, Yaping; Wang, Dong; Liu, Zhenming; Wang, Zhao-Yi

2010-04-01

Accumulating evidence suggested that an orphan G protein-coupled receptor (GPR)30, mediates nongenomic responses to estrogen. The present study was performed to investigate the molecular mechanisms underlying GPR30 function. We found that knockdown of GPR30 expression in breast cancer SK-BR-3 cells down-regulated the expression levels of estrogen receptor (ER)-alpha36, a variant of ER-alpha. Introduction of a GPR30 expression vector into GPR30 nonexpressing cells induced endogenous ER-alpha36 expression, and cotransfection assay demonstrated that GPR30 activated the promoter activity of ER-alpha36 via an activator protein 1 binding site. Both 17beta-estradiol (E2) and G1, a compound reported to be a selective GPR30 agonist, increased the phosphorylation levels of the MAPK/ERK1/2 in SK-BR-3 cells, which could be blocked by an anti-ER-alpha36-specific antibody against its ligand-binding domain. G1 induced activities mediated by ER-alpha36, such as transcription activation activity of a VP16-ER-alpha36 fusion protein and activation of the MAPK/ERK1/2 in ER-alpha36-expressing cells. ER-alpha36-expressing cells, but not the nonexpressing cells, displayed high-affinity, specific E2 and G1 binding, and E2- and G1-induced intracellular Ca(2+) mobilization only in ER-alpha36 expressing cells. Taken together, our results demonstrated that previously reported activities of GPR30 in response to estrogen were through its ability to induce ER-alpha36 expression. The selective G protein-coupled receptor (GPR)30 agonist G1 actually interacts with ER-alpha36. Thus, the ER-alpha variant ER-alpha36, not GPR30, is involved in nongenomic estrogen signaling.

Metabolite-Sensing G Protein-Coupled Receptors-Facilitators of Diet-Related Immune Regulation.

PubMed

Tan, Jian K; McKenzie, Craig; Mariño, Eliana; Macia, Laurence; Mackay, Charles R

2017-04-26

Nutrition and the gut microbiome regulate many systems, including the immune, metabolic, and nervous systems. We propose that the host responds to deficiency (or sufficiency) of dietary and bacterial metabolites in a dynamic way, to optimize responses and survival. A family of G protein-coupled receptors (GPCRs) termed the metabolite-sensing GPCRs bind to various metabolites and transmit signals that are important for proper immune and metabolic functions. Members of this family include GPR43, GPR41, GPR109A, GPR120, GPR40, GPR84, GPR35, and GPR91. In addition, bile acid receptors such as GPR131 (TGR5) and proton-sensing receptors such as GPR65 show similar features. A consistent feature of this family of GPCRs is that they provide anti-inflammatory signals; many also regulate metabolism and gut homeostasis. These receptors represent one of the main mechanisms whereby the gut microbiome affects vertebrate physiology, and they also provide a link between the immune and metabolic systems. Insufficient signaling through one or more of these metabolite-sensing GPCRs likely contributes to human diseases such as asthma, food allergies, type 1 and type 2 diabetes, hepatic steatosis, cardiovascular disease, and inflammatory bowel diseases.

Analysis of G-Protein Coupled Receptor 30 (GPR30) on Endothelial Inflammation.

PubMed

Chakrabarti, Subhadeep; Davidge, Sandra T

2016-01-01

The female sex hormone estrogen (the most common form 17-β-estradiol or E2) is known to have both anti-inflammatory and pro-inflammatory effects. Given the diversity of estrogen responses mediated through its three distinct receptors, namely, estrogen receptor α (ERα), ERβ, and the G-protein coupled receptor 30 (GPR30), it is plausible that different receptors have specific modulatory effects on inflammation in different tissues. We have shown that activation of GPR30 exerted anti-inflammatory effects as demonstrated by significant attenuation of tumor necrosis factor (TNF)-mediated upregulation of adhesion molecules in isolated human umbilical vein endothelial cells. Interestingly, estrogen alone had no such effect and blockade of classical ERs restored the anti-inflammatory effect, suggesting that this effect was dependent on GPR30 and opposed to classical ERs. These findings were further validated by the negation of anti-inflammatory GPR30 effects by classical ER agonists. This chapter focuses on multiple pharmacological options to activate GPR30 and the use of TNF activated endothelial cells as a model system for inflammatory response as assessed by adhesion molecule detection through western blotting.

Regulation of neuronal pH by the metabotropic Zn(2+)-sensing Gq-coupled receptor, mZnR/GPR39.

PubMed

Ganay, Thibault; Asraf, Hila; Aizenman, Elias; Bogdanovic, Milos; Sekler, Israel; Hershfinkel, Michal

2015-12-01

Synaptically released Zn(2+) acts as a neurotransmitter, in part, by activating the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39). In previous work using epithelial cells, we described crosstalk between Zn(2+) signaling and changes in intracellular pH and/or extracellular pH (pHe). As pH changes accompany neuronal activity under physiological and pathological conditions, we tested whether Zn(2+) signaling is involved in regulation of neuronal pH. Here, we report that up-regulation of a major H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), is induced by mZnR/GPR39 activation in an extracellular-regulated kinase 1/2-dependent manner in hippocampal neurons in vitro. We also observed that changes in pHe can modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. Similarly, Zn(2+)-dependent extracellular-regulated kinase 1/2 phosphorylation and up-regulation of NHE activity were absent at acidic pHe. Thus, our results suggest that when pHe is maintained within the physiological range, mZnR/GPR39 activation can up-regulate NHE-dependent recovery from intracellular acidification. During acidosis, as pHe drops, mZnR/GPR39-dependent NHE activation is inhibited, thereby attenuating further H(+) extrusion. This mechanism may serve to protect neurons from excessive decreases in pHe. Thus, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain. We show that the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39) activation induces up-regulation of a major neuronal H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), thereby enhancing neuronal recovery from intracellular acidification. Changes in extracellular pH (pHe), however, modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. This mechanism may serve to protect neurons from excessive decreases in pHe during acidosis. Hence, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain. © 2015 International Society for Neurochemistry.

Sex steroid receptors in male human bladder: expression and biological function.

PubMed

Chavalmane, Aravinda K; Comeglio, Paolo; Morelli, Annamaria; Filippi, Sandra; Fibbi, Benedetta; Vignozzi, Linda; Sarchielli, Erica; Marchetta, Matilde; Failli, Paola; Sandner, Peter; Saad, Farid; Gacci, Mauro; Vannelli, Gabriella B; Maggi, Mario

2010-08-01

In male, lower urinary tract symptoms (LUTS) have been associated, beside benign prostatic hyperplasia, to some unexpected comorbidities (hypogonadism, obesity, metabolic syndrome), which are essentially characterized by an unbalance between circulating androgens/estrogens. Within the bladder, LUTS are linked to RhoA/Rho-kinase (ROCK) pathway overactivity. To investigate the effects of changing sex steroids on bladder smooth muscle. ER α, ER β, GPR30/GPER1 and aromatase mRNA expression was analyzed in male genitourinary tract tissues, and cells isolated from bladder, prostate, and urethra. Estrogen and G1 effect on RhoA/ROCK signaling output like cell migration, gene expression, and cytoskeletal remodeling, and [Ca(2+) ](i) was also studied in hB cells. Contractile studies on bladder strips from castrated male rats supplemented with estradiol and testosterone was also performed. The effects of classical (ER α, ER β) and nonclassical (GPR30/GPER1) estrogen receptor ligands (17 β-estradiol and G1, respectively) and androgens on RhoA/ROCK-.mediated cell functions were studied in hB cells. Contractility studies were also performed in bladder strips from castrated male rats supplemented with testosterone or estradiol. Aromatase and sex steroid receptors, including GPR30, were expressed in human bladder and mediates several biological functions. Both 17 β-estradiol and G1 activated calcium transients and induced RhoA/ROCK signaling (cell migration, cytoskeleton remodeling and smooth muscle gene expression). RhoA/ROCK inhibitors blunted these effects. Estrogen-, but not androgen-supplementation to castrated rats increased sensitivity to the ROCK inhibitor, Y-27632 in isolated bladder strips. In hB cells, testosterone elicited effects similar to estrogen, which were abrogated by blocking its aromatization through letrozole. Our data indicate for the first time that estrogen-more than androgen-receptors up-regulate RhoA/ROCK signaling. Since an altered estrogen/androgen ratio characterizes conditions, such as aging, obesity and metabolic syndrome, often associated to LUTS, we speculate that a relative hyperestrogenism may induce bladder overactivity through the up-regulation of RhoA/ROCK pathway. © 2010 International Society for Sexual Medicine.

Nuclear alternate estrogen receptor GPR30 mediates 17beta-estradiol-induced gene expression and migration in breast cancer-associated fibroblasts.

PubMed

Madeo, Antonio; Maggiolini, Marcello

2010-07-15

Fibroblasts are the principal cellular component of connective tissue and are associated with cancer cells at all stages of tumor progression. Structural and functional contributions of fibroblasts to the growth, survival, and invasive capacity of cancer cells are beginning to emerge. In breast carcinoma, approximately 80% of stromal fibroblasts termed cancer-associated fibroblasts (CAF) are thought to manifest an activated phenotype that promotes cancer cell proliferation tumor growth at metastatic sites similar to the primary tumor. In this report, we show that CAFs respond to physiologic concentrations of 17beta-estradiol (E2) by rapidly inducing extracellular signal-regulated kinase phosphorylation and immediate early gene expression, including c-fos and connective tissue growth factor, and cyclin D1. Notably, the E2 response is mediated by the alternate estrogen receptor GPR30, which interfaces with the epidermal growth factor receptor (EGFR) signaling pathway. In particular, E2 stimulates a physical interaction between GPR30 and phosphorylated EGFR, recruiting them to the cyclin D1 gene promoter. Nuclear localization induced by E2 was confirmed by cellular immunofluorescence methods. GPR30 was required for CAF proliferation and migration induced by E2. Our results provide important new mechanistic insights into how CAFs are stimulated by estrogen through a GPR30-mediated nuclear signaling pathway. More generally, they define estrogenic GPR30 signaling as a functionally important component of the tumor microenvironment. (c)2010 AACR.

The role of parkinson's disease-associated receptor GPR37 in the hippocampus: functional interplay with the adenosinergic system.

PubMed

Lopes, João P; Morató, Xavier; Souza, Carolina; Pinhal, Cindy; Machado, Nuno J; Canas, Paula M; Silva, Henrique B; Stagljar, Igor; Gandía, Jorge; Fernández-Dueñas, Víctor; Luján, Rafael; Cunha, Rodrigo A; Ciruela, Francisco

2015-07-01

GPR37 is an orphan G protein-coupled receptor mostly enriched in brain areas such as the cerebellum, striatum, and hippocampus. Identified as a substrate of parkin, GPR37 has been suggested to play a role in Parkinson's disease. Distributed throughout the brain, the function of GPR37, however, remains unknown. We now provide the first mapping of GPR37 within the hippocampus, where GPR37 is widely expressed and localized at the level of the extrasynaptic plasma membrane of dendritic spines, dendritic shafts, and axon terminals. GPR37 per se does not appear to play a role in learning and memory, since knocking out GPR37 (GPR37-KO) did not alter the performance in different hippocampal-related memory tasks. This is in agreement with slice electrophysiology experiments showing no differences both in short-term plasticity paired-pulse facilitation and long-term potentiation between WT and GPR37-KO mice. However, we report a potential functional interaction between GPR37 and adenosine A2A receptors (A2 A R) in the hippocampus, with A2 A R modulating the GPR37-associated phenotype. Thus, the absence of GPR37 appeared to sensitize mice to hippocampal A2 A R-mediated signaling, as observed by the effect of the A2 A R antagonist SCH58261 increasing synaptic depotentiation, reducing novel object recognition memory and reverting the anxiolytic effect of GPR37 deletion. Collectively, these findings afford insight into the localization and role of the orphan GPR37 within the hippocampus with potential involvement in A2 A R function (i.e., A2 A R sensitization). GPR37 is an orphan G protein-coupled receptor widely expressed in the hippocampus and localized at the level of the extrasynaptic plasma membrane of dendritic spines, dendritic shafts and axon terminals. This orphan receptor per se does not appear to directly control the learning and memory processes; however knocking-out GPR37 triggers anxiolytic-like effects and sensitizes mice to hippocampal A2A R-mediated signalling. © 2015 International Society for Neurochemistry.

Antagonists for the orphan G-protein-coupled receptor GPR55 based on a coumarin scaffold.

PubMed

Rempel, Viktor; Volz, Nicole; Gläser, Franziska; Nieger, Martin; Bräse, Stefan; Müller, Christa E

2013-06-13

The orphan G-protein-coupled receptor GPR55, which is activated by 1-lysophosphatidylinositol and interacts with cannabinoid (CB) receptor ligands, has been proposed as a new potential drug target for the treatment of diabetes, Parkinson's disease, neuropathic pain, and cancer. We applied β-arrestin assays to identify 3-substituted coumarins as a novel class of antagonists and performed an extensive structure-activity relationship study for GPR55. Selectivity versus the related receptors CB1, CB2, and GPR18 was assessed. Among the 7-unsubstituted coumarins selective, competitive GPR55 antagonists were identified, such as 3-(2-hydroxybenzyl)-5-isopropyl-8-methyl-2H-chromen-2-one (12, PSB-SB-489, IC50 = 1.77 μM, pA2 = 0.547 μM). Derivatives with long alkyl chains in position 7 were potent, possibly allosteric GPR55 antagonists which showed ancillary CB receptor affinity. 7-(1,1-Dimethyloctyl)-5-hydroxy-3-(2-hydroxybenzyl)-2H-chromen-2-one (69, PSB-SB-487, IC50 = 0.113 μM, KB = 0.561 μM) and 7-(1,1-dimethylheptyl)-5-hydroxy-3-(2-hydroxybenzyl)-2H-chromen-2-one (67, PSB-SB-1203, IC50 = 0.261 μM) were the most potent GPR55 antagonists of the present series.

A neuropeptide ligand of the G protein-coupled receptor GPR103 regulates feeding, behavioral arousal, and blood pressure in mice.

PubMed

Takayasu, Shinobu; Sakurai, Takeshi; Iwasaki, Satoshi; Teranishi, Hitoshi; Yamanaka, Akihiro; Williams, S Clay; Iguchi, Haruhisa; Kawasawa, Yuka Imamura; Ikeda, Yukio; Sakakibara, Iori; Ohno, Kousaku; Ioka, Ryoichi X; Murakami, Saori; Dohmae, Naoshi; Xie, Jian; Suda, Toshihiro; Motoike, Toshiyuki; Ohuchi, Takashi; Yanagisawa, Masashi; Sakai, Juro

2006-05-09

Here, we report the isolation and characterization of an endogenous peptide ligand of GPR103 from rat brains. The purified peptide was found to be the 43-residue RF-amide peptide QRFP. We also describe two mouse homologues of human GPR103, termed mouse GPR103A and GPR103B. QRFP binds and activates the human GPR103, as well as mouse GPR103A and GPR103B, with nanomolar affinities in transfected cells. Systematic in situ hybridization analysis in mouse brains showed that QRFP is expressed exclusively in the periventricular and lateral hypothalamus, whereas the two receptor mRNAs are distinctly localized in various brain areas without an overlap to each other. When administered centrally in mice, QRFP induced feeding behavior, accompanied by increased general locomotor activity and metabolic rate. QRFP-induced food intake was abolished by preadministration of BIBP3226, a specific antagonist for the Y1 neuropeptide Y receptor. Hypothalamic prepro-QRFP mRNA expression was up-regulated upon fasting and in genetically obese ob/ob and db/db mice. Central QRFP administration also evoked highly sustained elevation of blood pressure and heart rate. Our findings suggest that QRFP and GPR103A/B may regulate diverse neuroendocrine and behavioral functions and implicate this neuropeptide system in metabolic syndrome.

The activation of G protein-coupled receptor 30 (GPR30) inhibits proliferation of estrogen receptor-negative breast cancer cells in vitro and in vivo.

PubMed

Wei, W; Chen, Z-J; Zhang, K-S; Yang, X-L; Wu, Y-M; Chen, X-H; Huang, H-B; Liu, H-L; Cai, S-H; Du, J; Wang, H-S

2014-10-02

There is an urgent clinical need for safe and effective treatment agents and therapy targets for estrogen receptor negative (ER-) breast cancer. G protein-coupled receptor 30 (GPR30), which mediates non-genomic signaling of estrogen to regulate cell growth, is highly expressed in ER--breast cancer cells. We here showed that activation of GPR30 by the receptor-specific agonist G-1 inhibited the growth of ER--breast cancer cells in vitro. Treatment of ER--breast cancer cells with G-1 resulted in G2/M-phase arrest, downregulation of G2-checkpoint regulator cyclin B, and induction of mitochondrial-related apoptosis. The G-1 treatment increased expression of p53 and its phosphorylation levels at Serine 15, promoted its nuclear translocation, and inhibited its ubiquitylation, which mediated the growth arrest effects on cell proliferation. Further, the G-1 induced sustained activation and nuclear translocation of ERK1/2, which was mediated by GPR30/epidermal growth factor receptor (EGFR) signals, also mediated its inhibition effects of G-1. With extensive use of siRNA-knockdown experiments and inhibitors, we found that upregulation of p21 by the cross-talk of GPR30/EGFR and p53 was also involved in G-1-induced cell growth arrest. In vivo experiments showed that G-1 treatment significantly suppressed the growth of SkBr3 xenograft tumors and increased the survival rate, associated with proliferation suppression and upregulation of p53, p21 while downregulation of cyclin B. The discovery of multiple signal pathways mediated the suppression effects of G-1 makes it a promising candidate drug and lays the foundation for future development of GPR30-based therapies for ER- breast cancer treatment.

G protein-coupled receptor 30 regulates trophoblast invasion and its deficiency is associated with preeclampsia.

PubMed

Tong, Chao; Feng, Xiang; Chen, Jun; Qi, Xingchen; Zhou, Liyuan; Shi, Shuming; Kc, Kamana; Stanley, Joanna L; Baker, Philip N; Zhang, Hua

2016-04-01

Preeclampsia is known to be associated with reduced circulating levels of estrogen. The effects of estrogen in preeclampsia are normally mediated by the classical estrogen receptors. Intriguingly, a novel estrogen receptor, G protein-coupled receptor 30 (GPR30), has been recently found to play an important role in several estrogenic effects. However, the mechanisms by which GPR30 may mediate the development of preeclampsia remain unknown. We observed that the expression of GPR30 in placental trophoblast cells is lower in preeclamptic placentas compared with normotensive controls. We then investigated the role of GPR30 in trophoblast cell invasion by utilizing placental explants and the immortalized human trophoblast cell line (HTR8/SVneo). The selective GPR30 agonist G1 and a general estrogen receptors agonist 17-β-estradiol (E2) both improved trophoblast cells invasion by upregulating MMP9 expression and the PI3K-Akt signaling pathway. This effect was abolished by a selective GPR30 inhibitor G15, implying that GPR30 may be involved in regulating trophoblast invasion, and that down-regulation of this receptor may result in the development of preeclampsia. The present study suggests that GPR30 is a critical regulator of trophoblast cell invasion, and as such may be a potential therapeutic interventional target for preeclampsia and other pregnancy complications resulting from impaired trophoblast invasion.

Expression and functional roles of estrogen receptor GPR30 in human intervertebral disc.

PubMed

Wei, Aiqun; Shen, Bojiang; Williams, Lisa A; Bhargav, Divya; Yan, Feng; Chong, Beng H; Diwan, Ashish D

2016-04-01

Estrogen withdrawal, a characteristic of female aging, is associated with age-related intervertebral disc (IVD) degeneration. The function of estrogen is mediated by two classic nuclear receptors, estrogen receptor (ER)-α and -β, and a membrane bound G-protein-coupled receptor 30 (GPR30). To date, the expression and function of GPR30 in human spine is poorly understood. This study aimed to evaluate GPR30 expression in IVD, and its role in estrogen-related regulation of proliferation and apoptosis of disc nucleus pulposus (NP) cells. GPR30 expression was examined in 30 human adult NP and 9 fetal IVD. Results showed that GPR30 was expressed in NP cells at both mRNA and protein levels. In human fetal IVD, GPR30 protein was expressed in the NP at 12-14 weeks gestation, but was undetectable at 8-11 weeks. The effect of 17β-estradiol (E2) on GPR30-mediated proliferation and interleukin-1β (IL-1β)-induced apoptosis of NP cells was investigated. Cultured NP cells were treated with or without E2, GPR30 antagonist G36, and ER antagonist ICI 182,780. NP cell viability was tested by MTS assay. Apoptosis was determined by flow cytometry using fluorescence labeled annexin-V, TUNEL assay and immumnocytochemical staining of activated caspase-3. E2 enhanced cell proliferation and prevented IL-1β-induced cell death, but the effect was partially blocked by G36 and completely abrogated by a combination of ICI 182,780 and G36. This study demonstrates that GPR30 is expressed in human IVD to transmit signals triggering E2-induced NP cell proliferation and protecting against IL-1β-induced apoptosis. The effects of E2 on NP cells require both GPR30 and classic estrogen receptors. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Estradiol and tamoxifen induce cell migration through GPR30 and activation of focal adhesion kinase (FAK) in endometrial cancers with low or without nuclear estrogen receptor α (ERα).

PubMed

Tsai, Chia-Lung; Wu, Hsien-Ming; Lin, Chiao-Yun; Lin, Yi-Jun; Chao, Angel; Wang, Tzu-Hao; Hsueh, Swei; Lai, Chyong-Huey; Wang, Hsin-Shih

2013-01-01

Estrogens and tamoxifen (an antiestrogen) exert their actions by activation of estrogen receptor (ER) through genomic and non-genomic mechanisms and are implicated in the development of endometrial cancer. Previous reports have demonstrated that estradiol and tamoxifen induce proliferation of human endometrial cancer cells through GPR30 (non-genomic ER) signaling pathway. Herein, we demonstrate that phosphorylation of focal adhesion kinase (FAK) is involved in cell migration induced by estradiol, tamoxifen and G1 (a GPR30 agonist) through the transmembrane ER (GPR30) in endometrial cancer cell lines with or without ERα (Ishikawa and RL95-2). Additionally, the GPR30-mediated cell migration was further abolished by administration of either specific RNA interference targeting GPR30 or an FAK inhibitor. Moreover, we have validated that the signaling between GPR30 and phosphorylated FAK is indeed mediated by the EGFR/PI3K/ERK pathway. Clinically, a significant correlation between levels of GPR30 and phophorylated FAK (pFAK) observed in human endometrial cancer tissues with low or without ERα further suggested that estrogen-induced phosphorylation of FAK and cell migration were most likely triggered by GPR30 activation. These results provided new insights for understanding the pathophysiological functions of GPR30 in human endometrial cancers.

Assessment of cell line competence for studies of pharmacological GPR30 modulation.

PubMed

Sousa, Cátia; Ribeiro, Madalena; Rufino, Ana Teresa; Leitão, Alcino Jorge; Mendes, Alexandrina Ferreira

2017-04-01

Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30. The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17β-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals. None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50-55 kDa previously identified in cell lines that respond to 17β-estradiol and G-1. Cells that do not express the 44 and 50-55 kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.

Atypical Responsiveness of the Orphan Receptor GPR55 to Cannabinoid Ligands*

PubMed Central

Kapur, Ankur; Zhao, Pingwei; Sharir, Haleli; Bai, Yushi; Caron, Marc G.; Barak, Larry S.; Abood, Mary E.

2009-01-01

The cannabinoid receptor 1 (CB1) and CB2 cannabinoid receptors, associated with drugs of abuse, may provide a means to treat pain, mood, and addiction disorders affecting widespread segments of society. Whether the orphan G-protein coupled receptor GPR55 is also a cannabinoid receptor remains unclear as a result of conflicting pharmacological studies. GPR55 has been reported to be activated by exogenous and endogenous cannabinoid compounds but surprisingly also by the endogenous non-cannabinoid mediator lysophosphatidylinositol (LPI). We examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a β-arrestin-green fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Our data demonstrate that AM251 and SR141716A (rimonabant), which are cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. They possess comparable efficacy in inducing β-arrestin trafficking and, moreover, activate the G-protein-dependent signaling of protein kinase CβII. Conversely, the potent synthetic cannabinoid agonist CP55,940 acts as a GPR55 antagonist/partial agonist. CP55,940 blocks GPR55 internalization, the formation of β-arrestin GPR55 complexes, and the phosphorylation of ERK1/2; CP55,940 produces only a slight amount of protein kinase CβII membrane recruitment but does not stimulate membrane remodeling like LPI, AM251, or rimonabant. Our studies provide a paradigm for measuring the responsiveness of GPR55 to a variety of ligand scaffolds comprising cannabinoid and novel compounds and suggest that at best GPR55 is an atypical cannabinoid responder. The activation of GPR55 by rimonabant may be responsible for some of the off-target effects that led to its removal as a potential obesity therapy. PMID:19723626

GPR30 Activation Contributes to the Puerarin-Mediated Neuroprotection in MPP+-Induced SH-SY5Y Cell Death.

PubMed

Cheng, Yue-Fa; Zhu, Guoqi; Wu, Qing-Wen; Xie, Yue-Sheng; Jiang, Yan; Guo, Lan; Guan, Ya-Li; Liu, Ying-Shuo; Zhang, Jun

2017-02-01

The neuroprotective action of puerarin in Parkinson's disease (PD) models has been well investigated. However, the mechanisms involved in protection have not been completely understood. G protein-coupled receptor 30 (GPR30) is a G protein-coupled estrogen receptor and considered a potential target in the neuroprotection against PD. In this study, we investigated whether puerarin prevented against 1-methyl-4-phenylpyridinium (MPP + )-induced cell death via GPR30. Our results showed that the GPR30 agonist, G1, exhibited puerarin-mediated neuroprotection against MPP + -induced cell death of SH-SY5Y cells. This protective action was reversed by the GPR30 antagonist. Moreover, a time- and concentration-dependent effect of puerarin on GPR30 expression was verified at the protein level but not at the mRNA level. Further, we showed that an mTor-dependent new GPR30 synthesis contributed to the protection conferred by puerarin. Finally, glial cell line-derived neurotrophic factor (GDNF) levels were enhanced by puerarin and G1 in both control and MPP + -lesioned cells via GPR30. Taken together, our data strongly suggest that puerarin prevents MPP + -induced cell death via facilitating GPR30 expression and GDNF release.

Baicalein suppresses 17-β-estradiol-induced migration, adhesion and invasion of breast cancer cells via the G protein-coupled receptor 30 signaling pathway.

PubMed

Shang, Dandan; Li, Zheng; Zhu, Zhuxia; Chen, Huamei; Zhao, Lujun; Wang, Xudong; Chen, Yan

2015-04-01

Flavonoids are structurally similar to steroid hormones, particularly estrogens, and therefore have been studied for their potential effects on hormone-dependent cancers. Baicalein is the primary flavonoid derived from the root of Scutellaria baicalensis Georgi. In the present study, we investigated the effects of baicalein on 17β-estradiol (E2)-induced migration, adhesion and invasion of MCF-7 and SK-BR-3 breast cancer cells. The results demonstrated that baicalein suppressed E2-stimulated wound-healing migration and cell‑Matrigel adhesion, and ameliorated E2-promoted invasion across a Matrigel-coated Transwell membrane. Furthermore, baicalein interfered with E2-induced novel G protein-coupled estrogen receptor (GPR30)-related signaling, including a decrease in tyrosine phosphorylation of epidermal growth factor receptor (EGFR) as well as phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine kinase Akt, without affecting GPR30 expression. The results also showed that baicalein suppressed the expression of GPR30 target genes, cysteine-rich 61 (CYR61) and connective tissue growth factor (CTGF) induced by E2. Furthermore, baicalein prevented GPR30-related signaling activation and upregulation of CYR61 and CTGF mRNA levels induced by G1, a specific GPR 30 agonist. The results suggest that baicalein inhibits E2-induced migration, adhesion and invasion through interfering with GPR30 signaling pathway activation, which indicates that it may act as a therapeutic candidate for the treatment of GPR30-positive breast cancer metastasis.

Embelin and its derivatives unravel the signaling, proinflammatory and antiatherogenic properties of GPR84 receptor.

PubMed

Gaidarov, Ibragim; Anthony, Todd; Gatlin, Joel; Chen, Xiaohua; Mills, David; Solomon, Michelle; Han, Sangdon; Semple, Graeme; Unett, David J

2018-05-01

GPR84 is an orphan G-protein coupled receptor, expressed on monocytes, macrophages and neutrophils and is significantly upregulated by inflammatory stimuli. The physiological role of GPR84 remains largely unknown. Medium chain fatty acids (MCFA) activate the receptor and have been proposed to be its endogenous ligands, although the high concentrations of MCFAs required for receptor activation generally exceed normal physiological levels. We identified the natural product embelin as a highly potent and selective surrogate GPR84 agonist (originally disclosed in patent application WO2007027661A2, 2007) and synthesized close structural analogs with widely varying receptor activities. These tools were used to perform a comprehensive study of GPR84 signaling and function in recombinant cells and in primary human macrophages and neutrophils. Activation of recombinant GPR84 by embelin in HEK293 cells results in G i/o as well as G12/13-Rho signaling. In human macrophages, GPR84 initiates PTX sensitive Erk1/2 and Akt phosphorylation, PI-3 kinase activation, calcium flux, and release of prostaglandin E2. In addition, GPR84 signaling in macrophages elicits G i Gβγ-mediated augmentation of intracellular cAMP, rather than the decrease expected from G iα engagement. GPR84 activation drives human neutrophil chemotaxis and primes them for amplification of oxidative burst induced by FMLP and C5A. Loss of GPR84 is associated with attenuated LPS-induced release of proinflammatory mediators IL-6, KC-GROα, VEGF, MIP-2 and NGAL from peritoneal exudates. While initiating numerous proinflammatory activities in macrophages and neutrophils, GPR84 also possesses GPR109A-like antiatherosclerotic properties in macrophages. Macrophage receptor activation leads to upregulation of cholesterol transporters ABCA1 and ABCG1 and stimulates reverse cholesterol transport. These data suggest that GPR84 may be a target of therapeutic value and that distinct modes of receptor modulation (inhibition vs. stimulation) may be required for inflammatory and atherosclerotic indications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Expression of the Fatty Acid Receptors GPR84 and GPR120 and Cytodifferentiation of Epithelial Cells in the Gastric Mucosa of Mouse Pups in the Course of Dietary Transition.

PubMed

Widmayer, Patricia; Kusumakshi, Soumya; Hägele, Franziska A; Boehm, Ulrich; Breer, Heinz

2017-01-01

During weaning, the ingested food of mouse pups changes from exclusively milk to solid food. In contrast to the protein- and carbohydrate-rich solid food, high fat milk is characterized primarily by fatty acids of medium chain length particularly important for the suckling pups. Therefore, it seems conceivable that the stomach mucosa may be specialized for detecting these important nutrients during the suckling phase. Here, we analyzed the expression of the G protein coupled receptors GPR84 and GPR120 (FFAR4), which are considered to be receptors for medium and long chain fatty acids (LCFAs), respectively. We found that the mRNA levels for GPR84 and GPR120 were high during the suckling period and progressively decreased in the course of weaning. Visualization of the receptor-expressing cells in 2-week-old mice revealed a high number of labeled cells, which reside in the apical as well as in the basal region of the gastric glands. At the base of the gastric glands, all GPR84-immunoreactive cells and some of the GPR120-positive cells also expressed chromogranin A (CgA), suggesting that they are enteroendocrine cells. We demonstrate that the majority of the CgA/GPR84 cells are X/A-like ghrelin cells. The high degree of overlap between ghrelin and GPR84 decreased post-weaning, whereas the overlap between ghrelin and GPR120 increased. At the apical region of the glands the fatty acid receptors were mainly expressed in unique cell types. These contain lipid-filled vacuole- and vesicle-like structures and may have absorptive functions. We detected decreased immunoreactivity for GPR84 and no lipid droplets in surface cells post-weaning. In conclusion, expression of GPR84 in ghrelin cells as well as in surface cells suggests an important role of medium chain fatty acids (MCFAs) in the developing gastric mucosa of suckling mice.

Expression of the Fatty Acid Receptors GPR84 and GPR120 and Cytodifferentiation of Epithelial Cells in the Gastric Mucosa of Mouse Pups in the Course of Dietary Transition

PubMed Central

Widmayer, Patricia; Kusumakshi, Soumya; Hägele, Franziska A.; Boehm, Ulrich; Breer, Heinz

2017-01-01

During weaning, the ingested food of mouse pups changes from exclusively milk to solid food. In contrast to the protein- and carbohydrate-rich solid food, high fat milk is characterized primarily by fatty acids of medium chain length particularly important for the suckling pups. Therefore, it seems conceivable that the stomach mucosa may be specialized for detecting these important nutrients during the suckling phase. Here, we analyzed the expression of the G protein coupled receptors GPR84 and GPR120 (FFAR4), which are considered to be receptors for medium and long chain fatty acids (LCFAs), respectively. We found that the mRNA levels for GPR84 and GPR120 were high during the suckling period and progressively decreased in the course of weaning. Visualization of the receptor-expressing cells in 2-week-old mice revealed a high number of labeled cells, which reside in the apical as well as in the basal region of the gastric glands. At the base of the gastric glands, all GPR84-immunoreactive cells and some of the GPR120-positive cells also expressed chromogranin A (CgA), suggesting that they are enteroendocrine cells. We demonstrate that the majority of the CgA/GPR84 cells are X/A-like ghrelin cells. The high degree of overlap between ghrelin and GPR84 decreased post-weaning, whereas the overlap between ghrelin and GPR120 increased. At the apical region of the glands the fatty acid receptors were mainly expressed in unique cell types. These contain lipid-filled vacuole- and vesicle-like structures and may have absorptive functions. We detected decreased immunoreactivity for GPR84 and no lipid droplets in surface cells post-weaning. In conclusion, expression of GPR84 in ghrelin cells as well as in surface cells suggests an important role of medium chain fatty acids (MCFAs) in the developing gastric mucosa of suckling mice. PMID:28871231

Free fatty acid receptors act as nutrient sensors to regulate energy homeostasis.

PubMed

Ichimura, Atsuhiko; Hirasawa, Akira; Hara, Takafumi; Tsujimoto, Gozoh

2009-09-01

Free fatty acids (FFAs) have been demonstrated to act as ligands of several G-protein-coupled receptors (GPCRs) (FFAR1, FFAR2, FFAR3, GPR84, and GPR120). These fatty acid receptors are proposed to play critical roles in a variety of types of physiological homeostasis. FFAR1 and GPR120 are activated by medium- and long-chain FFAs. GPR84 is activated by medium-chain, but not long-chain, FFAs. In contrast, FFAR2 and FFAR3 are activated by short-chain FFAs. FFAR1 is expressed mainly in pancreatic beta-cells and mediates insulin secretion, whereas GPR120 is expressed abundantly in the intestine and promotes the secretion of glucagon-like peptide-1 (GLP-1). FFAR3 is expressed in enteroendocrine cells and regulates host energy balance through effects that are dependent upon the gut microbiota. In this review, we summarize the identification, structure, and pharmacology of these receptors and present an essential overview of the current understanding of their physiological roles.

Attenuation of CXCR4 responses by CCL18 in acute lymphocytic leukemia B cells.

PubMed

Catusse, J; Wollner, S; Leick, M; Schröttner, P; Schraufstätter, I; Burger, M

2010-11-01

CCL18 and CXCL12 are homeostatic chemokines with high constitutive concentrations in serum. Elevated levels of CCL18 have been described in various diseases including childhood acute lymphocytic leukemia (ALL) but its functions remain poorly characterized. Its receptor has not been identified, but functional cellular responses like lymphocyte chemotaxis have been described. CXCL12 is a pivotal chemokine for hematopoiesis and B cell homing processes. We demonstrate that CCL18 interferes with CXCL12-mediated pre-B ALL cell activation. CXCL12-induced calcium mobilization, chemotaxis, pseudo-emperipolesis and cellular proliferation could be significantly reduced by CCL18 in pre-B ALL cell lines. The results could be observed in primary cells from patients suffering from pre-B ALL, but not in cells from patients suffering from common ALL. Direct effects of CCL18 on the receptor for CXCL12, CXCR4, could be excluded. Moreover, we found that CCL18 modulations of CXCL12-induced responses are mediated through the chemokine-like receptor GPR30. CCL18 bound to GPR30 expressing cells, and antibodies against GPR30 abolished this binding as well as CCL18-mediated functional effects. We also observed that, CCL18 interferes with the activation of GPR30 by previously identified ligands (17β-estradiol and chemical agonists). We therefore suggest that CCL18 is an important modulator of CXCR4-dependent responses in pre-B ALL cells via interactions with GPR30. © 2010 Wiley-Liss, Inc.

G-protein-coupled estrogen receptor GPR30 and tamoxifen resistance in breast cancer.

PubMed

Ignatov, Atanas; Ignatov, Tanja; Weissenborn, Christine; Eggemann, Holm; Bischoff, Joachim; Semczuk, Andrzej; Roessner, Albert; Costa, Serban Dan; Kalinski, Thomas

2011-07-01

Recently, we have shown that the new G-protein-coupled estrogen receptor GPR30 plays an important role in the development of tamoxifen resistance in vitro. This study was undertaken to evaluate the correlation between GPR30 and tamoxifen resistance in breast cancer patients. GPR30 protein expression was evaluated by immunohistochemical analysis in 323 patients with primary operable breast cancer. The association between GPR30 expression and tamoxifen resistance was confirmed in a second cohort of 103 patients treated only with tamoxifen. Additionally, we evaluated GPR30 expression in 33 primary tumors and in recurrent tumors from the same patients. GPR30 expression was detected in 56.7% of the breast cancer specimens investigated and it correlated with overexpression of HER-2 (P = 0.021), EGFR (P = 0.024) and lymph node status (P = 0.047). In a first cohort, survival analysis showed that GPR30 was negatively correlated with relapse-free survival (RFS) only in patients treated with tamoxifen (tamoxifen with or without chemotherapy). GPR30 expression was associated with shorter RFS (HR = 1.768; 95% CI, 1.156-2.703; P = 0.009). In a subset of patients treated only with tamoxifen, multivariate analysis revealed that GPR30 expression is an independent unfavorable factor for RFS (HR = 4.440; 95% CI, 1.408-13.997; P = 0.011). In contrast, GPR30 tended to be a favorable factor regarding RFS in patients who did not receive tamoxifen. In 33 paired biopsies obtained before and after adjuvant therapy, GPR30 expression significantly increased only under tamoxifen treatment (P = 0.001). GPR30 expression in breast cancer independently predicts a poor RFS in patients treated with tamoxifen.

GPR30 Activation Opposes Estrogen-Dependent Uterine Growth via Inhibition of Stromal ERK1/2 and Estrogen Receptor Alpha (ERα) Phosphorylation Signals

PubMed Central

Gao, Fei; Ma, Xinghong; Ostmann, Alicia B.

2011-01-01

Although estradiol-17β (E2)-regulated early and late phase uterine responses have been well defined, the molecular mechanisms linking the phases remain poorly understood. We have previously shown that E2-regulated early signals mediate cross talk with estrogen receptor (ER)-α to elicit uterine late growth responses. G protein-coupled receptor (GPR30) has been implicated in early nongenomic signaling mediated by E2, although its role in E2-dependent uterine biology is unclear. Using selective activation of GPR30 by G-1, we show here a new function of GPR30 in regulating early signaling events, including the inhibition of ERK1/2 and ERα (Ser118) phosphorylation signals and perturbation of growth regulation under the direction of E2 in the mouse uterus. We observed that GPR30 primarily localizes in the uterine epithelial cells, and its activation alters gene expression and mediates inhibition of ERK1/2 and ERα (Ser118) phosphorylation signals in the stromal compartment, suggesting a paracrine signaling is involved. Importantly, viral-driven manipulation of GPR30 or pharmacological inhibition of ERK1/2 activation effectively alters E2-dependent uterine growth responses. Overall, GPR30 is a negative regulator of ERα-dependent uterine growth in response to E2. Our work has uncovered a novel GPR30-regulated inhibitory event, which may be physiologically relevant in both normal and pathological situations to negatively balance ERα-dependent uterine growth regulatory functions induced by E2. PMID:21303939

The 17,18-epoxyeicosatetraenoic acid-G protein-coupled receptor 40 axis ameliorates contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques.

PubMed

Nagatake, Takahiro; Shiogama, Yumiko; Inoue, Asuka; Kikuta, Junichi; Honda, Tetsuya; Tiwari, Prabha; Kishi, Takayuki; Yanagisawa, Atsushi; Isobe, Yosuke; Matsumoto, Naomi; Shimojou, Michiko; Morimoto, Sakiko; Suzuki, Hidehiko; Hirata, So-Ichiro; Steneberg, Pär; Edlund, Helena; Aoki, Junken; Arita, Makoto; Kiyono, Hiroshi; Yasutomi, Yasuhiro; Ishii, Masaru; Kabashima, Kenji; Kunisawa, Jun

2017-12-27

Metabolites of eicosapentaenoic acid exert various physiologic actions. 17,18-Epoxyeicosatetraenoic acid (17,18-EpETE) is a recently identified new class of antiallergic and anti-inflammatory lipid metabolite of eicosapentaenoic acid, but its effects on skin inflammation and the underlying mechanisms remain to be investigated. We evaluated the effectiveness of 17,18-EpETE for control of contact hypersensitivity in mice and cynomolgus macaques. We further sought to reveal underlying mechanisms by identifying the responsible receptor and cellular target of 17,18-EpETE. Contact hypersensitivity was induced by topical application of 2,4-dinitrofluorobenzene. Skin inflammation and immune cell populations were analyzed by using flow cytometric, immunohistologic, and quantitative RT-PCR analyses. Neutrophil mobility was examined by means of imaging analysis in vivo and neutrophil culture in vitro. The receptor for 17,18-EpETE was identified by using the TGF-α shedding assay, and the receptor's involvement in the anti-inflammatory effects of 17,18-EpETE was examined by using KO mice and specific inhibitor treatment. We found that preventive or therapeutic treatment with 17,18-EpETE ameliorated contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques. 17,18-EpETE was recognized by G protein-coupled receptor (GPR) 40 (also known as free fatty acid receptor 1) and inhibited chemoattractant-induced Rac activation and pseudopod formation in neutrophils. Indeed, the antiallergic inflammatory effect of 17,18-EpETE was abolished in the absence or inhibition of GPR40. 17,18-EpETE inhibits neutrophil mobility through GPR40 activation, which is a potential therapeutic target to control allergic inflammatory diseases. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

G protein‐coupled receptor 37‐like 1 modulates astrocyte glutamate transporters and neuronal NMDA receptors and is neuroprotective in ischemia

PubMed Central

Jolly, Sarah; Bazargani, Narges; Quiroga, Alejandra C.; Pringle, Nigel P.

2017-01-01

Abstract We show that the G protein‐coupled receptor GPR37‐like 1 (GPR37L1) is expressed in most astrocytes and some oligodendrocyte precursors in the mouse central nervous system. This contrasts with GPR37, which is mainly in mature oligodendrocytes. Comparison of wild type and Gpr37l1–/– mice showed that loss of GPR37L1 did not affect the input resistance or resting potential of astrocytes or neurons in the hippocampus. However, GPR37L1‐mediated signalling inhibited astrocyte glutamate transporters and – surprisingly, given its lack of expression in neurons – reduced neuronal NMDA receptor (NMDAR) activity during prolonged activation of the receptors as occurs in ischemia. This effect on NMDAR signalling was not mediated by a change in the release of D‐serine or TNF‐α, two astrocyte‐derived agents known to modulate NMDAR function. After middle cerebral artery occlusion, Gpr37l1 expression was increased around the lesion. Neuronal death was increased by ∼40% in Gpr37l1–/– brain compared to wild type in an in vitro model of ischemia. Thus, GPR37L1 protects neurons during ischemia, presumably by modulating extracellular glutamate concentration and NMDAR activation. PMID:28795439

G-protein coupled estrogen receptor 1 expression in rat and human heart: Protective role during ischaemic stress.

PubMed

Patel, Vanlata H; Chen, Jing; Ramanjaneya, Manjunath; Karteris, Emmanouil; Zachariades, Elena; Thomas, Peter; Been, Martin; Randeva, Harpal S

2010-08-01

G-protein coupled estrogen receptor 1, GPER, formerly known as GPR30, is a seven transmembrane domain receptor that mediates rapid estrogen responses in a wide variety of cell types. To date, little is known about the role of GPER during ischaemia/reperfusion injury. In this study, we report both mRNA and protein expression of GPER in the rat and human heart. The role of GPER in estrogen protection against ischaemic stress in the rat heart was also assessed using the isolated Langendorff system. Pre-treatment with 17beta-estradiol (E2) significantly decreased infarct size, (61.48+/-2.2% to 27.92+/-2.9% (P<0.001). Similarly, treatment with the GPER agonist G1 prior to 30-min global ischaemia followed by 120-min reperfusion significantly reduced infarct size from 61.48+/-2.2% to 23.85+/-3.2% (P<0.001), whilst addition of GPR30 antibody, abolished the protective effect of G1 (infarct size: 55.42+/-1.3%). The results suggest that GPER under cardiac stress exerts direct protection in the heart and may serve as a potential therapeutic target for cardiac drug therapy.

Targeting of the Orphan Receptor GPR35 by Pamoic Acid: A Potent Activator of Extracellular Signal-Regulated Kinase and β-Arrestin2 with Antinociceptive ActivityS⃞

PubMed Central

Zhao, Pingwei; Sharir, Haleli; Kapur, Ankur; Cowan, Alan; Geller, Ellen B.; Adler, Martin W.; Seltzman, Herbert H.; Reggio, Patricia H.; Heynen-Genel, Susanne; Sauer, Michelle; Chung, Thomas D.Y.; Bai, Yushi; Chen, Wei; Caron, Marc G.; Barak, Larry S.

2010-01-01

Known agonists of the orphan receptor GPR35 are kynurenic acid, zaprinast, 5-nitro-2-(3-phenylproplyamino) benzoic acid, and lysophosphatidic acids. Their relatively low affinities for GPR35 and prominent off-target effects at other pathways, however, diminish their utility for understanding GPR35 signaling and for identifying potential therapeutic uses of GPR35. In a screen of the Prestwick Library of drugs and drug-like compounds, we have found that pamoic acid is a potent GPR35 agonist. Pamoic acid is considered by the Food and Drug Administration as an inactive compound that enables long-acting formulations of numerous drugs, such as the antihelminthics oxantel pamoate and pyrantel pamoate; the psychoactive compounds hydroxyzine pamoate (Vistaril) and imipramine pamoate (Tofranil-PM); and the peptide hormones triptorelin pamoate (Trelstar) and octreotide pamoate (OncoLar). We have found that pamoic acid induces a Gi/o-linked, GPR35-mediated increase in the phosphorylation of extracellular signal-regulated kinase 1/2, recruitment of β-arrestin2 to GPR35, and internalization of GPR35. In mice, it attenuates visceral pain perception, indicating an antinociceptive effect, possibly through GPR35 receptors. We have also identified in collaboration with the Sanford-Burnham Institute Molecular Libraries Probe Production Center new classes of GPR35 antagonist compounds, including the nanomolar potency antagonist methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID2745687). Pamoic acid and potent antagonists such as CID2745687 present novel opportunities for expanding the chemical space of GPR35, elucidating GPR35 pharmacology, and stimulating GPR35-associated drug development. Our results indicate that the unexpected biological functions of pamoic acid may yield potential new uses for a common drug constituent. PMID:20826425

IL-1β and TNFα inhibit GPR120 (FFAR4) and stimulate GPR84 (EX33) and GPR41 (FFAR3) fatty acid receptor expression in human adipocytes: implications for the anti-inflammatory action of n-3 fatty acids.

PubMed

Muredda, Laura; Kępczyńska, Małgorzata A; Zaibi, Mohamed S; Alomar, Suliman Y; Trayhurn, Paul

2018-05-01

Regulation of the expression of GPCR fatty acid receptor genes has been examined in human adipocytes differentiated in culture. TNFα and IL-1β induced a marked reduction in GPR120 expression, mRNA level falling 17-fold at 24 h in adipocytes incubated with TNFα. In contrast, GPR84 mRNA was dramatically increased by these cytokines (>500-fold for IL-1β at 4 h); GPR41 expression was also stimulated. Rosiglitazone did not affect GPR84 expression, but GPR120 and GPR41 expression increased. Dexamethasone, insulin, linoleic and docosahexaenoic acids (DHA), and TUG891 (GPR120 agonist) had little effect on GPR120 and GPR84 expression. TUG891 did not attenuate the pro-inflammatory actions of TNFα and IL-1β. DHA slightly countered the actions of IL-1β on CCL2, IL6 and ADIPOQ expression, though not on secretion of these adipokines. GPR120 and GP84 gene expression in human adipocytes is highly sensitive to pro-inflammatory mediators; the inflammation-induced inhibition of GPR120 expression may compromise the anti-inflammatory action of GPR120 agonists.

Pharmacology of GPR55 in yeast and identification of GSK494581A as a mixed-activity glycine transporter subtype 1 inhibitor and GPR55 agonist.

PubMed

Brown, Andrew J; Daniels, Dion A; Kassim, Mumta; Brown, Sue; Haslam, Carl P; Terrell, Victoria R; Brown, Jason; Nichols, Paula L; Staton, Penny C; Wise, Alan; Dowell, Simon J

2011-04-01

GPR55 is a G protein-coupled receptor activated by L-α-lysophosphatidylinositol and suggested to have roles in pain signaling, bone morphogenesis, and possibly in vascular endothelial cells. It has affinity for certain cannabinoids (molecules that interact with the cannabinoid CB(1) and CB(2) receptors), but investigation of its functional role in cell-based systems and in tissue has been limited by a lack of selective pharmacological tools. Here, we present our characterization of GPR55 in the yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK293) cells. We describe GSK494581A (1-{2-fluoro-4-[1-(methyloxy)ethyl]phenyl}-4-{[4'-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}piperazine), a selective small-molecule ligand of GPR55 identified through diversity screening. GSK494581A is one of a series of benzoylpiperazines originally identified and patented as inhibitors of the glycine transporter subtype 1 (GlyT1). The structure-activity relationship between GPR55 and GlyT1 is divergent across this series. The most GPR55-selective example is GSK575594A (3-fluoro-4-(4-{[4'-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}-1-piperazinyl)aniline), which is approximately 60-fold selective for GPR55 (pEC(50) = 6.8) over GlyT1 (pIC(50) = 5.0). Several exemplars with activity at GPR55 and GlyT1 have been profiled at a broad range of other molecular targets and are inactive at cannabinoid receptors and all other targets tested. The benzoylpiperazine agonists activate human GPR55 but not rodent GPR55, suggesting that the relatively low level of sequence identity between these orthologs (75%) translates to important functional differences in the ligand-binding site.

Roles of ERβ and GPR30 in Proliferative Response of Human Bladder Cancer Cell to Estrogen.

PubMed

Huang, Weiren; Chen, Yuanbin; Liu, Yuchen; Zhang, Qiaoxia; Yu, Zhou; Mou, Lisha; Wu, Hanwei; Zhao, Li; Long, Ting; Qin, Danian; Gui, Yaoting

2015-01-01

Bladder cancer belongs to one of the most common cancers and is a leading cause of deaths in our society. Urothelial carcinoma of the bladder (UCB) is the main type of this cancer, and the estrogen receptors in UCB remain to be studied. Our experiment aimed to investigate the possible biological effect of 17β-estradiol on human bladder-derived T24 carcinoma cells and to indicate its related mechanisms. T24 cells were treated with various doses of 17β-estradiol, and cell proliferation was detected using MTT assays. 17β-estradiol promoted T24 cell proliferation independent of ERβ/GPR30-regulated EGFR-MAPK pathway, while it inhibited cell growth via GPR30. Furthermore, the expression levels of downstream genes (c-FOS, BCL-2, and CYCLIN D1) were increased by 17β-estradiol and this effect was independently associated with activity of the EGFR-MAPK pathway. The two estrogen receptors might be potential therapeutic targets for the treatment of bladder cancer.

Acidosis Activation of the Proton-Sensing GPR4 Receptor Stimulates Vascular Endothelial Cell Inflammatory Responses Revealed by Transcriptome Analysis

PubMed Central

Dong, Lixue; Li, Zhigang; Leffler, Nancy R.; Asch, Adam S.; Chi, Jen-Tsan; Yang, Li V.

2013-01-01

Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by GPR4 small molecule inhibitors and hold potential therapeutic value. PMID:23613998

The novel estrogen receptor G-protein-coupled receptor 30 is expressed in human bone.

PubMed

Heino, Terhi J; Chagin, Andrei S; Sävendahl, Lars

2008-05-01

Estrogens have significant impact on bone mineral metabolism. Besides the classical estrogen receptors (ERalpha and ERbeta), a trans-membrane G-protein-coupled receptor (GPR30) has been demonstrated to mediate estrogenic effects. We aimed to study whether GPR30 is expressed in bone cells and if so, whether the level of expression is developmentally regulated. Metaphyseal bone biopsies were collected from the tibia in 14 boys and 6 girls, all at different stages of puberty. GPR30 protein expression was studied by immunohistochemistry in paraffin-embedded sections. GPR30-positive osteocytes and osteoblasts were quantified and linear regression analysis was applied. Cytoplasmic GPR30 expression was detected in osteoblasts, osteocytes, and osteoclasts. Osteocytes were more frequently positive for GPR30 than osteoblasts (58+/-4% vs 46+/-3% positive cells respectively, P<0.05). Detailed analysis demonstrated that GPR30 positivity declined during pubertal development in osteocytes (R=-0.56, P<0.01) but not in osteoblasts (R=-0.31, P>0.05). No sex difference was observed in the numbers of GPR30-positive osteoblasts or osteocytes. Furthermore, GPR30 expression did not correlate with chronological or bone age. In conclusion, the novel ER GPR30 is expressed in osteoblasts, osteocytes, and osteoclasts suggesting that non-genomic estrogen signaling via GPR30 may exist in bone. However, the functional role of GPR30 in bone tissue remains to be elucidated.

Utilization of the Tango beta-arrestin recruitment technology for cell-based EDG receptor assay development and interrogation.

PubMed

Wetter, Justin A; Revankar, Chetana; Hanson, Bonnie J

2009-10-01

Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. These LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. These receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. This complicates examination and comparison of these receptors across the entire family. The Tango technology uses the conserved beta-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. This method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. The authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG(1) and EDG(3) receptors.

P2X and P2Y receptors as possible targets of therapeutic manipulations in CNS illnesses.

PubMed

Köles, Laszlo; Furst, Susanna; Illes, Peter

2005-03-01

Adenine and/or uridine nucleotide-sensitive receptors are classified into two types belonging to the ligand-gated ionotropic family (P2X) and the metabotropic, G-protein-coupled family (P2Y). In humans, seven different P2X receptors (P2X(1-7)) and eight different P2Y receptors (P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11-14)) have been detected hitherto. All P2 receptors are expressed in the CNS, with the preferential expression of the P2X(2), P2X(4), P2X(6) and P2Y(1) receptors in neurons. In addition to the neurotransmitter and modulator functions, neurite outgrowth, proliferation of glial cells and the expression of transmitter receptors at target cells have also been suggested to be regulated by extracellular nucleotides in the nervous system. In spite of the expanding knowledge in the purinergic research field, the present therapeutic utilization of P2 receptor ligands is mostly related to peripheral diseases such as thromboembolic disorders and cystic fibrosis. In this review we provide some evidence that P2 receptors play an important role in the regulation of CNS functions related to hippocampal activity, the mesolimbic dopaminergic system and the nociceptive system. The role of purinergic receptors located on astrocytes/microglia and implications of these receptors for neurodegenerative/neuroinflammatory disorders, CNS injury and epilepsy will be highlighted as well. (c) 2005 Prous Science. All rights reserved.

Agonists and antagonists for P2 receptors

PubMed Central

Jacobson, Kenneth A.; Costanzi, Stefano; Joshi, Bhalchandra V.; Besada, Pedro; Shin, Dae Hong; Ko, Hyojin; Ivanov, Andrei A.; Mamedova, Liaman

2015-01-01

Recent work has identified nucleotide agonists selective for P2Y1, P2Y2 and P2Y6 receptors and nucleotide antagonists selective for P2Y1, P2Y12 and P2X1 receptors. Selective non-nucleotide antagonists have been reported for P2Y1, P2Y2, P2Y6, P2Y12, P2Y13, P2X2/3/P2X3 and P2X7 receptors. For example, the dinucleotide INS 37217 (Up4dC) potently activates the P2Y2 receptor, and the non-nucleotide antagonist A-317491 is selective for P2X2/3/P2X3 receptors. Nucleotide analogues in which the ribose moiety is substituted by a variety of novel ring systems, including conformation-ally locked moieties, have been synthesized as ligands for P2Y receptors. The focus on conformational factors of the ribose-like moiety allows the inclusion of general modifications that lead to enhanced potency and selectivity. At P2Y1,2,4,11 receptors, there is a preference for the North conformation as indicated with (N)-methanocarba analogues. The P2Y1 antagonist MRS2500 inhibited ADP-induced human platelet aggregation with an IC50 of 0.95 nM. MRS2365, an (N)-methanocarba analogue of 2-MeSADP, displayed potency (EC50) of 0.4 nM at the P2Y1 receptor, with >10 000-fold selectivity in comparison to P2Y12 and P2Y13 receptors. At P2Y6 receptors there is a dramatic preference for the South conformation. Three-dimensional structures of P2Y receptors have been deduced from structure activity relationships (SAR), mutagenesis and modelling studies. Detailed three-dimensional structures of P2X receptors have not yet been proposed. PMID:16805423

Deletion of the pH sensor GPR4 decreases renal acid excretion.

PubMed

Sun, Xuming; Yang, Li V; Tiegs, Brian C; Arend, Lois J; McGraw, Dennis W; Penn, Raymond B; Petrovic, Snezana

2010-10-01

Proton receptors are G protein-coupled receptors that accept protons as ligands and function as pH sensors. One of the proton receptors, GPR4, is relatively abundant in the kidney, but its potential role in acid-base homeostasis is unknown. In this study, we examined the distribution of GPR4 in the kidney, its function in kidney epithelial cells, and the effects of its deletion on acid-base homeostasis. We observed GPR4 expression in the kidney cortex, in the outer and inner medulla, in isolated kidney collecting ducts, and in cultured outer and inner medullary collecting duct cells (mOMCD1 and mIMCD3). Cultured mOMCD1 cells exhibited pH-dependent accumulation of intracellular cAMP, characteristic of GPR4 activation; GPR4 knockdown attenuated this accumulation. In vivo, deletion of GPR4 decreased net acid secretion by the kidney and resulted in a nongap metabolic acidosis, indicating that GPR4 is required to maintain acid-base homeostasis. Collectively, these findings suggest that GPR4 is a pH sensor with an important role in regulating acid secretion in the kidney collecting duct.

Regulation of P2Y1 receptor traffic by sorting Nexin 1 is retromer independent.

PubMed

Nisar, Shaista; Kelly, Eamonn; Cullen, Pete J; Mundell, Stuart J

2010-04-01

The activity and traffic of G-protein coupled receptors (GPCRs) is tightly controlled. Recent work from our laboratory has shown that P2Y(1) and P2Y(12) responsiveness is rapidly and reversibly modulated in human platelets and that the underlying mechanism requires receptor trafficking as an essential part of this process. However, little is known about the molecular mechanisms underlying P2Y receptor traffic. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. These functions may in part be due to interactions of SNX1 with the mammalian retromer complex. In this study, we investigated the role of SNX1 in P2Y receptor trafficking. We show that P2Y(1) receptors recycle via a slow recycling pathway that is regulated by SNX1, whereas P2Y(12) receptors return to the cell surface via a rapid route that is SNX1 independent. SNX1 inhibition caused a dramatic increase in the rate of P2Y(1) receptor recycling, whereas inhibition of Vps26 and Vps35 known to be present in retromer had no effect, indicating that SNX1 regulation of P2Y(1) receptor recycling is retromer independent. In addition, inhibition of SNX4, 6 and 17 proteins did not affect P2Y(1) receptor recycling. SNX1 has also been implicated in GPCR degradation; however, we provide evidence that P2Y receptor degradation is SNX1 independent. These data describe a novel function of SNX1 in the regulation of P2Y(1) receptor recycling and suggest that SNX1 plays multiple roles in endocytic trafficking of GPCRs.

Role of GPR30 in estrogen-induced prostate epithelial apoptosis and benign prostatic hyperplasia.

PubMed

Yang, Deng-Liang; Xu, Jia-Wen; Zhu, Jian-Guo; Zhang, Yi-Lin; Xu, Jian-Bang; Sun, Qing; Cao, Xiao-Nian; Zuo, Wu-Lin; Xu, Ruo-Shui; Huang, Jie-Hong; Jiang, Fu-Neng; Zhuo, Yang-Jia; Xiao, Bai-Quan; Liu, Yun-Zhong; Yuan, Dong-Bo; Sun, Zhao-Lin; He, Hui-Chan; Lun, Zhao-Rong; Zhong, Wei-De; Zhou, Wen-Liang

2017-06-03

Several studies have implicated estrogen and the estrogen receptor (ER) in the pathogenesis of benign prostatic hyperplasia (BPH); however, the mechanism underlying this effect remains elusive. In the present study, we demonstrated that estrogen (17β-estradiol, or E2)-induced activation of the G protein-coupled receptor 30 (GPR30) triggered Ca 2+ release from the endoplasmic reticulum, increased the mitochondrial Ca 2+ concentration, and thus induced prostate epithelial cell (PEC) apoptosis. Both E2 and the GPR30-specific agonist G1 induced a transient intracellular Ca 2+ release in PECs via the phospholipase C (PLC)-inositol 1, 4, 5-triphosphate (IP 3 ) pathway, and this was abolished by treatment with the GPR30 antagonist G15. The release of cytochrome c and activation of caspase-3 in response to GPR30 activation were observed. Data generated from the analysis of animal models and human clinical samples indicate that treatment with the GPR30 agonist relieves testosterone propionate (TP)-induced prostatic epithelial hyperplasia, and that the abundance of GPR30 is negatively associated with prostate volume. On the basis of these results, we propose a novel regulatory mechanism whereby estrogen induces the apoptosis of PECs via GPR30 activation. Inhibition of this activation is predicted to lead to abnormal PEC accumulation, and to thereby contribute to BPH pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Imbalance between proliferation and apoptosis-related impaired GPR30 expression is involved in preeclampsia.

PubMed

Li, Jianxin; Chen, Zhu; Zhou, Xiaobo; Shi, Shuming; Qi, Hongbo; Baker, Philip N; Zhang, Hua

2016-11-01

The proliferation and apoptosis of cells in the placenta play a critical role in preeclampsia (PE) in which estrogen has been implicated via estrogen receptors (ERs). A novel ER, G-protein-coupled receptor 30 (GPR30), has recently been shown to be involved in PE. We investigated the basic levels of proliferation and apoptosis in normal placentae and placentae with PE and compared GPR30 expression levels between the two groups. We demonstrated that low GPR30 expression levels, more apoptosis, and less proliferation were associated with PE. Moreover, our in vitro study showed that both the selective GPR30 agonist G1 and the general ER agonist 17-β-estradiol were able to protect the placenta from hypoxia-reoxygenation injuries, resulting in decreased apoptosis and increased proliferation. Furthermore, this protective effect was abolished by the addition of the selective GPR30 inhibitor G15. These results provide evidence that (1) GPR30 is involved in regulating cell proliferation and apoptosis; (2) pharmacologic upregulation of GPR30 is beneficial for PE management; (3) GPR30 may therefore be an interventional target for pregnancies complicated by PE.

Association of a reduction of G-protein coupled receptor 30 expression and the pathogenesis of preeclampsia

PubMed Central

Feng, Xiang; Zhou, Liyuan; Mao, Xun; Tong, Chao; Chen, Xuyang; Zhao, Diqi; Baker, Philip N.; Xia, Yinyin; Zhang, Hua

2017-01-01

Preeclampsia is a pregnancy-specific disorder, which is a leading cause of maternal and perinatal mortality and morbidity. A lower increase of estrogen, compared with the increase in progesterone, is associated with pathogenesis of the disease during pregnancy. G-protein-coupled receptor 30 (GPR30) mediates the action of estrogen, however remains to be investigated in preeclampsia. The levels of GPR30 were measured in placentae from uncomplicated pregnancies and pregnancies complicated by preeclampsia using immunohistochemistry and western blotting. GPR30 expression was additionally measured in placental HTR8/SVneo cells following 17β-estrogen (E2) treatment in normal or hypoxia-reoxygenation conditions by western blotting. In addition, the outgrowth of HTR8/SVneo cells following E2 treatment in hypoxia-reoxygenation conditions was measured. Levels of GPR30 were significantly reduced in placentae from women with preeclampsia as compared with uncomplicated pregnancies. Treatment with E2 significantly increased the expression of GPR30 in HTR8/SVneo cells, in normal and hypoxia-reoxygenation conditions. Furthermore, treatment with E2 increased the outgrowth of HTR8/SVneo cells in hypoxia-reoxygenation conditions. The present study demonstrated lowered placental expression of GPR30 in preeclampsia. Estrogen treatment increases GPR30 expression in extravillous trophoblast and GPR30 may be involved in extravillous trophoblast invasion. PMID:28849224

Activation of GPR30 attenuates chronic pain-related anxiety in ovariectomized mice.

PubMed

Liu, Shui-bing; Tian, Zhen; Guo, Yan-yan; Zhang, Nan; Feng, Bin; Zhao, Ming-gao

2015-03-01

Estrogen regulates neuroendocrine and inflammatory processes that play critical roles in neuroinflammation, anxiety, and chronic pain. Patients suffering from chronic pain often complain of anxiety. However, limited information is available regarding the neural circuitry of chronic pain-related anxiety and the related function of estrogen. Hindpaw injection of complete Freund's adjuvant (CFA) and chronic constriction injury (CCI) of the sciatic nerve induced notable pain sensitization and anxiety-like behavior in ovariectomized (OVX) mice. We found that the level of G-protein-coupled receptor 30 (GPR30), a membrane estrogen receptor, was significantly increased in the basolateral amygdala (BLA) of ovariectomized (OVX) mice suffering from chronic inflammatory and neuropathic pain. Subcutaneous injection or BLA local infusion of the GPR30 agonist G1 significantly reduced anxiety-like behavior in CFA-injected and CCI-OVX mice; however, this treatment did not alter the nociceptive threshold. GPR30 knock down by shRNA in the BLA of OVX mice inhibited the anxiolytic effects of GPR30 activation. G1 administration reversed the upregulation of GluR1 subunit in AMPA and NR2A-containing NMDA receptors and the downregulation of GABAA receptors in the BLA of CFA-injected and CCI-OVX mice. Electrophysiological recording revealed that GPR30 activation could prevent imbalance between excitatory and inhibitory transmissions in the BLA synapses of CFA-injected OVX mice. In conclusion, GPR30 activation induced anxiolytic effects but did not affect the nociceptive threshold of mice under chronic pain. The anxiolytic effects of GPR30 were partially due to maintaining the balance between excitatory and inhibitory transmissions in the BLA. Copyright © 2015 Elsevier Ltd. All rights reserved.

G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio.

PubMed

Majumder, Suravi; Das, Sumana; Moulik, Sujata Roy; Mallick, Buddhadev; Pal, Puja; Mukherjee, Dilip

2015-01-15

GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

Free fatty acids-sensing G protein-coupled receptors in drug targeting and therapeutics.

PubMed

Yonezawa, Tomo; Kurata, Riho; Yoshida, Kaori; Murayama, Masanori A; Cui, Xiaofeng; Hasegawa, Akihiko

2013-01-01

G protein-coupled receptor (GPCR) (also known as seven-transmembrane domain receptor) superfamily represents the largest protein family in the human genome. These receptors respond to various physiological ligands such as photons, odors, pheromones, hormones, ions, and small molecules including amines, amino acids to large peptides and steroids. Thus, GPCRs are involved in many diseases and the target of around half of all conventional drugs. The physiological roles of free fatty acids (FFAs), in particular, long-chain FFAs, are important for the development of many metabolic disease including obesity, diabetes, and atherosclerosis. In the past half decade, deorphanization of several GPCRs has revealed that GPR40, GPR41, GPR43, GPR84 and GPR120 sense concentration of extracellular FFAs with various carbon chain lengths. GPR40 and GPR120 are activated by medium- and long-chain FFAs. GPR84 is activated by medium- chain, but not long-chain, FFAs. GPR41 and GPR43 are activated by short-chain FFAs. GPR40 is highly expressed in pancreatic beta cells and plays a crucial role in FFAs-induced insulin secretion. GPR120 is mainly expressed in enteroendocrine cells and plays an important role for FFAs-induced glucagon-like peptide-1. GPR43 is abundant in leukocytes and adipose tissue, whilst GPR41 is highly expressed in adipose tissue, the pancreas and leukocytes. GPR84 is expressed in leukocytes and monocyte/macrophage. This review aims to shed light on the physiological roles and development of drugs targeting these receptors.

Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1*

PubMed Central

Tran, Quang-Kim; Firkins, Rachel; Giles, Jennifer; Francis, Sarah; Matnishian, Vahe; Tran, Phuong; VerMeer, Mark; Jasurda, Jake; Burgard, Michelle Ann; Gebert-Oberle, Briana

2016-01-01

Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca2+ signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca2+ and Ca2+-CaM indicated that E2 also increases free Ca2+-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca2+-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca2+ efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca2+ signals and promote Ca2+-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca2+/CaM signals and functional linkage in the endothelial CaM target network. PMID:26987903

Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1.

PubMed

Tran, Quang-Kim; Firkins, Rachel; Giles, Jennifer; Francis, Sarah; Matnishian, Vahe; Tran, Phuong; VerMeer, Mark; Jasurda, Jake; Burgard, Michelle Ann; Gebert-Oberle, Briana

2016-05-13

Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17β-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gβγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic deletion of GPR52 enhances the locomotor-stimulating effect of an adenosine A2A receptor antagonist in mice: A potential role of GPR52 in the function of striatopallidal neurons.

PubMed

Nishiyama, Keiji; Suzuki, Hirobumi; Maruyama, Minoru; Yoshihara, Tomoki; Ohta, Hiroyuki

2017-09-01

G protein-coupled receptor 52 (GPR52) is largely co-expressed with dopamine D 2 receptor (DRD2) in the striatum and nucleus accumbens, and this expression pattern is similar to that of adenosine A 2A receptor (ADORA2A). GPR52 has been proposed as a therapeutic target for positive symptoms of schizophrenia, based on observations from pharmacological and transgenic mouse studies. However, the physiological role of GPR52 in dopaminergic functions in the basal ganglia remains unclear. Here, we used GPR52 knockout (KO) mice to examine the role of GPR52 in dopamine receptor-mediated and ADORA2A-mediated locomotor activity and dopamine receptor signaling. High expression of GPR52 protein in the striatum, nucleus accumbens, and lateral globus pallidus of wild type (WT) littermates was confirmed by immunohistochemical analysis. GPR52 KO and WT mice exhibited almost identical locomotor responses to the dopamine releaser methamphetamine and the N-methyl-d-aspartate antagonist MK-801. In contrast, the locomotor response to the ADORA2A antagonist istradefylline was significantly augmented in GPR52 KO mice compared to WT mice. Gene expression analysis revealed that striatal expression of DRD2, but not of dopamine D 1 receptor and ADORA2A, was significantly decreased in GPR52 KO mice. Moreover, a significant reduction in the mRNA expression of enkephalin, a marker of the activity of striatopallidal neurons, was observed in the striatum of GPR52 KO mice, suggesting that GPR52 deletion could enhance DRD2 signaling. Taken together, these results imply the physiological relevance of GPR52 in modulating the function of striatopallidal neurons, possibly by interaction of GPR52 with ADORA2A and DRD2. Copyright © 2017 Elsevier B.V. All rights reserved.

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b*

PubMed Central

Tran, Quang-Kim; VerMeer, Mark; Burgard, Michelle A.; Hassan, Ali B.; Giles, Jennifer

2015-01-01

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca2+-ATPase (PMCA) is essential for removal of cytoplasmic Ca2+ and for shaping the time courses of Ca2+-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca2+ extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca2+ signaling and GPER/GPR30-mediated activities. PMID:25847233

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b.

PubMed

Tran, Quang-Kim; VerMeer, Mark; Burgard, Michelle A; Hassan, Ali B; Giles, Jennifer

2015-05-22

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca(2+)-ATPase (PMCA) is essential for removal of cytoplasmic Ca(2+) and for shaping the time courses of Ca(2+)-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca(2+) extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca(2+) signaling and GPER/GPR30-mediated activities. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Epidermal growth factor induces G protein-coupled receptor 30 expression in estrogen receptor-negative breast cancer cells.

PubMed

Albanito, Lidia; Sisci, Diego; Aquila, Saveria; Brunelli, Elvira; Vivacqua, Adele; Madeo, Antonio; Lappano, Rosamaria; Pandey, Deo Prakash; Picard, Didier; Mauro, Loredana; Andò, Sebastiano; Maggiolini, Marcello

2008-08-01

Different cellular receptors mediate the biological effects induced by estrogens. In addition to the classical nuclear estrogen receptors (ERs)-alpha and -beta, estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPR)-30. Using as a model system SkBr3 and BT20 breast cancer cells lacking the classical ER, the regulation of GPR30 expression by 17beta-estradiol, the selective GPR30 ligand G-1, IGF-I, and epidermal growth factor (EGF) was evaluated. Transient transfections with an expression plasmid encoding a short 5'-flanking sequence of the GPR30 gene revealed that an activator protein-1 site located within this region is required for the activating potential exhibited only by EGF. Accordingly, EGF up-regulated GPR30 protein levels, which accumulated predominantly in the intracellular compartment. The stimulatory role elicited by EGF on GPR30 expression was triggered through rapid ERK phosphorylation and c-fos induction, which was strongly recruited to the activator protein-1 site found in the short 5'-flanking sequence of the GPR30 gene. Of note, EGF activating the EGF receptor-MAPK transduction pathway stimulated a regulatory loop that subsequently engaged estrogen through GPR30 to boost the proliferation of SkBr3 and BT20 breast tumor cells. The up-regulation of GPR30 by ligand-activated EGF receptor-MAPK signaling provides new insight into the well-known estrogen and EGF cross talk, which, as largely reported, contributes to breast cancer progression. On the basis of our results, the action of EGF may include the up-regulation of GPR30 in facilitating a stimulatory role of estrogen, even in ER-negative breast tumor cells.

Synthesis and pharmacological profile of a new selective G protein-coupled receptor 119 agonist; 6-((2-fluoro-3-(1-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-4-yl)propyl)amino)-2,3-dihydro-1H-inden-1-one.

PubMed

Sakairi, Masao; Kogami, Masakazu; Torii, Masafumi; Makino, Mitsuhiro; Kataoka, Daisuke; Okamoto, Ryuji; Miyazawa, Toshiyuki; Inoue, Megumi; Takahashi, Naoki; Harada, Satoko; Watanabe, Nobuhide

2012-01-01

6-((2-Fluoro-3-(1-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-4-yl)propyl)amino)-2,3-dihydro-1H-inden-1-one is a potent drug-like G protein-coupled receptor 119 (GPR119) agonist. It is hoped that this compound would be instrumental in probing the pharmacological potential of GPR119 agonists.

Cloning and characterization of the rat free fatty acid receptor GPR120: in vivo effect of the natural ligand on GLP-1 secretion and proliferation of pancreatic beta cells.

PubMed

Tanaka, Toshiki; Yano, Takeaki; Adachi, Tetsuya; Koshimizu, Taka-aki; Hirasawa, Akira; Tsujimoto, Gozoh

2008-06-01

We have recently found that GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain free fatty acids (FFAs) and that GPR120 stimulation promotes the secretion of glucagons-like peptide-1 (GLP-1) in the mouse (Hirasawa et al., Nat Med 11:90-94, 2005). In this study, we cloned and characterized rat GPR120 (rGPR120), and then we examined the in vivo effects of acute and long-term administration of the natural ligand alpha-linolenic acid (alpha-LA). The cloned rat GPR120 complimentary DNA had a seven transmembrane structure, and a homology comparison of human, mouse, and rat GPR120 revealed that the rat GPR120 (rGPR120) shares 85 and 98% sequence identity with the human and mouse GPR120 proteins, respectively. The tissue distribution and ligand properties of rGPR120 were similar to those of mouse GPR120. In addition, alpha-LA provoked a transient increase in [Ca2+]i levels in HEK293 cells expressing rGPR120. Furthermore, administration of alpha-LA to the rat increased plasma GLP-1 levels, and long-term administration of alpha-LA led to proliferation of pancreatic beta cells, probably because of the enhanced GLP-1 secretion. These results show that rat GPR120 is a G-protein-coupled receptor whose ligand is a free fatty acid, and it may play an important role in the FFA-associated physiological responses.

Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell-matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis.

PubMed

Chen, Yan; Li, Zheng; He, Yan; Shang, Dandan; Pan, Jigang; Wang, Hongmei; Chen, Huamei; Zhu, Zhuxia; Wan, Lei; Wang, Xudong

2014-03-01

Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

GPR30: a seven-transmembrane-spanning estrogen receptor that triggers EGF release.

PubMed

Filardo, Edward J; Thomas, Peter

2005-10-01

Heterotrimeric G proteins and seven-transmembrane-spanning (7TM) receptors are implicated in rapid estrogen signaling. The orphan 7TM receptor GPR30 is linked to estrogen-mediated activation of adenylyl cyclase, release of epidermal growth factor (EGF)-related ligands, and specific estrogen binding. GPR30 acts independently of estrogen receptors, ERalpha and ERbeta, and probably functions as a heptahelical ER. 7TM receptors elicit signals that stimulate second messengers, and convey intracellular signals via EGF receptors. Identification of GPR30 as a Gs-coupled 7TM receptor that triggers release of heparin-binding EGF establishes its role in cell signaling cascades initiated by estrogens, and explains their capacity to activate second messengers and promote EGF-like effects. Thus, estrogen can signal by the same mechanism as various other hormones, through a specific 7TM receptor.

Genetic variants of the unsaturated fatty acid receptor GPR120 relating to obesity in dogs.

PubMed

Miyabe, Masahiro; Gin, Azusa; Onozawa, Eri; Daimon, Mana; Yamada, Hana; Oda, Hitomi; Mori, Akihiro; Momota, Yutaka; Azakami, Daigo; Yamamoto, Ichiro; Mochizuki, Mariko; Sako, Toshinori; Tamura, Katsutoshi; Ishioka, Katsumi

2015-10-01

G protein-coupled receptor (GPR) 120 is an unsaturated fatty acid receptor, which is associated with various physiological functions. It is reported that the genetic variant of GPR120, p.Arg270His, is detected more in obese people, and this genetic variation functionally relates to obesity in humans. Obesity is a common nutritional disorder also in dogs, but the genetic factors have not ever been identified in dogs. In this study, we investigated the molecular structure of canine GPR120 and searched for candidate genetic variants which may relate to obesity in dogs. Canine GPR120 was highly homologous to those of other species, and seven transmembrane domains and two N-glycosylation sites were conserved. GPR120 mRNA was expressed in lung, jejunum, ileum, colon, hypothalamus, hippocampus, spinal cord, bone marrow, dermis and white adipose tissues in dogs, as those in mice and humans. Genetic variants of GPR120 were explored in client-owned 141 dogs, resulting in that 5 synonymous and 4 non-synonymous variants were found. The variant c.595C>A (p.Pro199Thr) was found in 40 dogs, and the gene frequency was significantly higher in dogs with higher body condition scores, i.e. 0.320 in BCS4-5 dogs, 0.175 in BCS3 dogs and 0.000 in BCS2 dogs. We conclude that c.595C>A (p.Pro199Thr) is a candidate variant relating to obesity, which may be helpful for nutritional management of dogs.

GPR120: Mechanism of action, role and potential for medical applications.

PubMed

Karakuła-Juchnowicz, Hanna; Róg, Joanna; Juchnowicz, Dariusz; Morylowska-Topolska, Justyna

2017-11-19

G protein-coupled receptors (GPCRs) constitute a family of transmembrane proteins that mediate many cellular processes. GPR120/FFAR4, a receptor from this family that is activated by fatty acids, has received considerable attention recently. This paper presents a literature review concerning the role of GPR120 and its mechanism of action in animal and human studies as well as the potential use of GPR120 for the treatment of chronic diseases. Two electronic databases - Medline and Google Scholar - were searched for available studies addressing the review topic that were written in English and published from 2000 to June 2017. The following key terms were used in the search: GPR120, FFA4, GPR120 agonist, PUFAs, EPA, DHA, adipocyte, obesity, hyperlipidemia, inflammation, cancer, diabetes, insulin resistance, taste, atherogenesis, hepatis, central nervous system. In humans, GPR120 expression is expressed in macrophages, eosinophils, and adipose tissue, in cells of the tongue, liver, lungs, small and large intestine, gastric mucosa, and pancreas, in the central nervous system and placental microvilli. Medium- and long-chain fatty acids act as ligands for the receptor. Through the internalization of beta-arrestin-2 complex and the inhibition of NF-κB, GPR120 mediates the activation of the cell's anti-inflammatory mechanisms. The receptor is also involved in the maturation of adipocytes, the modulation of insulin signalling pathways, the regulation of glucose metabolism, and the secretion of intestinal hormones. GPR120 is a promising target for the treatment of numerous diseases, whose pathophysiology is associated with low-grade inflammation. As a result of intensive searches, a likely group of synthetic agonists of the receptor was determined with potential therapeutic applications in conditions such as obesity, impaired carbohydrate metabolism, inflammatory bowel diseases, cancer, mental disorders.

Expression of fatty acid sensing G-protein coupled receptors in peripartal Holstein cows.

PubMed

Agrawal, Alea; Alharthi, Abdulrahman; Vailati-Riboni, Mario; Zhou, Zheng; Loor, Juan J

2017-01-01

G-protein coupled receptors (GPCR), also referred as Free Fatty Acid Receptors (FFAR), are widely studied within human medicine as drug targets for metabolic disorders. To combat metabolic disorders prevalent in dairy cows during the transition period, which co-occur with negative energy balance and changes to lipid and glucose metabolism, it may be helpful to identify locations and roles of FFAR and other members of the GPCR family in bovine tissues. Quantitative RT-PCR (qPCR) of subcutaneous adipose, liver, and PMNL samples during the transition period (-10, +7, and +20 or +30 d) were used for expression profiling of medium- (MCFA) and long-chain fatty acid (LCFA) receptors GPR120 and GPR40 , MCFA receptor GPR84 , and niacin receptor HCAR2/3 . Adipose samples were obtained from cows with either high (HI; BCS ≥ 3.75) or low (LO; BCS ≤ 3.25) body condition score (BCS) to examine whether FFAR expression is correlated with this indicator of health and body reserves. Supplementation of rumen-protected methionine (MET), which may improve immune function and production postpartum, was also compared with unsupplemented control (CON) cows for liver and blood polymorphonuclear leukocytes (PMNL) samples. In adipose tissue, GPR84 and GPR120 were differentially expressed over time, while GPR40 was not expressed; in PMNL, GPR40 was differentially expressed over time and between MET vs. CON, GPR84 expression differed only between dietary groups, and GPR120 was not expressed; in liver, GPCR were either not expressed or barely detectable. The data indicate that there is likely not a direct role in liver for the selected GPCR during the transition period, but they do play variable roles in adipose and PMN. In future, these receptors may prove useful targets and/or markers for peripartal metabolism and immunity.

G protein-coupled receptor 30 ligand G-1 increases aryl hydrocarbon receptor signalling by inhibition of tubulin assembly and cell cycle arrest in human MCF-7 cells.

PubMed

Tarnow, Patrick; Tralau, Tewes; Luch, Andreas

2016-08-01

Regulatory crosstalk between the aryl hydrocarbon receptor (AHR) and oestrogen receptor α (ERα) is well established. Apart from the nuclear receptors ERα and ERβ, oestrogen signalling further involves an unrelated G protein-coupled receptor termed GPR30. In order to investigate potential regulatory crosstalk, this study investigated the influence of G-1 as one of the few GPR30-specific ligands on the AHR regulon in MCF-7 cells. As a well-characterised model system, these human mammary carcinoma cells co-express all three receptors (AHR, ERα and GPR30) and are thus ideally suited to study corresponding regulatory pathway interactions on transcript level. Indeed, treatment with micromolar concentrations of the GPR30-specific agonist G-1 resulted in up-regulation of AHR as well as the transcripts for cytochromes P450 1A1 and 1B1, two well-known targets of the AHR regulon. While this was partly attributable to G-1-mediated inhibition of tubulin assembly and subsequent cell cycle arrest in the G2/M phase, the effects nevertheless required functional AHR. However, G-1-induced up-regulation of CYP 1A1 was not mediated by GPR30, as G15 antagonist treatment as well as a knockdown of GPR30 and AHR failed to inhibit this effect.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Jun; Byrne, Noel; Wang, John

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational couplingmore » between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.« less

Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yan; Li, Zheng; He, Yan

2014-03-01

Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but notmore » calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. - Highlights: • Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells. • GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2. • Calpain is required in the cell–matrigel adhesion induced by E2 and ICI.« less

Activation of G-protein-coupled receptor 30 is sufficient to enhance spatial recognition memory in ovariectomized rats.

PubMed

Hawley, Wayne R; Grissom, Elin M; Moody, Nicole M; Dohanich, Gary P; Vasudevan, Nandini

2014-04-01

In ovariectomized rats, administration of estradiol, or selective estrogen receptor agonists that activate either the α or β isoforms, have been shown to enhance spatial cognition on a variety of learning and memory tasks, including those that capitalize on the preference of rats to seek out novelty. Although the effects of the putative estrogen G-protein-coupled receptor 30 (GPR30) on hippocampus-based tasks have been reported using food-motivated tasks, the effects of activation of GPR30 receptors on tasks that depend on the preference of rats to seek out spatial novelty remain to be determined. Therefore, the aim of the current study was to determine if short-term treatment of ovariectomized rats with G-1, an agonist for GPR30, would mimic the effects on spatial recognition memory observed following short-term estradiol treatment. In Experiment 1, ovariectomized rats treated with a low dose (1 μg) of estradiol 48 h and 24 h prior to the information trial of a Y-maze task exhibited a preference for the arm associated with the novel environment on the retention trial conducted 48 h later. In Experiment 2, treatment of ovariectomized rats with G-1 (25 μg) 48 h and 24 h prior to the information trial of a Y-maze task resulted in a greater preference for the arm associated with the novel environment on the retention trial. Collectively, the results indicated that short-term treatment of ovariectomized rats with a GPR30 agonist was sufficient to enhance spatial recognition memory, an effect that also occurred following short-term treatment with a low dose of estradiol. Copyright © 2014 Elsevier B.V. All rights reserved.

The cannabinoid receptor CB1 modulates the signaling properties of the lysophosphatidylinositol receptor GPR55.

PubMed

Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P; Brown, Andrew J; Heinemann, Akos; Waldhoer, Maria

2012-12-28

The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors.

The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55*

PubMed Central

Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P.; Brown, Andrew J.; Heinemann, Akos; Waldhoer, Maria

2012-01-01

The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors. PMID:23161546

Expression analysis of G Protein-Coupled Receptors in mouse macrophages.

PubMed

Lattin, Jane E; Schroder, Kate; Su, Andrew I; Walker, John R; Zhang, Jie; Wiltshire, Tim; Saijo, Kaoru; Glass, Christopher K; Hume, David A; Kellie, Stuart; Sweet, Matthew J

2008-04-29

Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery.

Estrogens Induce Rapid Cytoskeleton Re-Organization in Human Dermal Fibroblasts via the Non-Classical Receptor GPR30

PubMed Central

Carnesecchi, Julie; Malbouyres, Marilyne; de Mets, Richard; Balland, Martial; Beauchef, Gallic; Vié, Katell; Chamot, Christophe; Lionnet, Claire; Ruggiero, Florence; Vanacker, Jean-Marc

2015-01-01

The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation. PMID:25781607

Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

PubMed

Carnesecchi, Julie; Malbouyres, Marilyne; de Mets, Richard; Balland, Martial; Beauchef, Gallic; Vié, Katell; Chamot, Christophe; Lionnet, Claire; Ruggiero, Florence; Vanacker, Jean-Marc

2015-01-01

The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

Differential local tissue permissiveness influences the final fate of GPR17‐expressing oligodendrocyte precursors in two distinct models of demyelination

PubMed Central

Coppolino, Giusy T.; Marangon, Davide; Negri, Camilla; Menichetti, Gianluca; Fumagalli, Marta; Gelosa, Paolo; Dimou, Leda; Furlan, Roberto; Lecca, Davide

2018-01-01

Abstract Promoting remyelination is recognized as a novel strategy to foster repair in neurodegenerative demyelinating diseases, such as multiple sclerosis. In this respect, the receptor GPR17, recently emerged as a new target for remyelination, is expressed by early oligodendrocyte precursors (OPCs) and after a certain differentiation stage it has to be downregulated to allow progression to mature myelinating oligodendrocytes. Here, we took advantage of the first inducible GPR17 reporter mouse line (GPR17‐iCreERT2xCAG‐eGFP mice) allowing to follow the final fate of GPR17+ cells by tamoxifen‐induced GFP‐labeling to unveil the destiny of these cells in two demyelination models: experimental autoimmune encephalomyelitis (EAE), characterized by marked immune cell activation and inflammation, and cuprizone induced demyelination, where myelin dysfunction is achieved by a toxic insult. In both models, demyelination induced a strong increase of fluorescent GFP+ cells at damaged areas. However, only in the cuprizone model reacting GFP+ cells terminally differentiated to mature oligodendrocytes, thus contributing to remyelination. In EAE, GFP+ cells were blocked at immature stages and never became myelinating oligodendrocytes. We suggest these strikingly distinct fates be due to different permissiveness of the local CNS environment. Based on previously reported GPR17 activation by emergency signals (e.g., Stromal Derived Factor‐1), we propose that a marked inflammatory milieu, such as that reproduced in EAE, induces GPR17 overactivation resulting in impaired downregulation, untimely and prolonged permanence in OPCs, leading, in turn, to differentiation blockade. Combined treatments with remyelinating agents and anti‐inflammatory drugs may represent new potential adequate strategies to halt neurodegeneration and foster recovery. PMID:29424466

Differential local tissue permissiveness influences the final fate of GPR17-expressing oligodendrocyte precursors in two distinct models of demyelination.

PubMed

Coppolino, Giusy T; Marangon, Davide; Negri, Camilla; Menichetti, Gianluca; Fumagalli, Marta; Gelosa, Paolo; Dimou, Leda; Furlan, Roberto; Lecca, Davide; Abbracchio, Maria P

2018-05-01

Promoting remyelination is recognized as a novel strategy to foster repair in neurodegenerative demyelinating diseases, such as multiple sclerosis. In this respect, the receptor GPR17, recently emerged as a new target for remyelination, is expressed by early oligodendrocyte precursors (OPCs) and after a certain differentiation stage it has to be downregulated to allow progression to mature myelinating oligodendrocytes. Here, we took advantage of the first inducible GPR17 reporter mouse line (GPR17-iCreER T2 xCAG-eGFP mice) allowing to follow the final fate of GPR17 + cells by tamoxifen-induced GFP-labeling to unveil the destiny of these cells in two demyelination models: experimental autoimmune encephalomyelitis (EAE), characterized by marked immune cell activation and inflammation, and cuprizone induced demyelination, where myelin dysfunction is achieved by a toxic insult. In both models, demyelination induced a strong increase of fluorescent GFP + cells at damaged areas. However, only in the cuprizone model reacting GFP + cells terminally differentiated to mature oligodendrocytes, thus contributing to remyelination. In EAE, GFP + cells were blocked at immature stages and never became myelinating oligodendrocytes. We suggest these strikingly distinct fates be due to different permissiveness of the local CNS environment. Based on previously reported GPR17 activation by emergency signals (e.g., Stromal Derived Factor-1), we propose that a marked inflammatory milieu, such as that reproduced in EAE, induces GPR17 overactivation resulting in impaired downregulation, untimely and prolonged permanence in OPCs, leading, in turn, to differentiation blockade. Combined treatments with remyelinating agents and anti-inflammatory drugs may represent new potential adequate strategies to halt neurodegeneration and foster recovery. © 2018 The Authors GLIA Published by Wiley Periodicals, Inc.

Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis.

PubMed

Hill, Jeremy D; Zuluaga-Ramirez, Viviana; Gajghate, Sachin; Winfield, Malika; Persidsky, Yuri

2018-06-11

The cannabinoid system exerts functional regulation of neural stem cell (NSC) proliferation and adult neurogenesis, yet not all effects of cannabinoid-like compounds seen can be attributed to the cannabinoid 1 receptor (CB 1 R) or cannabinoid 2 receptor (CB 2 R). The recently de-orphaned GPR55 has been shown to be activated by numerous cannabinoid ligands suggesting that GPR55 is a third cannabinoid receptor. Here we examined the role of GPR55 activation in NSC proliferation and early adult neurogenesis. The effects of GPR55 agonists (LPI, O-1602, ML184) on human NSC proliferation in vitro were assessed by flow cytometry. hNSC differentiation was determined by flow cytometry, qPCR, and immunohistochemistry. Immature neuron formation in the hippocampus of C57BL/6 and GPR55 -/- mice was evaluated by immunohistochemistry. Activation of GPR55 significantly increased proliferation rates of hNSCs in vitro. These effects were attenuated by ML193, a selective GPR55 antagonist. ML184 significantly promoted neuronal differentiation in vitro while ML193 reduced differentiation rates as compared to vehicle treatment. Continuous administration into the hippocampus of O-1602 via cannula connected to osmotic pump resulted in increased Ki67+ cells within the dentate gyrus. O-1602 increased immature neuron generation as assessed by DCX+ and BrdU+ cells as compared to vehicle treated animals. GPR55 -/- animals displayed reduced rates of proliferation and neurogenesis within the hippocampus while O-1602 had no effect as compared to vehicle controls. Together, these findings suggest GPR55 activation as a novel target and strategy to regulate NSC proliferation and adult neurogenesis. This article is protected by copyright. All rights reserved.

Genetic variants of the unsaturated fatty acid receptor GPR120 relating to obesity in dogs

PubMed Central

MIYABE, Masahiro; GIN, Azusa; ONOZAWA, Eri; DAIMON, Mana; YAMADA, Hana; ODA, Hitomi; MORI, Akihiro; MOMOTA, Yutaka; AZAKAMI, Daigo; YAMAMOTO, Ichiro; MOCHIZUKI, Mariko; SAKO, Toshinori; TAMURA, Katsutoshi; ISHIOKA, Katsumi

2015-01-01

G protein-coupled receptor (GPR) 120 is an unsaturated fatty acid receptor, which is associated with various physiological functions. It is reported that the genetic variant of GPR120, p.Arg270His, is detected more in obese people, and this genetic variation functionally relates to obesity in humans. Obesity is a common nutritional disorder also in dogs, but the genetic factors have not ever been identified in dogs. In this study, we investigated the molecular structure of canine GPR120 and searched for candidate genetic variants which may relate to obesity in dogs. Canine GPR120 was highly homologous to those of other species, and seven transmembrane domains and two N-glycosylation sites were conserved. GPR120 mRNA was expressed in lung, jejunum, ileum, colon, hypothalamus, hippocampus, spinal cord, bone marrow, dermis and white adipose tissues in dogs, as those in mice and humans. Genetic variants of GPR120 were explored in client-owned 141 dogs, resulting in that 5 synonymous and 4 non-synonymous variants were found. The variant c.595C>A (p.Pro199Thr) was found in 40 dogs, and the gene frequency was significantly higher in dogs with higher body condition scores, i.e. 0.320 in BCS4–5 dogs, 0.175 in BCS3 dogs and 0.000 in BCS2 dogs. We conclude that c.595C>A (p.Pro199Thr) is a candidate variant relating to obesity, which may be helpful for nutritional management of dogs. PMID:25960032

GPR3 Stimulates Aβ Production via Interactions with APP and β-Arrestin2

PubMed Central

Nelson, Christopher D.; Sheng, Morgan

2013-01-01

The orphan G protein-coupled receptor (GPCR) GPR3 enhances the processing of Amyloid Precursor Protein (APP) to the neurotoxic beta-amyloid (Aβ) peptide via incompletely understood mechanisms. Through overexpression and shRNA knockdown experiments in HEK293 cells, we show that β-arrestin2 (βarr2), a GPCR-interacting scaffold protein reported to bind γ-secretase, is an essential factor for GPR3-stimulated Aβ production. For a panel of GPR3 receptor mutants, the degree of stimulation of Aβ production correlates with receptor-β-arrestin binding and receptor trafficking to endocytic vesicles. However, GPR3’s recruitment of βarr2 cannot be the sole explanation, because interaction with βarr2 is common to most GPCRs, whereas GPR3 is relatively unique among GPCRs in enhancing Aβ production. In addition to β-arrestin, APP is present in a complex with GPR3 and stimulation of Aβ production by GPR3 mutants correlates with their level of APP binding. Importantly, among a broader selection of GPCRs, only GPR3 and prostaglandin E receptor 2 subtype EP2 (PTGER2; another GPCR that increases Aβ production) interact with APP, and PTGER2 does so in an agonist-stimulated manner. These data indicate that a subset of GPCRs, including GPR3 and PTGER2, can associate with APP when internalized via βarr2, and thereby promote the cleavage of APP to generate Aβ. PMID:24069330

G-protein-coupled estrogen receptor-30 gene polymorphisms are associated with uterine leiomyoma risk.

PubMed

Kasap, Burcu; Öztürk Turhan, Nilgün; Edgünlü, Tuba; Duran, Müzeyyen; Akbaba, Eren; Öner, Gökalp

2016-01-06

The G-protein-coupled estrogen receptor (GPR30, GPER-1) is a member of the G-protein-coupled receptor 1 family and is expressed significantly in uterine leiomyomas. To understand the relationship between GPR30 single nucleotide polymorphisms and the risk of leiomyoma, we measured the follicle-stimulating hormone (FSH) and estradiol (E2) levels of 78 perimenopausal healthy women and 111 perimenopausal women with leiomyomas. The participants' leiomyoma number and volume were recorded. DNA was extracted from whole blood with a GeneJET Genomic DNA Purification Kit. An amplification-refractory mutation system polymerase chain reaction approach was used for genotyping of the GPR30 gene (rs3808350, rs3808351, and rs11544331). The differences in genotype and allele frequencies between the leiomyoma and control groups were calculated using the chi-square (χ2) and Fischer's exact test. The median FSH level was higher in controls (63 vs. 10 IU/L, p=0.000), whereas the median E2 level was higher in the leiomyoma group (84 vs. 9.1 pg/mL, p=0.000). The G allele of rs3808351 and the GG genotype of both the rs3808350 and rs3808351 polymorphisms and the GGC haplotype increased the risk of developing leiomyoma. There was no significant difference in genotype frequencies or leiomyoma volume. However, the GG genotype of the GPR30 rs3808351 polymorphism and G allele of the GPR30 rs3808351 polymorphism were associated with the risk of having a single leiomyoma. Our results suggest that the presence of the GG genotype of the GPR30 rs3808351 polymorphism and the G allele of the GPR30 rs3808351 polymorphism affect the characteristics and development of leiomyomas in the Turkish population.

Synthetic estrogen derivatives demonstrate the functionality of intracellular GPR30.

PubMed

Revankar, Chetana M; Mitchell, Hugh D; Field, Angela S; Burai, Ritwik; Corona, Cesear; Ramesh, Chinnasamy; Sklar, Larry A; Arterburn, Jeffrey B; Prossnitz, Eric R

2007-08-17

Estrogen mediates its effects through multiple cellular receptors. In addition to the classical nuclear estrogen receptors (ERalpha and ERbeta), estrogen also signals through the seven-transmembrane G-protein-coupled receptor (GPCR) GPR30. Although estrogen is a cell-permeable ligand, it is often assumed that all GPCRs function solely as cell surface receptors. Our previous results showed that GPR30 appeared to be expressed predominantly in the endoplasmic reticulum. A critical question that arises is whether this localization represents the site of functional receptor. To address this question, we synthesized a collection of cell-permeable and cell-impermeable estrogen derivatives. We hypothesized that if functional GPR30 were expressed at the cell surface, both permeable and impermeable derivatives would show activity. However, if functional GPR30 were predominantly intracellular, like ERalpha, only the permeable ligands should show activity. Cell permeability was assessed using cells expressing ERalpha as a model intracellular estrogen-binding receptor. Our results reveal that despite exhibiting similar binding affinities for GPR30, only the cell-permeable ligands are capable of stimulating rapid calcium mobilization and phosphoinositide 3-kinase (PI3K) activation. We conclude that GPR30 expressed intracellularly is capable of initiating cellular signaling and that there is insufficient GPR30 expressed on the cell surface to initiate signaling in response to impermeable ligands in the cell lines examined. To our knowledge, this is the first definitive demonstration of a functional intracellular transmembrane estrogen receptor.

Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mochimaru, Yuta; Azuma, Morio; Oshima, Natsuki

2015-02-20

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1more » overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.« less

The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells.

PubMed

Du, Gui-Qiang; Zhou, Long; Chen, Xiao-Yue; Wan, Xiao-Ping; He, Yin-Yan

2012-04-06

The selective ER modulator tamoxifen (TAM(1)) is the most widely used ER antagonist for treatment of women with hormone-dependent breast tumor. However, long-term treatment is associated with an increased risk of endometrial cancer. The aim of the present study was to demonstrate new insight into the role of G-protein coupled receptor 30 (GPR30) in the activity of TAM, which promoted endometrial cancer. In endometrial cancer cell lines ISHIKAWA and KLE, the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, 17β-estradiol (E2) and G1, a non-steroidal GPR30-specific agonist to promote cell proliferation and invasion was evaluated. All agents above induced high proliferative and invasive effects, while the down-regulation of GPR30 or the interruption of MAPK signal pathway partly or completely prevented the action of the regent. Moreover, the RNA and protein expression of GPR30 was up-regulated by G1, E2 or OHT in both cell lines. The present study provided a new insight into the mechanism involved in the agonistic activity exerted by TAM in the uterus. Copyright © 2012 Elsevier Inc. All rights reserved.

G Protein-Coupled Receptor 30 (GPR30) Expression Pattern in Inflammatory Bowel Disease Patients Suggests its Key Role in the Inflammatory Process. A Preliminary Study.

PubMed

Włodarczyk, Marcin; Sobolewska-Włodarczyk, Aleksandra; Cygankiewicz, Adam I; Jacenik, Damian; Piechota-Polańczyk, Aleksandra; Stec-Michalska, Krystyna; Krajewska, Wanda M; Fichna, Jakub; Wiśniewska-Jarosińska, Maria

2017-03-01

G protein-coupled receptor 30 (GPR30) is a recently de-orphanized estrogen receptor that mediates the effects of estrogens on different cells. It has been postulated that in inflammatory bowel diseases (IBD) activation of GPR30 blocks the pathways dependent on pro-inflammatory cytokines. The aim of our study was to investigate GPR30 expression in patients with IBD and its potential implication in future therapies. Fifty-seven patients were enrolled in our study: 20 subjects with Crohn's disease (CD), 22 with ulcerative colitis (UC) and 15 controls. In each subject, biopsies were taken from various left-colonic locations. Gene and protein expression of GPR30 was quantified using real time RT-PCR or Western blot. GPR30 mRNA and protein expression were detected in all tested colonic tissues. No significant differences in GPR30 gene expression were observed. In non-inflamed areas, GPR30 protein was strongly increased in CD patients, but moderately in UC patients (p= 0.014 and p=0.143, respectively, vs. controls). In CD patients, a significantly lower GPR30 protein content in inflamed than in non-inflamed tissue was observed (p=0.039). The change was independent of patient gender. Our observations indicate that GPR30 may play a role in the development and progression of inflammatory lesions in IBD, thus affecting disease severity, and consequently IBD treatment. Therefore, GPR30 may become an attractive target for novel anti-IBD drugs, particularly in CD.

Endothelium-dependent relaxation evoked by ATP and UTP in the aorta of P2Y2-deficient mice

PubMed Central

Guns, Pieter-Jan D F; Van Assche, Tim; Fransen, Paul; Robaye, Bernard; Boeynaems, Jean-Marie; Bult, Hidde

2006-01-01

Based on pharmacological criteria, we previously suggested that in the mouse aorta, endothelium-dependent relaxation by nucleotides is mediated by P2Y1 (adenosine diphosphate (ADP)), P2Y2 (adenosine triphosphate (ATP)) and P2Y6 (uridine diphosphate (UDP)) receptors. For UTP, it was unclear whether P2Y2, P2Y6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists, we used P2Y2-deficient mice to clarify the action of UTP. Thoracic aorta segments (width 2 mm) of P2Y2-deficient and wild-type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling. Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice, indicating that the receptors for these agonists function normally. P2Y2-deficient mice showed impaired ATP- and adenosine 5′[γ-thio] triphosphate (ATPγS)-evoked relaxation, suggesting that in WT mice, ATP and ATPγS activate predominantly the P2Y2 subtype. The ATP/ATPγS-evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y1, as they were sensitive to 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate (MRS2179), a P2Y1-selective antagonist. In contrast to ATP, the UTP-evoked relaxation was not different between knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y6(like) receptor subtype. In conclusion, we demonstrated that ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y2. But this P2Y2 receptor has apparently no major role in UTP-evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y6(like) receptor. PMID:16415908

Regulation of Glucose Homeostasis by GLP-1

PubMed Central

Nadkarni, Prashant; Chepurny, Oleg G.; Holz, George G.

2014-01-01

Glucagon-like peptide-1(7–36)amide (GLP-1) is a secreted peptide that acts as a key determinant of blood glucose homeostasis by virtue of its abilities to slow gastric emptying, to enhance pancreatic insulin secretion, and to suppress pancreatic glucagon secretion. GLP-1 is secreted from L cells of the gastrointestinal mucosa in response to a meal, and the blood glucose-lowering action of GLP-1 is terminated due to its enzymatic degradation by dipeptidyl-peptidase-IV (DPP-IV). Released GLP-1 activates enteric and autonomic reflexes while also circulating as an incretin hormone to control endocrine pancreas function. The GLP-1 receptor (GLP-1R) is a G protein-coupled receptor that is activated directly or indirectly by blood glucose-lowering agents currently in use for the treatment of type 2 diabetes mellitus (T2DM). These therapeutic agents include GLP-1R agonists (exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, and langlenatide) and DPP-IV inhibitors (sitagliptin, vildagliptin, saxagliptin, linagliptin, and alogliptin). Investigational agents for use in the treatment of T2DM include GPR119 and GPR40 receptor agonists that stimulate the release of GLP-1 from L cells. Summarized here is the role of GLP-1 to control blood glucose homeo-stasis, with special emphasis on the advantages and limitations of GLP-1-based therapeutics. PMID:24373234

The seven-transmembrane receptor Gpr1 governs processes relevant for the antagonistic interaction of Trichoderma atroviride with its host.

PubMed

Omann, Markus R; Lehner, Sylvia; Escobar Rodríguez, Carolina; Brunner, Kurt; Zeilinger, Susanne

2012-01-01

Mycoparasitic Trichoderma species are applied as biocontrol agents in agriculture to guard plants against fungal diseases. During mycoparasitism, Trichoderma directly interacts with phytopathogenic fungi, preceded by a specific recognition of the host and resulting in its disarming and killing. In various fungal pathogens, including mycoparasites, signalling via heterotrimeric G proteins plays a major role in regulating pathogenicity-related functions. However, the corresponding receptors involved in the recognition of host-derived signals are largely unknown. Functional characterization of Trichoderma atroviride Gpr1 revealed a prominent role of this seven-transmembrane protein of the cAMP-receptor-like family of fungal G-protein-coupled receptors in the antagonistic interaction with the host fungus and governing of mycoparasitism-related processes. Silencing of gpr1 led to an avirulent phenotype accompanied by an inability to attach to host hyphae. Furthermore, gpr1-silenced transformants were unable to respond to the presence of living host fungi with the expression of chitinase- and protease-encoding genes. Addition of exogenous cAMP was able to restore host attachment in gpr1-silenced transformants but could not restore mycoparasitic overgrowth. A search for downstream targets of the signalling pathway(s) involving Gpr1 resulted in the isolation of genes encoding e.g. a member of the cyclin-like superfamily and a small secreted cysteine-rich protein. Although silencing of gpr1 caused defects similar to those of mutants lacking the Tga3 Gα protein, no direct interaction between Gpr1 and Tga3 was observed in a split-ubiquitin two-hybrid assay.

The seven-transmembrane receptor Gpr1 governs processes relevant for the antagonistic interaction of Trichoderma atroviride with its host

PubMed Central

Omann, Markus R.; Lehner, Sylvia; Escobar Rodríguez, Carolina; Brunner, Kurt

2012-01-01

Mycoparasitic Trichoderma species are applied as biocontrol agents in agriculture to guard plants against fungal diseases. During mycoparasitism, Trichoderma directly interacts with phytopathogenic fungi, preceded by a specific recognition of the host and resulting in its disarming and killing. In various fungal pathogens, including mycoparasites, signalling via heterotrimeric G proteins plays a major role in regulating pathogenicity-related functions. However, the corresponding receptors involved in the recognition of host-derived signals are largely unknown. Functional characterization of Trichoderma atroviride Gpr1 revealed a prominent role of this seven-transmembrane protein of the cAMP-receptor-like family of fungal G-protein-coupled receptors in the antagonistic interaction with the host fungus and governing of mycoparasitism-related processes. Silencing of gpr1 led to an avirulent phenotype accompanied by an inability to attach to host hyphae. Furthermore, gpr1-silenced transformants were unable to respond to the presence of living host fungi with the expression of chitinase- and protease-encoding genes. Addition of exogenous cAMP was able to restore host attachment in gpr1-silenced transformants but could not restore mycoparasitic overgrowth. A search for downstream targets of the signalling pathway(s) involving Gpr1 resulted in the isolation of genes encoding e.g. a member of the cyclin-like superfamily and a small secreted cysteine-rich protein. Although silencing of gpr1 caused defects similar to those of mutants lacking the Tga3 Gα protein, no direct interaction between Gpr1 and Tga3 was observed in a split-ubiquitin two-hybrid assay. PMID:22075023

Differential free fatty acid receptor-1 (FFAR1/GPR40) signalling is associated with gene expression or gelatinase granule release in bovine neutrophils.

PubMed

Mena, Sandra J; Manosalva, Carolina; Carretta, Maria D; Teuber, Stefanie; Olmo, Iván; Burgos, Rafael A; Hidalgo, Maria A

2016-08-01

Fatty acids have been recognized as regulators of immune function in addition to their known metabolic role. Long-chain fatty acids bind free fatty acid receptor (FFAR)-1/GPR40, which is expressed on bovine neutrophils, and increase responses such as granule release and gene expression. In this study, we investigated the molecular mechanisms governing the up-regulation of cyclooxygenase-2 (COX-2) and IL-8, as well as matrix metalloproteinase (MMP)-9 granule release in FFAR1/GPR40 agonist-stimulated neutrophils. Our results showed that natural (oleic and linoleic acid) and synthetic (GW9508) FFAR1/GPR40 agonists increased ERK1/2, p38 MAPK and Akt phosphorylation, and that the FFAR1/GPR40 antagonist GW1100 reduced these responses. We evaluated the levels of IκBα, a component of the classical activation pathway of the transcription factor NF-κB, and we observed IκBα reduction after stimulation with FFAR1/GPR40 agonists, an effect that was inhibited by GW1100 or the inhibitors UO126, SB203580 or LY294002. FFAR1/GPR40 agonists increased COX-2 and IL-8 expression, which was inhibited by GW1100 and an NF-κB inhibitor. Finally, the FFAR1/GPR40 agonist-induced MMP-9 granule release was reduced by GW1100 and UO126. In conclusion, FFAR1/GPR40 agonists differentially stimulate neutrophil functions; COX-2 and IL-8 are expressed after FFAR1/GPR40 activation via NF-κB, IκBα reduction is FFAR1/GPR40- and PI3K/MAPK-dependent, and MMP-9 granule release is FFAR1/GPR40- and ERK1/2-dependent. © The Author(s) 2016.

Evidence of alterations in the expression of orphan receptors GPR26 and GPR39 due to the etiology of the metabolic syndrome.

PubMed

Romero-Nava, Rodrigo; Zhou, De-Shan; García, Noemí; Ruiz-Hernández, Armando; Si, Yin-Chu; Sánchez-Muñoz, Fausto; Huang, Fengyang; Hong, Enrique; Villafaña, Santiago

2017-08-01

Metabolic syndrome (MS) is composed of several metabolic abnormalities that increase the risk of cardiovascular diseases and diabetes. Although there are treatments for the components of MS, this pathology maintains a high mortality, suggesting that there are other mechanisms in which orphan receptors such as GPR26 and GPR39 may be involved. For this reason, the aim of this work was to evaluate the expression of GPR26 and GPR39 orphan receptors in two models of MS (diet and genetics). We used male Wistar rats, which received 70% fructose in drinking water for 9 weeks, and obese Zucker rats. We measured weight, blood pressure, glucose, triglycerides, total cholesterol, HDL cholesterol, LDL cholesterol to determine the MS and the expression of the orphan receptors GPR26 and GPR39 in brain, heart, aorta, liver, and kidney by RT-PCR. The analysis of the expression of the orphan receptors GPR26 and GPR39 showed that the receptors are expressed in some tissues, but the expression of the GPR26 tends to decrease in the heart and aorta, whereas in the brain, no changes were observed, this receptor is not expressed in the liver and kidney of both strains. The expression of GPR39 isoforms depends on the tissue and MS model. We conclude that the orphan receptors GPR26, GPR39v1, and GPR39v2 are expressed in different tissues and their profile expression is dependent on the etiology of the MS.

17-Beta-estradiol inhibits transforming growth factor-beta signaling and function in breast cancer cells via activation of extracellular signal-regulated kinase through the G protein-coupled receptor 30.

PubMed

Kleuser, Burkhard; Malek, Daniela; Gust, Ronald; Pertz, Heinz H; Potteck, Henrik

2008-12-01

Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-beta plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-beta signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-beta signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-beta signaling network as well as TGF-beta functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-beta-estradiol (E2) to inhibit the TGF-beta pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-beta signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-beta signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-beta pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-beta signaling. These findings are of great clinical relevance, because down-regulation of TGF-beta signaling is associated with the development of breast cancer resistance in response to antiestrogens.

Molecular and functional interaction between GPR18 and cannabinoid CB2 G-protein-coupled receptors. Relevance in neurodegenerative diseases.

PubMed

Reyes-Resina, Irene; Navarro, Gemma; Aguinaga, David; Canela, Enric I; Schoeder, Clara T; Zaluski, Michal; Kiec-Kononowicz, Katarzyna; Saura, Carlos A; Müller, Christa E; Franco, Rafael

2018-06-02

GPR18, still considered an orphan receptor, may respond to endocannabinoids, whose canonical receptors are CB 1 and CB 2 . GPR18 and CB 2 receptors share a role in peripheral immune response regulation and are co-expressed in microglia, which are immunocompetent cells in the central nervous system (CNS). We aimed at identifying heteroreceptor complexes formed by GPR18 and CB 1 R or CB 2 R in resting and activated microglia. Receptor-receptor interaction was assessed using energy-transfer approaches, and receptor function by determining cAMP levels and ERK1/2 phosphorylation in heterologous cells and primary cultures of microglia. Heteroreceptor identification in primary cultures of microglia was achieved by in situ proximity ligation assays. Energy transfer results showed interaction of GPR18 with CB 2 R but not with CB 1 R. CB 2 -GPR18 heteroreceptor complexes displayed particular functional properties (heteromer prints) often consisting of negative cross-talk (activation of one receptor reduces signaling arising from the partner receptor) and cross-antagonism (the response of one of the receptors is blocked by a selective antagonist of the partner receptor). Activated microglia showed the heteromer print (negative cross-talk and bidirectional cross-antagonism) and increased expression of CB 2 R and GPR18. Due to the important role of CB 2 R in neuroprotection, we further investigated heteroreceptor occurrence in primary cultures of microglia from transgenic mice overexpressing human APP Sw,Ind , an Alzheimer's disease model. Microglial cells from transgenic mice showed the heteromer print and functional interactions that were similar to those found in cells from wild-type animals that were activated by treatment with lipopolysaccharide and interferon-ɤ. Our results show that GPR18 and its heteromers may play important roles in neurodegenerative processes. Copyright © 2018. Published by Elsevier Inc.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G Protein–Coupled Receptors

PubMed Central

Aust, Gabriela; Araç, Demet; Engel, Felix B.; Formstone, Caroline; Fredriksson, Robert; Hall, Randy A.; Harty, Breanne L.; Kirchhoff, Christiane; Knapp, Barbara; Krishnan, Arunkumar; Liebscher, Ines; Lin, Hsi-Hsien; Martinelli, David C.; Monk, Kelly R.; Peeters, Miriam C.; Piao, Xianhua; Prömel, Simone; Schöneberg, Torsten; Schwartz, Thue W.; Singer, Kathleen; Stacey, Martin; Ushkaryov, Yuri A.; Vallon, Mario; Wolfrum, Uwe; Wright, Mathew W.; Xu, Lei; Langenhan, Tobias

2015-01-01

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein–coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential. PMID:25713288

Bisphenol A at a low concentration boosts mouse spermatogonial cell proliferation by inducing the G protein-coupled receptor 30 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheng, Zhi-Guo; Huang, Wei; Liu, Yu-Xiang

Bisphenol A (BPA) is one of the most prevalent chemicals in daily-use materials, therefore, human exposure to BPA is ubiquitous. We found that low concentrations of BPA stimulate the spermatogonial GC-1 cells proliferation by G protein-coupled receptor 30 (GPR30)-mediated epidermal growth factor receptor (EGFR)-extracellular regulated kinase (ERK)-c-Fos pathway. However, through the same pathway GPR30 expression has been shown to be induced by EGF, an EGFR ligand. Thus, we want to know if low concentrations of BPA are able to induce the GPR30 expression and the possible mechanism(s) in GC-1 cells. By transient transfection with expression plasmids, 10{sup −9} M BPAmore » significantly transactivates the Gpr30-5′-flanking region through activating the GPR30, cGMP-dependent protein kinase (PKG), estrogen receptor-α (ER-α), and EFGR-ERK pathways. Furthermore, an activator protein-1 (AP-1) site located within this region is found to be responsible for the transactivation of BPA. Expectedly, through the same pathways, BPA significantly induces the gene and protein expression of GPR30. c-Fos is further observed to be strongly recruited to the AP-1 site in a chromatin immunoprecipitation assay and its dysfunction on the AP-1 site markedly suppresses the expression of GPR30, p-ERK1/2, p-Ser118-ER-α and cell proliferation by BPA. Our results demonstrate that a low-concentration BPA induces GPR30 expression through the GPR30-EFGR-ERK-c-Fos, ER-α, and PKG pathways, presumably boosting the cells proliferation via a regulatory loop. The present study provides a novel insight into the potential role of GPR30 in the initiation and progression of male germ cell cancer induced by environmentally relevant BPA. - Highlights: ► Low concentrations of BPA activate the PKG and GPR30-EFGR-ERK-ER-α pathways. ► Low concentrations of BPA activate the AP-1 site of Gpr30-5′-flanking region. ► Low concentrations of BPA induce the expression of GPR30 gene and protein. ► Low concentrations of BPA boost GC-1 cells proliferation via a regulatory loop.« less

Evidence for the putative cannabinoid receptor, GPR55, mediated inhibitory effects on intestinal contractility in mice

PubMed Central

Ross, Gracious R; Lichtman, Aron; Dewey, William L; Akbarali, Hamid I

2012-01-01

Background Cannabinoids inhibit intestinal motility via presynaptic cannabinoid receptor type I(CB1) in enteric neurons while cannabinoid receptor type II (CB2) receptors are located mainly in immune cells. The recently deorphanized G-protein-coupled receptor, GPR55, has been proposed to be the “third” cannabinoid receptor. Although gene expression of GPR55 is evident in the gut, functional evidence for GPR55 in the gut is unknown. In this study, we tested the hypothesis that GPR55 activation inhibits neurogenic contractions in the gut. Methods We assessed the inhibitory effect of the atypical cannabinoid O-1602, a GPR55 agonist, in mouse colon. Isometric tension recordings in colonic tissue strips were used from either wild type, GPR55−/− or CB1−/−/CB2−/−knock-out mice. Results O-1602 inhibited the electrical field-induced contractions in the colon strips from wild type and CB1−/−/CB2−/− in a concentration–dependent manner, suggesting a non-CB1/CB2-receptor mediated prejunctional effect. The concentration–dependent response of O-1602 was significantly inhibited in GPR55−/− mice. O-1602 did not relax colonic strips pre-contracted with high K+ (80 mmol/l), indicating no involvement of Ca2+ channel blockade in O-1602–induced relaxation. However, 10 μmol/l O-1602 partially inhibited the exogenous acetylcholine (10 μmol/l) –induced contractions. Moreover, we also assessed the inhibitory effects of JWH 015, a CB2/GPR55 agonist on neurogenic contractions of mouse ileum. Surprisingly, the effects of JWH015 were independent of the known cannabinoid receptors. Conclusion These findings taken together suggest that activation of GPR55 leads to inhibition of neurogenic contractions in the gut, and are predominantly prejunctional. PMID:22759743

GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Zhuo; Department of Cardiology, Jinan Central Hospital, Affiliated with Shandong University, 105 Jiefang Road, Jinan, 250013; Wang, Hao

2015-03-27

Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, andmore » immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression.« less

Angiotensin II-aldosterone interaction in human coronary microarteries involves GPR30, EGFR, and endothelial NO synthase.

PubMed

Batenburg, Wendy W; Jansen, Pieter M; van den Bogaerdt, Antoon J; J Danser, Alexander H

2012-04-01

The aim of this study was to investigate the aldosterone-angiotensin (Ang) II interaction in human coronary microarteries (HCMAs). HCMAs, obtained from 75 heart-beating organ donors, were mounted in myographs and exposed to Ang II, either directly or following a 30-min pre-incubation with aldosterone, 17β-oestradiol, hydrocortisone, the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, the extracellular regulated kinase 1/2 (ERK1/2) inhibitor PD98059, the GPR30 antagonist G15, or the epidermal growth factor receptor (EGFR) antagonist AG1478. Ang II constricted HCMAs in a concentration-dependent manner. All steroids, at nanomolar levels, potentiated Ang II and G15 prevented this effect. The potentiation disappeared or was reversed into Ang II antagonism at micromolar steroid levels. NO synthase (NOS) inhibition prevented the latter antagonism in the case of 17β-oestradiol, whereas both aldosterone and 17β-oestradiol at micro- (but not nano-) molar levels induced endothelial NOS phosphorylation in human umbilical vein endothelial cells. AG1478, but not SB203580 or PD98059, abolished the Ang II-induced contraction in the presence of aldosterone or 17β-oestradiol, and none of these drugs affected Ang II alone. Steroids including aldosterone affect Ang II-induced vasoconstriction in a biphasic manner. Potentiation occurs at nanomolar steroid levels and depends on GPR30 and EGFR transactivation. At micromolar steroid levels, this potentiation either disappears (aldosterone and hydrocortisone) or is reversed into an inhibition (17β-oestradiol), and this is due to the endothelial NOS activation that occurs at such concentrations.

G protein-coupled receptor 30 down-regulates cofactor expression and interferes with the transcriptional activity of glucocorticoid.

PubMed

Ylikomi, Timo; Vienonen, Annika; Ahola, Tytti M

2004-11-01

G protein-coupled receptor 30 (GPR30) has previously been described to be important in steroid-mediated growth and to inhibit cell proliferation. Here we investigated whether the effect of GPR30 on cell growth is dependent on steroid hormone receptors. We stably introduced GPR30 in immortalized normal mammary epithelial (HME) cells using retroviruses for gene delivery. GPR30 inhibited the growth and proliferation of the cells. They expressed glucocorticoid receptor, but not estrogen or progesterone receptor. GPR30 down-regulated the expression of cofactor transcription intermediary factor 2 (TIF2) analyzed using quantitative RT-PCR analysis, and also diminished the expression of TIF2 at protein level analyzed by Western blotting using nuclear extracts from mammary epithelial cells. When HME cells were transiently transfected with the glucocorticoid response element MMTV-luc reporter plasmid, stable expression of GPR30 resulted in the abolition of ligand-induced transactivation of the promoter. In COS cells, transient transfection of GPR30 with glucocorticoid receptor alpha resulted in an abrogation of the MMTV-luc and GRE-luc reporter activities induced by dexamethasone. The results suggest a novel mechanism by which membrane-initiated signaling interferes with steroid signaling.

Analysis of GPR101 and AIP genes mutations in acromegaly: a multicentric study.

PubMed

Ferraù, Francesco; Romeo, P D; Puglisi, S; Ragonese, M; Torre, M L; Scaroni, C; Occhi, G; De Menis, E; Arnaldi, G; Trimarchi, F; Cannavò, S

2016-12-01

This multicentric study aimed to investigate the prevalence of the G protein-coupled receptor 101 (GPR101) p.E308D variant and aryl hydrocarbon receptor interacting protein (AIP) gene mutations in a representative cohort of Italian patients with acromegaly. 215 patients with GH-secreting pituitary adenomas, referred to 4 Italian referral centres for pituitary diseases, have been included. Three cases of gigantism were present. Five cases were classified as FIPA. All the patients have been screened for germline AIP gene mutations and GPR101 gene p.E308D variant. Heterozygous AIP gene variants have been found in 7 patients (3.2 %). Five patients carried an AIP mutation (2.3 %; 4 females): 3 patients harboured the p.R3O4Q mutation, one had the p.R304* mutation and the last one the IVS3+1G>A mutation. The prevalence of AIP mutations was 3.3 % and 2.8 % when considering only the patients diagnosed when they were <30 or <40-year old, respectively. Furthermore, 2.0 % of the patients with a pituitary macroadenoma and 4.2 % of patients resistant to somatostatin analogues treatment were found to harbour an AIP gene mutation. None of the patients was found to carry the GPR101 p.E308D variant. The prevalence of AIP gene mutations among our sporadic and familial acromegaly cases was similar to that one reported in previous studies, but lower when considering only the cases diagnosed before 40 years of age. The GPR101 p.E308D change is unlikely to have a role in somatotroph adenomas tumorigenesis, since none of our sporadic or familial patients tested positive for this variant.

Expression analysis of G Protein-Coupled Receptors in mouse macrophages

PubMed Central

Lattin, Jane E; Schroder, Kate; Su, Andrew I; Walker, John R; Zhang, Jie; Wiltshire, Tim; Saijo, Kaoru; Glass, Christopher K; Hume, David A; Kellie, Stuart; Sweet, Matthew J

2008-01-01

Background Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic micro-array analysis of GPCR expression in primary mouse macrophages to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS). Results Several members of the P2RY family had striking expression patterns in macrophages; P2ry6 mRNA was essentially expressed in a macrophage-specific fashion, whilst P2ry1 and P2ry5 mRNA levels were strongly down-regulated by LPS. Expression of several other GPCRs was either restricted to macrophages (e.g. Gpr84) or to both macrophages and neural tissues (e.g. P2ry12, Gpr85). The GPCR repertoire expressed by bone marrow-derived macrophages and thioglycollate-elicited peritoneal macrophages had some commonality, but there were also several GPCRs preferentially expressed by either cell population. Conclusion The constitutive or regulated expression in macrophages of several GPCRs identified in this study has not previously been described. Future studies on such GPCRs and their agonists are likely to provide important insights into macrophage biology, as well as novel inflammatory pathways that could be future targets for drug discovery. PMID:18442421

The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Gui-Qiang; Zhou, Long; Chen, Xiao-Yue

2012-04-06

Highlights: Black-Right-Pointing-Pointer We assessed hydroxytamoxifen (OHT) effects in two endometrial cancer cell lines. Black-Right-Pointing-Pointer GPR30 mediates the proliferative effects induced by OHT. Black-Right-Pointing-Pointer GPR30 mediates the invasive effects induced by OHT. Black-Right-Pointing-Pointer GPR30 expression was up-regulated by OHT in endometrial cancer cell line. -- Abstract: The selective ER modulator tamoxifen (TAM) is the most widely used ER antagonist for treatment of women with hormone-dependent breast tumor. However, long-term treatment is associated with an increased risk of endometrial cancer. The aim of the present study was to demonstrate new insight into the role of G-protein coupled receptor 30 (GPR30) in themore » activity of TAM, which promoted endometrial cancer. In endometrial cancer cell lines ISHIKAWA and KLE, the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, 17{beta}-estradiol (E2) and G1, a non-steroidal GPR30-specific agonist to promote cell proliferation and invasion was evaluated. All agents above induced high proliferative and invasive effects, while the down-regulation of GPR30 or the interruption of MAPK signal pathway partly or completely prevented the action of the regent. Moreover, the RNA and protein expression of GPR30 was up-regulated by G1, E2 or OHT in both cell lines. The present study provided a new insight into the mechanism involved in the agonistic activity exerted by TAM in the uterus.« less

Differential expression of GPR15 on T cells during ulcerative colitis

PubMed Central

Adamczyk, Alexandra; Gageik, Daniel; Frede, Annika; Pastille, Eva; Hansen, Wiebke; Rueffer, Andreas; Buer, Jan; Büning, Jürgen; Langhorst, Jost

2017-01-01

G protein–coupled receptor 15 (GPR15) was recently highlighted as a colon-homing receptor for murine and human CD4+ T cells. The aim of this study was to explore the functional phenotype of human GPR15+CD4+ T cells, focusing on Tregs and effector T cells (Teffs), and to determine whether GPR15 is the driver for the migration of T cells to the colon during ulcerative colitis (UC). In the peripheral blood, GPR15 was expressed on Tregs and Teffs; both GPR15+ T cell subsets produced less IFN-γ and IL-4 but more IL-17 after stimulation and showed a higher migration activity compared with GPR15–CD4+ T cells. In UC patients, GPR15 expression was increased on Tregs in the peripheral blood but not on Teffs. Interestingly, the expression of GPR15 was significantly enhanced on colonic T cells of UC patients in noninflamed biopsies but not in inflamed biopsies. The differential expression of GPR15 in UC patients was accompanied by a significant reduction of bacterial immunoregulatory metabolites in the feces. In conclusion, GPR15 expression on CD4+ T cells is altered in UC patients, which may have implications for the development of therapeutic approaches to target T cell trafficking to the colon. PMID:28422750

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

PubMed

Brunner, Kurt; Omann, Markus; Pucher, Marion E; Delic, Marizela; Lehner, Sylvia M; Domnanich, Patrick; Kratochwill, Klaus; Druzhinina, Irina; Denk, Dagmar; Zeilinger, Susanne

2008-12-01

Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

Modulation of l-α-Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids*

PubMed Central

Anavi-Goffer, Sharon; Baillie, Gemma; Irving, Andrew J.; Gertsch, Jürg; Greig, Iain R.; Pertwee, Roger G.; Ross, Ruth A.

2012-01-01

GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55. PMID:22027819

GPR 120: The Potential Target for Obesity Treatment.

PubMed

Tanagho, Peter A; Shohdy, Kyrillus S

2016-01-01

G protein coupled receptor 120 (GPR120) is a class of receptors in the gastrointestinal tract (GIT) that is implicated in nutrient sensing and body weight regulation. Functions of GPR120 are thought to be mediated by the release of a group of hormones known as incretins, such as glucagon like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). We have searched PubMed with the keywords "GPR120","GLP-1" and "obesity". Relevant studies were retrieved and included in the review. Recently, many exogenous compounds have been investigated in their role in the release of GLP-1 and in causing weight loss in obese rats. However, some results question the putative role of GPR120 in metabolic homeostasis. Herein, we evaluate the potential use of GPR120 as a target receptor in obesity and found it to be ubiquitous throughout the GIT, with various functions in each site. In order to find the optimal drug, the role of GPR120 in each site needs to be defined and selectivity of the potential drug needs to be studied to ensure the success of this growing line of obesity management.

G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy.

PubMed

White, James P; Wrann, Christiane D; Rao, Rajesh R; Nair, Sreekumaran K; Jedrychowski, Mark P; You, Jae-Sung; Martínez-Redondo, Vicente; Gygi, Steven P; Ruas, Jorge L; Hornberger, Troy A; Wu, Zhidan; Glass, David J; Piao, Xianhua; Spiegelman, Bruce M

2014-11-04

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 (PGC-1α4) is a protein isoform derived by alternative splicing of the PGC1α mRNA and has been shown to promote muscle hypertrophy. We show here that G protein-coupled receptor 56 (GPR56) is a transcriptional target of PGC-1α4 and is induced in humans by resistance exercise. Furthermore, the anabolic effects of PGC-1α4 in cultured murine muscle cells are dependent on GPR56 signaling, because knockdown of GPR56 attenuates PGC-1α4-induced muscle hypertrophy in vitro. Forced expression of GPR56 results in myotube hypertrophy through the expression of insulin-like growth factor 1, which is dependent on Gα12/13 signaling. A murine model of overload-induced muscle hypertrophy is associated with increased expression of both GPR56 and its ligand collagen type III, whereas genetic ablation of GPR56 expression attenuates overload-induced muscle hypertrophy and associated anabolic signaling. These data illustrate a signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise.

G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy

PubMed Central

White, James P.; Wrann, Christiane D.; Rao, Rajesh R.; Nair, Sreekumaran K.; Jedrychowski, Mark P.; You, Jae-Sung; Martínez-Redondo, Vicente; Gygi, Steven P.; Ruas, Jorge L.; Hornberger, Troy A.; Wu, Zhidan; Glass, David J.; Piao, Xianhua; Spiegelman, Bruce M.

2014-01-01

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 (PGC-1α4) is a protein isoform derived by alternative splicing of the PGC1α mRNA and has been shown to promote muscle hypertrophy. We show here that G protein-coupled receptor 56 (GPR56) is a transcriptional target of PGC-1α4 and is induced in humans by resistance exercise. Furthermore, the anabolic effects of PGC-1α4 in cultured murine muscle cells are dependent on GPR56 signaling, because knockdown of GPR56 attenuates PGC-1α4–induced muscle hypertrophy in vitro. Forced expression of GPR56 results in myotube hypertrophy through the expression of insulin-like growth factor 1, which is dependent on Gα12/13 signaling. A murine model of overload-induced muscle hypertrophy is associated with increased expression of both GPR56 and its ligand collagen type III, whereas genetic ablation of GPR56 expression attenuates overload-induced muscle hypertrophy and associated anabolic signaling. These data illustrate a signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise. PMID:25336758

G-protein-coupled estrogen receptor-30 gene polymorphisms are associated with uterine leiomyoma risk

PubMed Central

Kasap, Burcu; Turhan, Nilgün Öztürk; Edgünlü, Tuba; Duran, Müzeyyen; Akbaba, Eren; Öner, Gökalp

2016-01-01

The G-protein-coupled estrogen receptor, GPER-1) is a member of the G-protein-coupled receptor 1 family and is expressed significantly in uterine leiomyomas. To understand the relationship between GPR30 single nucleotide polymorphisms and the risk of leiomyoma, we measured the follicle-stimulating hormone (FSH) and estradiol (E2) levels of 78 perimenopausal healthy women and 111 perimenopausal women with leiomyomas. The participants’ leiomyoma number and volume were recorded. DNA was extracted from whole blood with a GeneJET Genomic DNA Purification Kit. An amplification-refractory mutation system polymerase chain reaction approach was used for genotyping of the GPR30 gene (rs3808350, rs3808351, and rs11544331). The differences in genotype and allele frequencies between the leiomyoma and control groups were calculated using the chi-square (χ2) and Fischer’s exact test. The median FSH level was higher in controls (63 vs. 10 IU/L, p=0.000), whereas the median E2 level was higher in the leiomyoma group (84 vs. 9.1 pg/mL, p=0.000). The G allele of rs3808351 and the GG genotype of both the rs3808350 and rs3808351 polymorphisms and the GGC haplotype increased the risk of developing leiomyoma. There was no significant difference in genotype frequencies or leiomyoma volume. However, the GG genotype of the GPR30 rs3808351 polymorphism and G allele of the GPR30 rs3808351 polymorphism were associated with the risk of having a single leiomyoma. Our results suggest that the presence of the GG genotype of the GPR30 rs3808351 polymorphism and the G allele of the GPR30 rs3808351 polymorphism affect the characteristics and development of leiomyomas in the Turkish population. PMID:26773178

Targeting GPR120 and other fatty acid sensing GPCRs ameliorates insulin resistance and inflammatory diseases

PubMed Central

Talukdar, Saswata; Olefsky, Jerrold M; Osborn, Olivia

2011-01-01

The last decade has seen great progress in the understanding of the molecular pharmacology, physiological function and therapeutic potential of the G protein-coupled receptors. Free Fatty acids (FFAs) have been demonstrated to act as ligands of several GPCRs including GPR40, GPR43, GPR84, GPR119 and GPR120. We have recently shown that GPR120 acts as a physiological receptor of ω3 fatty acids in macrophages and adipocytes, which mediate potent anti-inflammatory and insulin sensitizing effects. The important role GPR120 plays in the control of inflammation raises the possibility that targeting this receptor could have therapeutic potential in many inflammatory diseases including obesity and type 2 diabetes. In this review, we discuss lipid-sensing GPCRs and highlight potential outcomes of targeting such receptors in ameliorating disease. PMID:21663979

Alterations in Gene and Protein Expression of Cannabinoid CB2 and GPR55 Receptors in the Dorsolateral Prefrontal Cortex of Suicide Victims.

PubMed

García-Gutiérrez, María S; Navarrete, Francisco; Navarro, Gemma; Reyes-Resina, Irene; Franco, Rafael; Lanciego, Jose Luis; Giner, Salvador; Manzanares, Jorge

2018-02-12

Recent studies point to the cannabinoid CB 2 receptors (CB 2 r) and the non-cannabinoid receptor GPR55 as potential key targets involved in the response to stress, anxiety, and depression. Considering the close relationship between neuropsychiatric disorders and suicide, the purpose of this study was to evaluate the potential alterations of CB 2 r and GPR55 in suicide victims. We analyzed gene and protein expression of both receptors by real-time PCR and western blot, respectively, in the dorsolateral prefrontal cortex (DLPFC) of 18 suicide victims with no clinical psychiatric history or treatment with anxiolytics or antidepressants, and 15 corresponding controls. We used in situ proximity ligation assay to evaluate whether the receptors formed heteromeric complexes and to determine the expression level of these heteromers, also assessing the co-expression of heteromers in neurons, astroglia, or microglia cells. CB 2 r and GPR55 gene expressions were significantly lower (by 33 and 41%, respectively) in the DLPFC of suicide cases. CB 2 r protein expression was higher, as were CB 2 -GPR55 heteroreceptor complexes. The results also revealed the presence of CB 2 -GPR55 receptor heteromers in both neurons and astrocytes, whereas microglial cells showed no expression. We did not observe any significant alterations of GPR55 protein expression. Additional studies will be necessary to evaluate if these alterations are reproducible in suicide victims diagnosed with different psychiatric disorders. Taken together, the results suggest that CB 2 r and GPR55 may play a relevant role in the neurobiology of suicide.

Identification of two new mutations in the GPR98 and the PDE6B genes segregating in a Tunisian family.

PubMed

Hmani-Aifa, Mounira; Benzina, Zeineb; Zulfiqar, Fareeha; Dhouib, Houria; Shahzadi, Amber; Ghorbel, Abdelmonem; Rebaï, Ahmed; Söderkvist, Peter; Riazuddin, Sheikh; Kimberling, William J; Ayadi, Hammadi

2009-04-01

Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous disorder. ARRP could be associated with extraocular manifestations that define specific syndromes such as Usher syndrome (USH) characterized by retinal degeneration and congenital hearing loss (HL). The USH type II (USH2) associates RP and mild-to-moderate HL with preserved vestibular function. At least three genes USH2A, the very large G-protein-coupled receptor, GPR98, and DFNB31 are responsible for USH2 syndrome. Here, we report on the segregation of non-syndromic ARRP and USH2 syndrome in a consanguineous Tunisian family, which was previously used to define USH2B locus. With regard to the co-occurrence of these two different pathologies, clinical and genetic reanalysis of the extended family showed (i) phenotypic heterogeneity within USH2 patients and (ii) excluded linkage to USH2B locus. Indeed, linkage analysis disclosed the cosegregation of the USH2 phenotype with the USH2C locus markers, D5S428 and D5S618, whereas the ARRP perfectly segregates with PDE6B flanking markers D4S3360 and D4S2930. Molecular analysis revealed two new missense mutations, p.Y6044C and p.W807R, occurring in GPR98 and PDE6B genes, respectively. In conclusion, our results show that the USH2B locus at chromosome 3p23-24.2 does not exist, and we therefore withdraw this locus designation. The combination of molecular findings for GPR98 and PDE6B genes enable us to explain the phenotypic heterogeneity and particularly the severe ocular affection first observed in one USH2 patient. This report presents an illustration of how consanguinity could increase familial clustering of multiple hereditary diseases within the same family.

4-Hydroxytamoxifen-stimulated processing of cyclin E is mediated via G protein-coupled receptor 30 (GPR30) and accompanied by enhanced migration in MCF-7 breast cancer cells.

PubMed

Li, Yang; Chen, Yan; Zhu, Zhu-Xia; Liu, Xiao-Hong; Yang, Li; Wan, Lei; Lei, Ting-Wen; Wang, Xu-Dong

2013-07-05

Over-expression of cleaved cyclin E in breast tumors is closely associated with tumor progression and resistance to antiestrogens. 17β-Estradiol (E2) has been recently shown to induce cyclin E processing in breast cancer cells. Tamoxifen has been used in patients with estrogen-sensitive breast cancer, yet resistance to antiestrogens and recurrence will appear in some of the patients after its continued use. We therefore addressed possible effects of tamoxifen on the generation of cleaved cyclin E and its signal mechanism(s) in estrogen-responsive MCF-7 breast cancer cells that express both G protein-coupled protein (GPR) 30 and estrogen receptor α (ERα). 4-Hydroxytamoxifen (OHT, tamoxifen's active form) failed to prevent E2-induced proteolysis of cyclin E and migration, but rather triggered cyclin E cleavage coincident with augmented migration. OHT-induced cyclin E truncation also occurred in SK-BR-3 cells that express GPR30 and lack ERα, but not in MDA-MB-231 cells that express neither GPR30 nor ERα. G1, a specific GPR 30 agonist, caused dramatic proteolysis of cyclin E and enhanced migration. Furthermore, OHT-stimulated cleavage of cyclin E and migration were tremendously attenuated by G15, a GPR30 antagonist, or siRNA against GPR30. In addition, inhibitors for EGFR or ERK1/2 remarkably suppressed OHT-induced truncation of cyclin E, suggesting involvement of EGFR signaling. Collectively, our data indicate that OHT contributes to the production of proteolyzed cyclin E via GPR30 with augmented migration in MCF-7 cells. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

N-Arachidonyl Glycine Does Not Activate G Protein–Coupled Receptor 18 Signaling via Canonical Pathways

PubMed Central

Lu, Van B.; Puhl, Henry L.

2013-01-01

Recent studies propose that N-arachidonyl glycine (NAGly), a carboxylic analogue of anandamide, is an endogenous ligand of the Gαi/o protein–coupled receptor 18 (GPR18). However, a high-throughput β-arrestin–based screen failed to detect activation of GPR18 by NAGly (Yin et al., 2009; JBC, 18:12328). To address this inconsistency, this study investigated GPR18 coupling in a native neuronal system with endogenous signaling pathways and effectors. GPR18 was heterologously expressed in rat sympathetic neurons, and the modulation of N-type (Cav2.2) calcium channels was examined. Proper expression and trafficking of receptor were confirmed by the “rim-like” fluorescence of fluorescently tagged receptor and the positive staining of external hemagglutinin-tagged GPR18-expressing cells. Application of NAGly on GPR18-expressing neurons did not inhibit calcium currents but instead potentiated currents in a voltage-dependent manner, similar to what has previously been reported (Guo et al., 2008; J Neurophysiol, 100:1147). Other proposed agonists of GPR18, including anandamide and abnormal cannabidiol, also failed to induce inhibition of calcium currents. Mutants of GPR18, designed to constitutively activate receptors, did not tonically inhibit calcium currents, indicating a lack of GPR18 activation or coupling to endogenous G proteins. Other downstream effectors of Gαi/o-coupled receptors, G protein–coupled inwardly rectifying potassium channels and adenylate cyclase, were not modulated by GPR18 signaling. Furthermore, GPR18 did not couple to other G proteins tested: Gαs, Gαz, and Gα15. These results suggest NAGly is not an agonist for GPR18 or that GPR18 signaling involves noncanonical pathways not examined in these studies. PMID:23104136

Soluble Fibre Meal Challenge Reduces Airway Inflammation and Expression of GPR43 and GPR41 in Asthma.

PubMed

Halnes, Isabel; Baines, Katherine J; Berthon, Bronwyn S; MacDonald-Wicks, Lesley K; Gibson, Peter G; Wood, Lisa G

2017-01-10

Short chain fatty acids (SCFAs) are produced following the fermentation of soluble fibre by gut bacteria. In animal models, both dietary fibre and SCFAs have demonstrated anti-inflammatory effects via the activation of free fatty acid receptors, such as G protein-coupled receptor 41 and 43 (GPR41 and GPR43). This pilot study examined the acute effect of a single dose of soluble fibre on airway inflammation-including changes in gene expression of free fatty acid receptors-in asthma. Adults with stable asthma consumed a soluble fibre meal ( n = 17) containing 3.5 g inulin and probiotics, or a control meal ( n = 12) of simple carbohydrates. Exhaled nitric oxide (eNO) was measured and induced sputum was collected at 0 and 4 h for differential cell counts, measurement of interleukin-8 (IL-8) protein concentration, and GPR41 and GPR43 gene expression. At 4 h after meal consumption, airway inflammation biomarkers, including sputum total cell count, neutrophils, macrophages, lymphocytes, sputum IL-8, and eNO significantly decreased compared to baseline in the soluble fibre group only. This corresponded with upregulated GPR41 and GPR43 sputum gene expression and improved lung function in the soluble fibre group alone. Soluble fibre has acute anti-inflammatory effects in asthmatic airways. Long-term effects of soluble fibre as an anti-inflammatory therapy in asthma warrants further investigation.

Differential requirements of arrestin-3 and clathrin for ligand-dependent and -independent internalization of human G protein-coupled receptor 40.

PubMed

Qian, Jing; Wu, Chun; Chen, Xiaopan; Li, Xiangmei; Ying, Guoyuan; Jin, Lili; Ma, Qiang; Li, Guo; Shi, Ying; Zhang, Guozheng; Zhou, Naiming

2014-11-01

G protein-coupled receptor 40 (GPR40) is believed to be an attractive target to enhance insulin secretion in patients with type 2 diabetes. GPR40 has been found to couple to Gq protein, leading to the activation of phospholipase C and subsequent increases in the intracellular Ca(2+) level. However, the underlying mechanisms that regulate the internalization and desensitization of GPR40 remain to be elucidated. In the present study, a construct of GPR40 fused with enhanced green fluorescent protein (EGFP) at its C-terminus was constructed for direct imaging of the localization and internalization of GPR40 by confocal microscopy. In stably transfected HEK-293 cells, GPR40 receptors underwent rapid agonist-induced internalization and constitutive ligand-independent internalization. Our data demonstrated that the agonist-mediated internalization of GPR40 was significantly blocked by hypertonic sucrose treatment and by siRNA mediated depletion of the heavy chain of clathrin. In contrast, constitutive GPR40 internalization was not affected by hypertonic sucrose or by knock-down of clathrin expression, but it was affected by treatment with methyl-β-cyclodextrin (MβCD) and nystatin. Furthermore, our results using an arrestin-3-EGFP redistribution assay and siRNA-mediated knock-down of arrestin-3 and GRK2 expression revealed that arrestin-3 and GRK2 play an essential role in the regulation of agonist-mediated GPR40 internalization, but are not involved in the regulation of constitutive GPR40 internalization. Additionally, our observation showed that upon activation by agonist, the internalized GPR40 receptors were rapidly recycled back to the plasma membrane via Rab4/Rab5 positive endosomes, whereas the constitutively internalized GPR40 receptors were recycled back to the cell surface through Rab5 positive endosomes. Because FFA receptors exhibit a high level of homology, our observations could be applicable to other members of this family. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of GPR30 in the mechanisms of tamoxifen resistance in breast cancer MCF-7 cells.

PubMed

Ignatov, Atanas; Ignatov, Tanja; Roessner, Albert; Costa, Serban Dan; Kalinski, Thomas

2010-08-01

Tamoxifen is the most frequently used anti-hormonal drug for treatment of women with hormone-dependent breast cancer. The aim of this study is to investigate the mechanism of tamoxifen resistance and the impact of the new estrogen G-protein coupled receptor (GPR30). MCF-7 cells were continuously exposed to tamoxifen for 6 months to induce resistance to the inhibitory effect of tamoxifen. These tamoxifen-resistant cells (TAM-R) exhibited enhanced sensitivity to 17-ss-estradiol and GPR30 agonist, G1, when compared to the parental cells. In TAM-R cells, tamoxifen was able to stimulate the cell growth and MAPK phosphorylation. These effects were abolished by EGFR inhibitor AG1478, GPR30 anti-sense oligonucleotide, and the selective c-Src inhibitor PP2. Only EGFR basal expression was slightly elevated in the TAM-R cells, whereas GPR30 expression and the basal phosphorylation of Akt and MAPK remained unchanged when compared to the parental cells. Interestingly, estrogen treatment significantly increased GPR30 translocation to the cell surface, which was stronger in TAM-R cells. Continuous treatment of MCF-7 cells with GPR30 agonist G1 mimics the long-term treatment with tamoxifen and increases drastically its agonistic activity. This data suggests the important role of GPR30/EGFR receptor signaling in the development of tamoxifen resistance. The inhibition of this pathway is a valid option to improve anti-hormone response in breast cancer.

Targeting GPR120 and other fatty acid-sensing GPCRs ameliorates insulin resistance and inflammatory diseases.

PubMed

Talukdar, Saswata; Olefsky, Jerrold M; Osborn, Olivia

2011-09-01

The past decade has seen great progress in the understanding of the molecular pharmacology, physiological function and therapeutic potential of G-protein-coupled receptors (GPCRs). Free fatty acids (FFAs) have been demonstrated to act as ligands of several GPCRs including GPR40, GPR43, GPR84, GPR119 and GPR120. We have recently shown that GPR120 acts as a physiological receptor of ω3 fatty acids in macrophages and adipocytes, which mediate potent anti-inflammatory and insulin sensitizing effects. The important role GPR120 plays in the control of inflammation raises the possibility that targeting this receptor could have therapeutic potential in many inflammatory diseases including obesity and type 2 diabetes. In this review paper, we discuss lipid-sensing GPCRs and highlight potential outcomes of targeting such receptors in ameliorating disease. Copyright © 2011 Elsevier Ltd. All rights reserved.

Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/GPR41 as new potential therapeutic targets.

PubMed

Ulven, Trond

2012-01-01

The deorphanization of the free fatty acid (FFA) receptors FFA1 (GPR40), FFA2 (GPR43), FFA3 (GPR41), GPR84, and GPR120 has made clear that the body is capable of recognizing and responding directly to nonesterified fatty acid of virtually any chain length. Colonic fermentation of dietary fiber produces high concentrations of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, a process which is important to health. The phylogenetically related 7-transmembrane (7TM) receptors free fatty acid receptor 2 (FFA2) and FFA3 are activated by these SCFAs, and several lines of evidence indicate that FFA2 and FFA3 mediate beneficial effects associated with a fiber-rich diet, and that they may be of interest as targets for treatment of inflammatory and metabolic diseases. FFA2 is highly expressed on immune cells, in particular neutrophils, and several studies suggest that the receptor plays a role in diseases involving a dysfunctional neutrophil response, such as inflammatory bowel disease (IBD). Both FFA2 and FFA3 have been implicated in metabolic diseases such as type 2 diabetes and in regulation of appetite. More research is however required to clarify the potential of the receptors as drug targets and establish if activation or inhibition would be the preferred mode of action. The availability of potent and selective receptor modulators is a prerequisite for these studies. The few modulators of FFA2 or FFA3 that have been published hitherto in the peer-reviewed literature in general have properties that make them less than ideal as such tools, but published patent applications indicate that better tool compounds might soon become available which should enable studies critical to validate the receptors as new drug targets.

Gpr132 sensing of lactate mediates tumor–macrophage interplay to promote breast cancer metastasis

PubMed Central

Chen, Peiwen; Zuo, Hao; Xiong, Hu; Kolar, Matthew J.; Chu, Qian; Saghatelian, Alan; Siegwart, Daniel J.; Wan, Yihong

2017-01-01

Macrophages are prominent immune cells in the tumor microenvironment that exert potent effects on cancer metastasis. However, the signals and receivers for the tumor–macrophage communication remain enigmatic. Here, we show that G protein-coupled receptor 132 (Gpr132) functions as a key macrophage sensor of the rising lactate in the acidic tumor milieu to mediate the reciprocal interaction between cancer cells and macrophages during breast cancer metastasis. Lactate activates macrophage Gpr132 to promote the alternatively activated macrophage (M2)-like phenotype, which, in turn, facilitates cancer cell adhesion, migration, and invasion. Consequently, Gpr132 deletion reduces M2 macrophages and impedes breast cancer lung metastasis in mice. Clinically, Gpr132 expression positively correlates with M2 macrophages, metastasis, and poor prognosis in patients with breast cancer. These findings uncover the lactate-Gpr132 axis as a driver of breast cancer metastasis by stimulating tumor–macrophage interplay, and reveal potential new therapeutic targets for breast cancer treatment. PMID:28049847

G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

PubMed

Wu, Melissa P; Doyle, Jamie R; Barry, Brenda; Beauvais, Ariane; Rozkalne, Anete; Piao, Xianhua; Lawlor, Michael W; Kopin, Alan S; Walsh, Christopher A; Gussoni, Emanuela

2013-12-01

Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities. © 2013 FEBS.

Association of the membrane estrogen receptor, GPR30, with breast tumor metastasis and transactivation of the epidermal growth factor receptor.

PubMed

Filardo, Edward J; Quinn, Jeffrey A; Sabo, Edmond

2008-10-01

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases function as a common signaling conduit for membrane receptors that lack intrinsic enzymatic activity, such as G-protein coupled receptors and integrins. GPR30, an orphan member of the seven transmembrane receptor (7TMR) superfamily has been linked to specific estrogen binding, rapid estrogen-mediated activation of adenylyl cyclase and the release of membrane-tethered proHB-EGF. More recently, GPR30 expression in primary breast adenocarcinoma has been associated with pathological parameters commonly used to assess breast cancer progression, including the development of extramammary metastases. This newly appreciated mechanism of cross communication between estrogen and EGF is consistent with the observation that 7TMR-mediated transactivation of the EGFR is a recurrent signaling paradigm and may explain prior data reporting the EGF-like effects of estrogen. The molecular details surrounding GPR30-mediated release of proHB-EGF, the involvement of integrin beta1 as a signaling intermediary in estrogen-dependent EGFR action, and the possible implications of these data for breast cancer progression are discussed herein.

In vivo effects of a GPR30 antagonist.

PubMed

Dennis, Megan K; Burai, Ritwik; Ramesh, Chinnasamy; Petrie, Whitney K; Alcon, Sara N; Nayak, Tapan K; Bologa, Cristian G; Leitao, Andrei; Brailoiu, Eugen; Deliu, Elena; Dun, Nae J; Sklar, Larry A; Hathaway, Helen J; Arterburn, Jeffrey B; Oprea, Tudor I; Prossnitz, Eric R

2009-06-01

Estrogen is central to many physiological processes throughout the human body. We have previously shown that the G protein-coupled receptor GPR30 (also known as GPER), in addition to classical nuclear estrogen receptors (ER and ER), activates cellular signaling pathways in response to estrogen. In order to distinguish between the actions of classical estrogen receptors and GPR30, we have previously characterized G-1 (1), a selective agonist of GPR30. To complement the pharmacological properties of G-1, we sought to identify an antagonist of GPR30 that displays similar selectivity against the classical estrogen receptors. Here we describe the identification and characterization of G15 (2), a G-1 analog that binds to GPR30 with high affinity and acts as an antagonist of estrogen signaling through GPR30. In vivo administration of G15 revealed that GPR30 contributes to both uterine and neurological responses initiated by estrogen. The identification of this antagonist will accelerate the evaluation of the roles of GPR30 in human physiology.

Identification of G-protein-coupled receptor 120 as a tumor-promoting receptor that induces angiogenesis and migration in human colorectal carcinoma.

PubMed

Wu, Q; Wang, H; Zhao, X; Shi, Y; Jin, M; Wan, B; Xu, H; Cheng, Y; Ge, H; Zhang, Y

2013-12-05

G-protein-coupled receptor 120 (GPR120) functions as a receptor for unsaturated long-chain free fatty acids and has an important role in regulating lipid and glucose metabolism. However, a role for GPR120 in the development of tumors has not yet been clarified. Here, we show that GPR120 signaling promotes angiogenic switching and motility of human colorectal carcinoma (CRC) cells. We show that the expression of GPR120 is significantly induced in CRC tissues and cell lines, which is associated with tumor progression. Activation of GPR120 signaling in human CRC promotes angiogenesis in vitro and in vivo, largely by inducing the expression and secretion of proangiogenic mediators such as vascular endothelial growth factor (VEGF), interleukin-8 and cyclooxygenase-2-derived prostaglandin E2. The PI3K/Akt-NF-κB pathway is activated by GPR120 signaling and is required for GPR120 signaling-induced angiogenic switching in CRC cells. And, GPR120 activation enhances the motility of CRC cells and induces epithelial-mesenchymal transition. Furthermore, in vivo study shows that activation of GPR120 promotes angiogenesis and tumor growth. Finally, we find that GPR120 expression is positively correlated with VEGF expression and inversely correlated with the epithelial marker E-cadherin in CRC tissues. Collectively, our results demonstrate that GPR120 functions as a tumor-promoting receptor in CRC and, therefore, shows promise as a new potential target for cancer therapeutics.

Immune malfunction in the GPR39 zinc receptor of knockout mice: Its relationship to depressive disorder.

PubMed

Młyniec, Katarzyna; Trojan, Ewa; Ślusarczyk, Joanna; Głombik, Katarzyna; Basta-Kaim, Agnieszka; Budziszewska, Bogusława; Skrzeszewski, Jakub; Siwek, Agata; Holst, Birgitte; Nowak, Gabriel

2016-02-15

Depression is a serious psychiatric disorder affecting not only the monaminergic, glutamatergic, and GABAergic neurosystems, but also the immune system. Patients suffering from depression show disturbance in the immune parameters as well as increased susceptibility to infections. Zinc is well known as an anti-inflammatory agent, and its link with depression has been proved, zinc deficiency causing depression- and anxiety-like behavior with immune malfunction. It has been discovered that trace-element zinc acts as a neurotransmitter in the central nervous system via zinc receptor GPR39. In this study we investigated whether GPR39 knockout would cause depressive-like behavior as measured by the forced swim test, and whether these changes would coexist with immune malfunction. In GPR39 knockout mice versus a wild-type control we found: i) depressive-like behavior; ii) significantly reduced thymus weight; (iii) reduced cell viability of splenocytes; iv) reduced proliferative response of splenocytes; and v) increased IL-6 production of splenocytes after ConA stimulation and decreased IL-1b and IL-6 release after LPS stimulation. The results indicate depressive-like behavior in GPR39 KO animals with an immune response similar to that observed in depressive disorder. Here for the first time we show immunological changes under GPR39-deficient conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Modulation of K+ currents in Xenopus spinal neurons by p2y receptors: a role for ATP and ADP in motor pattern generation

PubMed Central

Brown, Paul; Dale, Nicholas

2002-01-01

We have investigated the pharmacological properties and targets of p2y purinoceptors in Xenopus embryo spinal neurons. ATP reversibly inhibited the voltage-gated K+ currents by 10 ± 3 %. UTP and the analogues α,β-methylene-ATP and 2-methylthio-ATP also inhibited K+ currents. This agonist profile is similar to that reported for a p2y receptor cloned from Xenopus embryos. Voltage-gated K+ currents could be inhibited by ADP (9 ± 0.8 %) suggesting that a further p2y1-like receptor is also present in the embryo spinal cord. Unexpectedly we found that α,β-methylene-ADP, often used to block the ecto-5′-nucleotidase, also inhibited voltage-gated K+ currents (7 ± 2.3 %). This inhibition was occluded by ADP, suggesting that α,β-methylene-ADP is an agonist at p2y1 receptors. We have directly studied the properties of the ecto-5′-nucleotidase in Xenopus embryo spinal cord. Although ADP inhibited this enzyme, α,β-methylene-ADP had no action. Caution therefore needs to be used when interpreting the actions of α,β-methylene-ADP as it has previously unreported agonist activity at P2 receptors. Xenopus spinal neurons possess fast and slow voltage-gated K+ currents. By using catechol to selectively block the fast current, we completely occluded the actions of ATP and ADP. Furthermore, the purines appeared to block only the fast relaxation component of the tail currents. We therefore conclude that the p2y receptors target only the fast component of the delayed rectifier. As ATP breakdown to ADP is rapid and ADP may accumulate at higher levels than ATP, the contribution of ADP acting through p2y1-like receptors may be an important additional mechanism for the control of spinal motor pattern generation. PMID:11986373

Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R.

PubMed

Sundqvist, Martina; Christenson, Karin; Holdfeldt, André; Gabl, Michael; Mårtensson, Jonas; Björkman, Lena; Dieckmann, Regis; Dahlgren, Claes; Forsman, Huamei

2018-05-01

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo. In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo. Copyright © 2018 Elsevier B.V. All rights reserved.

Activation of G protein-coupled receptor 30 by thiodiphenol promotes proliferation of estrogen receptor α-positive breast cancer cells.

PubMed

Lei, Bingli; Peng, Wei; Xu, Gang; Wu, Minghong; Wen, Yu; Xu, Jie; Yu, Zhiqiang; Wang, Yipei

2017-02-01

Many studies have been shown that environmental estrogen bisphenol A (BPA) can activate nuclear receptor (estrogen receptor alpha, ERα) or membrane receptor (G-protein-coupled receptor, GPR30) in breast cancer cells and exerts genomic or nongenomic actions inducing cell proliferation. 4,4'-thiodiphenol (TDP) as one of BPA derivatives exhibits more potent estrogenic activity than BPA does. However, comparatively little is known about the ways in which TDP interferes with these signaling pathways and produces cell biological changes. This study evaluated the effect of TDP on cell viability, reactive oxygen species (ROS) formation, and intercellular calcium (Ca 2+ ) fluctuation in MCF-7 breast cancer cells. The underlying molecular mechanism of cell proliferation induced by TDP was analyzed by examining the activation of ERα and GPR30-mediated phosphatidylinotidol 3-kinase/protein kinase B (PI3K/AKT) and extracellular-signa1regulated kinase (ERK1/2) signaling pathways. The results showed that exposure to 0.1-10 μM TDP for 24, 48, and 72 h significantly increased viability of MCF-7 cells. At the same concentration range, TDP exposure for 3 and 24 h markedly elevated ROS production and intracellular Ca 2+ levels. In addition, 0.01-1 μM TDP significantly increased the expression of ERα, GPR30, p-AKT and p-ERK1/2 protein. Specific protein inhibitors blocked phosphorylation of ERK1/2 and AKT and decreased TDP-induced cell proliferation. These findings show that TDP activated the GPR30-PI3K/AKT and ERK1/2 pathways, and the resulting interaction with ERα stimulated MCF-7 cell proliferation. Our results indicate a novel mechanism through which TDP may exert relevant estrogenic action in ERα positive cancer cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Lysophosphatidylinositol-induced activation of the cation channel TRPV2 triggers glucagon-like peptide-1 secretion in enteroendocrine L cells.

PubMed

Harada, Kazuki; Kitaguchi, Tetsuya; Kamiya, Taichi; Aung, Kyaw Htet; Nakamura, Kazuaki; Ohta, Kunihiro; Tsuboi, Takashi

2017-06-30

The lysophosphatidylinositol (LPI) has crucial roles in multiple physiological processes, including insulin exocytosis from pancreatic islets. However, the role of LPI in secretion of glucagon-like peptide-1 (GLP-1), a hormone that enhances glucose-induced insulin secretion, is unclear. Here, we used the murine enteroendocrine L cell line GLUTag and primary murine small intestinal cells to elucidate the mechanism of LPI-induced GLP-1 secretion. Exogenous LPI addition increased intracellular Ca 2+ concentrations ([Ca 2+ ] i ) in GLUTag cells and induced GLP-1 secretion from both GLUTag and acutely prepared primary intestinal cells. The [Ca 2+ ] i increase was suppressed by an antagonist for G protein-coupled receptor 55 (GPR55) and by silencing of GPR55 expression, indicating involvement of G q and G 12/13 signaling pathways in the LPI-induced increased [Ca 2+ ] i levels and GLP-1 secretion. However, GPR55 agonists did not mimic many of the effects of LPI. We also found that phospholipase C inhibitor and Rho-associated kinase inhibitor suppressed the [Ca 2+ ] i increase and that LPI increased the number of focal adhesions, indicating actin reorganization. Of note, blockage or silencing of transient receptor potential cation channel subfamily V member 2 (TRPV2) channels suppressed both the LPI-induced [Ca 2+ ] i increase and GLP-1 secretion. Furthermore, LPI accelerated TRPV2 translocation to the plasma membrane, which was significantly suppressed by a GPR55 antagonist. These findings suggest that TRPV2 activation via actin reorganization induced by G q and G 12/13 signaling is involved in LPI-stimulated GLP-1 secretion in enteroendocrine L cells. Because GPR55 agonists largely failed to mimic the effects of LPI, its actions on L cells are at least partially independent of GPR55 activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Proton receptor GPR68 expression in dendritic-cell-like S100β-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification.

PubMed

Horiguchi, Kotaro; Higuchi, Masashi; Yoshida, Saishu; Nakakura, Takashi; Tateno, Kozue; Hasegawa, Rumi; Takigami, Shu; Ohsako, Shunji; Kato, Takako; Kato, Yukio

2014-11-01

S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.

Activation of GPR30 inhibits growth of prostate cancer cells via sustained activation of Erk1/2, c-jun/c-fos-dependent upregulation of p21, and induction of G2 cell-cycle arrest

PubMed Central

Chan, Queeny K.Y.; Lam, Hung-Ming; Ng, Chi-Fai; Lee, Amy Y.Y.; Chan, Eddie S.Y.; Ng, Ho-Keung; Ho, Shuk-Mei; Lau, Kin-Mang

2010-01-01

G protein–coupled receptor 30 (GPR30) exhibits estrogen-binding affinity and mediates nongenomic signaling of estrogen to regulate cell growth. We here demonstrated for the first time, in contrast to the reported promoting action of GPR30 on the growth of breast and ovarian cancer cells, that activation of GPR30 by the receptor-specific, non-estrogenic ligand G-1 inhibited growth of androgen-dependent and -independent prostate cancer (PCa) cells in vitro and PC-3 xenografts in vivo. However, G-1 elicited no growth or histological changes in the prostates of intact mice and did not inhibit growth in quiescent BPH-1, an immortalized benign prostatic epithelial cell line. Treatment of PC-3 cells with G-1-induced cell-cycle arrest at the G2 phase and reduced the expression of G2-checkpoint regulators (cyclin A2, cyclin B1, cdc25c, and cdc2) and the phosphorylation of their common transcriptional regulator NF-YA in PC-3 cells. With the extensive use of siRNA knockdown experiments and the MEK inhibitor PD98059 in the present study, we dissected the mechanism underlying G-1–induced inhibition of PC-3 cell growth, which was mediated through GPR30, followed by a sustained activation of Erk1/2 and a c-jun/c-fos-dependent upregulation of p21, resulting in the arrest of PC-3 growth at the G2 phase. The discovery of this signaling pathway lays the foundation for future development of GPR30-based therapies for PCa. PMID:20203690

Rapid activation of ERK1/2 and AKT in human breast cancer cells by cadmium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Zhiwei; Yu Xinyuan; Shaikh, Zahir A.

2008-05-01

Cadmium (Cd), an endocrine disruptor, can induce a variety of signaling events including the activation of ERK1/2 and AKT. In this study, the involvement of estrogen receptors (ER) in these events was evaluated in three human breast caner cell lines, MCF-7, MDA-MB-231, and SK-BR-3. The Cd-induced signal activation patterns in the three cell lines mimicked those exhibited in response to 17{beta}-estradiol. Specifically, treatment of MCF-7 cells, that express ER{alpha}, ER{beta} and GPR30, to 0.5-10 {mu}M Cd for only 2.5 min resulted in transient phosphorylation of ERK1/2. Cd also triggered a gradual increase and sustained activation of AKT during the 60more » min treatment period. In SK-BR-3 cells, that express only GPR30, Cd also caused a transient activation of ERK1/2, but not of AKT. In contrast, in MDA-MB-231 cells, that express only ER{beta}, Cd was unable to cause rapid activation of either ERK1/2 or AKT. A transient phosphorylation of ER{alpha} was also observed within 2.5 min of Cd exposure in the MCF-7 cells. While the estrogen receptor antagonist, ICI 182,780, did not prevent the effect of Cd on these signals, specific siRNA against hER{alpha} significantly reduced Cd-induced activation of ERK1/2 and completely blocked the activation of AKT. It is concluded that Cd, like estradiol, can cause rapid activation of ERK1/2 and AKT and that these signaling events are mediated by possible interaction with membrane ER{alpha} and GPR30, but not ER{beta}.« less

Targeting of free fatty acid receptor 1 in EOC: A novel strategy to restrict the adipocyte-EOC dependence.

PubMed

Munkarah, Adnan; Mert, Ismail; Chhina, Jasdeep; Hamid, Suhail; Poisson, Laila; Hensley-Alford, Sharon; Giri, Shailendra; Rattan, Ramandeep

2016-04-01

Adipocyte derived free fatty acids (FFA) promote epithelial ovarian cancer (EOC) by acting as a fuel source to support the energy requirement of the cancer cells. FFA may also exert biological effects through signaling pathways. Recently, a family of FFA activated G-protein coupled receptors (FFAR/GPCRs) was identified. Our objective was to investigate the role of FFAR/GPCRs in EOC and assess their potential as therapeutic targets. The mRNA (RT-PCR) expression of FFAR/GPCR family members (FFAR1/GPR40; FFAR2/GPR43, FFAR3/GPR41, FFAR4/GPR120 and GPR84) was examined in: (1) a syngeneic mouse model of EOC fed high energy diet (60% fat) or regular diet (30% fat), (2) EOC cell lines exposed to free fatty acids and (3) specimens from 13 histologically normal ovaries and 28 high grade ovarian serous carcinomas. The GPR 40 antagonist, GW1100, was used to inhibit FFAR1/GPR40 and cell survival was assayed by MTT in various cell lines. High Grade Serous carcinoma specimens expressed significantly increased GPR40 compared to normal ovaries (p=0.0020). Higher expression was noted in advanced stage disease. ID8 ovarian tumors from mice fed with high fat diet also showed higher GPR40 expression. Exposing EOC cells to FFAs, increased GPR40 expression. Treatment of EOC cell lines with GW100 resulted in growth inhibition and was associated with an alteration in their energy metabolism. FFA-induced cancer cell growth may be partly mediated through FFAR1/GPR40. Targeting of FFAR1/GPR40 may be an attractive treatment strategy in EOC, and possibly offers a targeted treatment for a subset of EOC patients. Copyright © 2016 Elsevier Inc. All rights reserved.

Bacterial metabolite S-equol modulates glucagon-like peptide-1 secretion from enteroendocrine L cell line GLUTag cells via actin polymerization.

PubMed

Harada, Kazuki; Sada, Shoko; Sakaguchi, Hidekazu; Takizawa, Mai; Ishida, Rika; Tsuboi, Takashi

2018-07-02

S-equol is one of gut bacterial metabolites produced from soybean isoflavone daizein. While S-equol is known to promote glucose-induced insulin secretion from pancreatic β cells, whether S-equol affects glucagon-like peptide-1 (GLP-1) secretion from enteroendoceine L cells remains unclear. Here we assessed the effect of S-equol on GLP-1 secretion from mouse enteroendocrine L cell line GLUTag cells. GLUTag cells expressed GPR30 and estrogen receptors, which are putative S-equol receptors. Application of S-equol induced an increase in intracellular Ca 2+ levels via GPR30. However, S-equol did not enhance GLP-1 exocytosis, and long-term treatment of S-equol suppressed GLP-1 secretion. Moreover, immunocytochemistry revealed that S-equol increased the density of cortical actin filaments via G 12/13 signaling under GPR30. These data suggest that S-equol prevents GLP-1 secretion as a result of competing regulation between Ca 2+ mobilization and actin reorganization. Copyright © 2018 Elsevier Inc. All rights reserved.

The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects

PubMed Central

Johns, D G; Behm, D J; Walker, D J; Ao, Z; Shapland, E M; Daniels, D A; Riddick, M; Dowell, S; Staton, P C; Green, P; Shabon, U; Bao, W; Aiyar, N; Yue, T-L; Brown, A J; Morrison, A D; Douglas, S A

2007-01-01

Background and purpose: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified ‘CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator ‘CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents. Experimental approach: Human recombinant GPR55 was expressed in HEK293T cells and specific GTPγS activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses. Key results: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTPγS activity (EC50 approximately 2 nM), whereas the CB1 and CB2-selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20±2%, KO 26±5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 μ M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains. Conclusions: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents. PMID:17704827

17β-estradiol-induced growth of triple-negative breast cancer cells is prevented by the reduction of GPER expression after treatment with gefitinib.

PubMed

Girgert, Rainer; Emons, Günter; Gründker, Carsten

2017-02-01

Triple-negative breast cancers (TNBCs) are neither susceptible to endocrine therapy due to a lack of estrogen receptor α expression nor trastuzumab. TNBCs frequently overexpress epidermal growth factor receptor (EGFR) and membrane bound estrogen receptor, GPER. To a certain extent the growth of TNBCs is stimulated by 17β-estradiol via GPER. We analyzed whether inhibition of EGFR by gefitinib reduces the expression of GPER and subsequent signal transduction in TNBC cells. Dependence of proliferation on 17β-estradiol was determined using Alamar Blue assay. Expression of GPR30 and activation of c-src, EGFR and cAMP-responsive element binding (CREB) protein by 17β-estradiol was analyzed by western blotting. Expression of c-fos, cyclin D1 and aromatase was determined using RT-PCR. Gefitinib reduced GPER expression concentration‑ and time‑dependently. In HCC70 cells, GPER expression was reduced to 15±11% (p<0.05) after treatment with 200 nM gefitinib for four days, and in HCC1806 cells GPER expression was reduced to 39±5% (p<0.01) of the control. 17β-estradiol significantly increased the percentage of HCC1806 cells within 7 days to 145±29% of the control (HCC70, 110±8%). This increase in cell growth was completely prevented in both TNBC cell lines after GPR30 expression was downregulated by treatment with 200 nM gefitinib. In HCC1806 cells, activation of c-src was increased by 17β-estradiol to 350±50% (p<0.01), and gefitinib reduced src activation to 110%. Similar results were obtained in the HCC70 cells. Phosphorylation of EGFR increased to 240±40% (p<0.05) in the HCC1806 cells treated with 17β-estradiol (HCC70, 147±25%). Gefitinib completely prevented this activation. Phosphorylation of CREB and induction of c-fos, cyclin D1 and aromatase expression by 17β-estradiol were all prevented by gefitinib. These experiments conclusively show that reduction of GPER expression is a promising therapeutic approach for TNBC.

17A, a novel non-coding RNA, regulates GABA B alternative splicing and signaling in response to inflammatory stimuli and in Alzheimer disease.

PubMed

Massone, Sara; Vassallo, Irene; Fiorino, Gloria; Castelnuovo, Manuele; Barbieri, Federica; Borghi, Roberta; Tabaton, Massimo; Robello, Mauro; Gatta, Elena; Russo, Claudio; Florio, Tullio; Dieci, Giorgio; Cancedda, Ranieri; Pagano, Aldo

2011-02-01

Alternative splicing is a central component of human brain complexity; nonetheless, its regulatory mechanisms are still largely unclear. In this work, we describe a novel non-coding (nc) RNA (named 17A) RNA polymerase (pol) III-dependent embedded in the human G-protein-coupled receptor 51 gene (GPR51, GABA B2 receptor). The stable expression of 17A in SHSY5Y neuroblastoma cells induces the synthesis of an alternative splicing isoform that abolish GABA B2 intracellular signaling (i.e., inhibition of cAMP accumulation and activation of K(+) channels). Indeed, 17A is expressed in human brain, and we report that it is upregulated in cerebral tissues derived from Alzheimer disease patients. We demonstrate that 17A expression in neuroblastoma cells enhances the secretion of amyloid β peptide (Aβ) and the Aβ x-42/Αβ x-40 peptide ratio and that its synthesis is induced in response to inflammatory stimuli. These data correlate, for the first time, the activity of a novel pol III-dependent ncRNA to alternative splicing events and, possibly, to neurodegeneration induced by abnormal GABA B function. We anticipate that further analysis of pol III-dependent regulation of alternative splicing will disclose novel regulatory pathways associated to brain physiology and/or pathology. Copyright © 2010 Elsevier Inc. All rights reserved.

CD36- and GPR120-mediated Ca²⁺ signaling in human taste bud cells mediates differential responses to fatty acids and is altered in obese mice.

PubMed

Ozdener, Mehmet Hakan; Subramaniam, Selvakumar; Sundaresan, Sinju; Sery, Omar; Hashimoto, Toshihiro; Asakawa, Yoshinori; Besnard, Philippe; Abumrad, Nada A; Khan, Naim Akhtar

2014-04-01

It is important to increase our understanding of gustatory detection of dietary fat and its contribution to fat preference. We studied the roles of the fat taste receptors CD36 and GPR120 and their interactions via Ca(2+) signaling in fungiform taste bud cells (TBC). We measured Ca(2+) signaling in human TBC, transfected with small interfering RNAs against messenger RNAs encoding CD36 and GPR120 (or control small interfering RNAs). We also studied Ca(2+) signaling in TBC from CD36(-/-) mice and from wild-type lean and obese mice. Additional studies were conducted with mouse enteroendocrine cell line STC-1 that express GPR120 and stably transfected with human CD36. We measured release of serotonin and glucagon-like peptide-1 from human and mice TBC in response to CD36 and GPR120 activation. High concentrations of linoleic acid induced Ca(2+) signaling via CD36 and GPR120 in human and mice TBC, as well as in STC-1 cells, and low concentrations induced Ca(2+) signaling via only CD36. Incubation of human and mice fungiform TBC with lineoleic acid down-regulated CD36 and up-regulated GPR120 in membrane lipid rafts. Obese mice had decreased spontaneous preference for fat. Fungiform TBC from obese mice had reduced Ca(2+) and serotonin responses, but increased release of glucagon-like peptide-1, along with reduced levels of CD36 and increased levels of GPR120 in lipid rafts. CD36 and GPR120 have nonoverlapping roles in TBC signaling during orogustatory perception of dietary lipids; these are differentially regulated by obesity. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis.

PubMed

Perry, Kimberly J; Johnson, Verity R; Malloch, Erica L; Fukui, Lisa; Wever, Jason; Thomas, Alvin G; Hamilton, Paul W; Henry, Jonathan J

2010-11-01

G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina, and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84's importance in lens, cornea, and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino-injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. © 2010 Wiley-Liss, Inc.

The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis

PubMed Central

Perry, Kimberly J.; Johnson, Verity R.; Malloch, Erica L.; Fukui, Lisa; Wever, Jason; Thomas, Alvin G.; Hamilton, Paul W.; Henry, Jonathan J.

2010-01-01

G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84’s importance in lens, cornea and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. PMID:20925114

Medium-chain fatty acids as ligands for orphan G protein-coupled receptor GPR84.

PubMed

Wang, Jinghong; Wu, Xiaosu; Simonavicius, Nicole; Tian, Hui; Ling, Lei

2006-11-10

Free fatty acids (FFAs) play important physiological roles in many tissues as an energy source and as signaling molecules in various cellular processes. Elevated levels of circulating FFAs are associated with obesity, dyslipidemia, and diabetes. Here we show that GPR84, a previously orphan G protein-coupled receptor, functions as a receptor for medium-chain FFAs with carbon chain lengths of 9-14. Medium-chain FFAs elicit calcium mobilization, inhibit 3',5'-cyclic AMP production, and stimulate [35S]guanosine 5'-O-(3-thiotriphosphate) binding in a GPR84-dependent manner. The activation of GPR84 by medium-chain FFAs couples primarily to a pertussis toxin-sensitive G(i/o) pathway. In addition, we show that GPR84 is selectively expressed in leukocytes and markedly induced in monocytes/macrophages upon activation by lipopolysaccharide. Furthermore, we demonstrate that medium-chain FFAs amplify lipopolysaccharide-stimulated production of the proinflammatory cytokine interleukin-12 p40 through GPR84. Our results indicate a role for GPR84 in directly linking fatty acid metabolism to immunological regulation.

Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor.

PubMed

Ecke, Denise; Hanck, Theodor; Tulapurkar, Mohan E; Schäfer, Rainer; Kassack, Matthias; Stricker, Rolf; Reiser, Georg

2008-01-01

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.

Mutations in the pH-Sensing G-protein-Coupled Receptor GPR68 Cause Amelogenesis Imperfecta.

PubMed

Parry, David A; Smith, Claire E L; El-Sayed, Walid; Poulter, James A; Shore, Roger C; Logan, Clare V; Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu; Harada, Akihiro; Zhang, Hong; Koruyucu, Mine; Seymen, Figen; Hu, Jan C-C; Simmer, James P; Ahmed, Mushtaq; Jafri, Hussain; Johnson, Colin A; Inglehearn, Chris F; Mighell, Alan J

2016-10-06

Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the pH of the developing enamel matrix. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with amelogenesis imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion, and a missense variant) were predicted to result in loss of function. GPR68 encodes a proton-sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localization of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The Proton-Sensing G-Protein Coupled Receptor GPR4 Promotes Angiogenesis in Head and Neck Cancer

PubMed Central

Chen, Xiaohong; Zhong, Qi; Huang, Junwei; Zhang, Yang; Guo, Wei; Yang, Zheng; Ding, Shuo; Chen, Ping

2016-01-01

Squamous cell carcinoma of the head and neck (SCCHN) is an aggressive disease with poor survival and is the sixth most common cancer worldwide. Gastroesophageal reflux is a common event in SCCHN patients. GPR4 is a proton-sensing G-protein coupled receptor, which can be activated by acidosis. The objective of this study was to explore the role of GPR4 in acid exposure and tumor angiogenesis in SCCHN. In this study, we confirmed that overexpressing GPR4 in SCCHN cells could increase the expression and secretion of IL6, IL8 and VEGFA at pH 5.9. This effect could be inhibited by SB203580 (a p38 inhibitor). Western blot analysis indicated that phosphorylation of p38 increased in GPR4 infected cells at pH 5.9, which could be inhibited by SB203580. In tube formation assay, HMEC-1 cells were incubated with conditioned medium (CM, pH 5.9, 6.5, 7.4) derived from control and GPR4 infected SCCHN cells. Tube length was significantly increased in HMEC-1 cells incubated with CM from GPR4 infected cells compared with control cells at pH5.9, which indicated the pro-angiogenic effect of GPR4 in acidic pH. The neutralizing antibodies of IL6, IL8 and VEGFA could inhibit tube formation of HMEC-1 cells. In vivo, the effect of GPR4 on angiogenesis was investigated with the chick chorioallantoic membrane (CAM) model. Control and GPR4 infected SCCHN cells were seeded onto the upper CAM surface (n = 5 in each group) and 5 μL DMEM/F12 (pH 5.9, 6.5, 7.4) was added to the surface of the cell every 24 h. Four days later, the upper CAM were harvested and the ratio of the vascular area to the CAM area was quantified using Image-Pro Plus 6.0 software. GPR4 infected cells could recruit more vascular than control cells at pH5.9. In conclusion, we suggested that GPR4 induces angiogenesis via GPR4-induced p38-mediated IL6, IL8 and VEGFA secretion at acidic extracellular pH in SCCHN. PMID:27078157

17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the g protein-coupled receptor GPR30.

PubMed

Vivacqua, Adele; Bonofiglio, Daniela; Albanito, Lidia; Madeo, Antonio; Rago, Vittoria; Carpino, Amalia; Musti, Anna Maria; Picard, Didier; Andò, Sebastiano; Maggiolini, Marcello

2006-10-01

The higher incidence of thyroid carcinoma (TC) in women during reproductive years compared with men and the increased risk associated with the therapeutic use of estrogens have suggested a pathogenetic role exerted by these steroids in the development of TC. In the present study, we evaluated the potential of 17beta-estradiol (E2), genistein (G), and 4-hydroxyta-moxifen (OHT) to regulate the expression of diverse estrogen target genes and the proliferation of human WRO, FRO, and ARO thyroid carcinoma cells, which were used as a model system. We have ascertained that ARO cells are devoid of estrogen receptors (ERs), whereas both WRO and FRO cells express a single variant of ERalpha that was neither transactivated, modulated, nor translocated into the nucleus upon treatment with ligands. However, E2, G, and OHT were able either to induce the transcriptional activity of c-fos promoter constructs, including those lacking the estrogen-responsive elements, or to increase c-fos, cyclin A, and D1 expression. It is noteworthy that we have demonstrated that the G protein-coupled receptor 30 (GPR30) and the mitogen-activated protein kinase (MAPK) pathway mediate both the up-regulation of c-fos and the growth response to E2, G, and OHT in TC cells studied, because these stimulatory effects were prevented by silencing GPR30 and using the MEK inhibitor 2'-amino-3'-methoxyflavone (PD 98059). Our findings provide new insight into the molecular mechanisms through which estrogens may induce the progression of TC.

GPR30 as an initiator of tamoxifen resistance in hormone-dependent breast cancer.

PubMed

Mo, Zhiqiang; Liu, Manran; Yang, Fangfang; Luo, Haojun; Li, Zhenhua; Tu, Gang; Yang, Guanglun

2013-11-29

Tamoxifen is widely used to treat hormone-dependent breast cancer, but its therapeutic benefit is limited by the development of drug resistance. Here, we investigated the role of estrogen G-protein coupled receptor 30 (GPR30) on Tamoxifen resistance in breast cancer. Primary tumors (PTs) of breast cancer and corresponding metastases (MTs) were used to evaluate the expression of GPR30 and epidermal growth factor receptor (EGFR) immunohistochemically. Tamoxifen-resistant (TAM-R) subclones derived from parent MCF-7 cells were used to investigate the role of GPR30 in the development of tamoxifen resistance, using MTT assay, western blot, RT-PCR, immunofluorescence, ELISA and flow cytometry. TAM-R xenografts were established to assess anti-tumor effects of combination therapy with GPR30 antagonist G15 plus 4-hydroxytamoxifen (Tam), using tumor volume measurement and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). In 53 human breast cancer specimens, GPR30 expression in MTs increased compared to matched PTs; in MTs, the expression patterns of GPR30 and EGFR were closely related. Compared to parent MCF-7 cells, TAM-R cells had greater growth responses to 17β-estradiol (E2), GPR30 agonist G1 and Tam, and significantly higher activation of Mitogen-activated protein (MAP) kinases; but this increased activity was abolished by G15 or AG1478. In TAM-R cells, GPR30 cell-surface translocation facilitated crosstalk with EGFR, and reduced cAMP generation, attenuating inhibition of EGFR signaling. Combination therapy both promoted apoptosis in TAM-R cells and decreased drug-resistant tumor progression. Long-term endocrine treatment facilitates the translocation of GPR30 to cell surfaces, which interferes with the EGFR signaling pathway; GPR30 also attenuates the inhibition of MAP kinases. These factors contribute to tamoxifen resistance development in breast cancer. Combination therapy with GPR30 inhibitors and tamoxifen may provide a new therapeutic option for drug-resistant breast cancer.

Mice lacking GPR3 receptors display late-onset obese phenotype due to impaired thermogenic function in brown adipose tissue

PubMed Central

Godlewski, Grzegorz; Jourdan, Tony; Szanda, Gergő; Tam, Joseph; Resat Cinar; Harvey-White, Judith; Liu, Jie; Mukhopadhyay, Bani; Pacher, Pál; Ming Mo, Fong; Osei-Hyiaman, Douglas; George Kunos

2015-01-01

We report an unexpected link between aging, thermogenesis and weight gain via the orphan G protein-coupled receptor GPR3. Mice lacking GPR3 and maintained on normal chow had similar body weights during their first 5 months of life, but gained considerably more weight thereafter and displayed reduced total energy expenditure and lower core body temperature. By the age of 5 months GPR3 KO mice already had lower thermogenic gene expression and uncoupling protein 1 protein level and showed impaired glucose uptake into interscapular brown adipose tissue (iBAT) relative to WT littermates. These molecular deviations in iBAT of GPR3 KO mice preceded measurable differences in body weight and core body temperature at ambient conditions, but were coupled to a failure to maintain thermal homeostasis during acute cold challenge. At the same time, the same cold challenge caused a 17-fold increase in Gpr3 expression in iBAT of WT mice. Thus, GPR3 appears to have a key role in the thermogenic response of iBAT and may represent a new therapeutic target in age-related obesity. PMID:26455425

Cannabinoid effects on β amyloid fibril and aggregate formation, neuronal and microglial-activated neurotoxicity in vitro.

PubMed

Janefjord, Emelie; Mååg, Jesper L V; Harvey, Benjamin S; Smid, Scott D

2014-01-01

Cannabinoid (CB) ligands have demonstrated neuroprotective properties. In this study we compared the effects of a diverse set of CB ligands against β amyloid-mediated neuronal toxicity and activated microglial-conditioned media-based neurotoxicity in vitro, and compared this with a capacity to directly alter β amyloid (Aβ) fibril or aggregate formation. Neuroblastoma (SH-SY5Y) cells were exposed to Aβ1-42 directly or microglial (BV-2 cells) conditioned media activated with lipopolysaccharide (LPS) in the presence of the CB1 receptor-selective agonist ACEA, CB2 receptor-selective agonist JWH-015, phytocannabinoids Δ(9)-THC and cannabidiol (CBD), the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide or putative GPR18/GPR55 ligands O-1602 and abnormal-cannabidiol (Abn-CBD). TNF-α and nitrite production was measured in BV-2 cells to compare activation via LPS or albumin with Aβ1-42. Aβ1-42 evoked a concentration-dependent loss of cell viability in SH-SY5Y cells but negligible TNF-α and nitrite production in BV-2 cells compared to albumin or LPS. Both albumin and LPS-activated BV-2 conditioned media significantly reduced neuronal cell viability but were directly innocuous to SH-SY5Y cells. Of those CB ligands tested, only 2-AG and CBD were directly protective against Aβ-evoked SH-SY5Y cell viability, whereas JWH-015, THC, CBD, Abn-CBD and O-1602 all protected SH-SY5Y cells from BV-2 conditioned media activated via LPS. While CB ligands variably altered the morphology of Aβ fibrils and aggregates, there was no clear correlation between effects on Aβ morphology and neuroprotective actions. These findings indicate a neuroprotective action of CB ligands via actions at microglial and neuronal cells.

Enhancement of Astroglial Aerobic Glycolysis by Extracellular Lactate-Mediated Increase in cAMP

PubMed Central

Vardjan, Nina; Chowdhury, Helena H.; Horvat, Anemari; Velebit, Jelena; Malnar, Maja; Muhič, Marko; Kreft, Marko; Krivec, Špela G.; Bobnar, Saša T.; Miš, Katarina; Pirkmajer, Sergej; Offermanns, Stefan; Henriksen, Gjermund; Storm-Mathisen, Jon; Bergersen, Linda H.; Zorec, Robert

2018-01-01

Besides being a neuronal fuel, L-lactate is also a signal in the brain. Whether extracellular L-lactate affects brain metabolism, in particular astrocytes, abundant neuroglial cells, which produce L-lactate in aerobic glycolysis, is unclear. Recent studies suggested that astrocytes express low levels of the L-lactate GPR81 receptor (EC50 ≈ 5 mM) that is in fat cells part of an autocrine loop, in which the Gi-protein mediates reduction of cytosolic cyclic adenosine monophosphate (cAMP). To study whether a similar signaling loop is present in astrocytes, affecting aerobic glycolysis, we measured the cytosolic levels of cAMP, D-glucose and L-lactate in single astrocytes using fluorescence resonance energy transfer (FRET)-based nanosensors. In contrast to the situation in fat cells, stimulation by extracellular L-lactate and the selective GPR81 agonists, 3-chloro-5-hydroxybenzoic acid (3Cl-5OH-BA) or 4-methyl-N-(5-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-4-(2-thienyl)-1,3-thiazol-2-yl)cyclohexanecarboxamide (Compound 2), like adrenergic stimulation, elevated intracellular cAMP and L-lactate in astrocytes, which was reduced by the inhibition of adenylate cyclase. Surprisingly, 3Cl-5OH-BA and Compound 2 increased cytosolic cAMP also in GPR81-knock out astrocytes, indicating that the effect is GPR81-independent and mediated by a novel, yet unidentified, excitatory L-lactate receptor-like mechanism in astrocytes that enhances aerobic glycolysis and L-lactate production via a positive feedback mechanism. PMID:29867342

G-protein-coupled receptor 30 mediates the effects of estrogen on endothelial cell tube formation in vitro.

PubMed

Zhou, Liyuan; Chen, Hong; Mao, Xun; Qi, Hongbo; Baker, Philip N; Zhang, Hua

2017-06-01

The placenta is the exchange organ between the mother and the fetus. The inadequate function of this organ is associated with a number of pregnancy disorders. Hypoxia and oxidative stress during placental development may induce endothelial dysfunction, resulting in the reduction in the perfusion of the placenta. During pregnancy, the levels of estrogen are increased. Decreased estrogen levels have been reported in women with preeclampsia. However, whether estrogen is involved in placental angiogenesis remains unclear. In this study, we aimed to investigate the effects of estrogen on endothelial cell tube formation and to elucidate the underlying mechanisms. For this purpose, human umbilical vein endothelial cells (HUVECs) were cultured with 17‑β‑estradiol under conditions of hypoxia/reoxygenation (H/R). The total pipe length of the tube‑like structure on endothelial cells was measured. The expression levels of G‑protein‑coupled receptor 30 (GPR30) and endothelial nitric oxide synthase (eNOS) and Akt were also measured in the endothelial cells following treatment with 17‑β‑estradiol under H/R conditions by western blot analysis and immunostaining. We found that the total pipe length of the tube‑like structure on endothelial cells was significantly reduced. This reduction was reversed by treatment with 17‑β‑estradiol. The expression of GPR30 in endothelial cells was significantly increased following treatment with 17‑β‑estradiol under H/R conditions. Furthermore, the levels of eNOS and Akt in endothelial cells were also significantly increased following treatment with 17-β-estradiol under H/R conditions. The activation of eNOS was inhibited by wortmannin, an inhibitor of PI3K/Akt. Our data thus demonstrate that estrogen prevents the failure of endothelial cell tube formation induced by H/R. GPR30 plays an important role in these protective effects through the activation of eNOS and Akt in endothelial cells. Our data suggest that increased levels of estrogen are important for placental angiogenesis.

Changes in obestatin gene and GPR39 receptor expression in peripheral tissues of rat models of obesity, type 1 and type 2 diabetes.

PubMed

Kolodziejski, Pawel Antoni; Pruszynska-Oszmalek, Ewa; Sassek, Maciej; Kaczmarek, Przemyslaw; Szczepankiewicz, Dawid; Billert, Maria; Mackowiak, Paweł; Strowski, Mathias Z; Nowak, Krzysztof W

2017-04-01

Obestatin has a role in regulating food intake and energy expenditure, but the roles of obestatin and the GPR39 receptor in obesity and type 1 and type 2 diabetes mellitus (T1DM and T2DM, respectively) are not well understood. The aim of the present study was to investigate changes in obestatin and GPR39 in pathophysiological conditions like obesity, T1DM, and T2DM. Using rat models of diet-induced obesity (DIO), T1DM and T2DM (n = 14 per group), obestatin, its precursor protein preproghrelin, and GPR39 expression was investigated in tissues involved in glucose and lipid homeostasis regulation. Furthermore, serum obestatin and ghrelin concentrations were determined. Serum obestatin concentrations were positively correlated with glucagon (r = 0.6456; P < 0.001) and visfatin (r = 0.5560; P < 0.001), and negatively correlated with insulin (r = -0.4362; P < 0.05), adiponectin (r = -0.3998; P < 0.05), and leptin (r = -0.4180; P < 0.05). There were differences in GPR39 and preproghrelin expression in the three animal models. Hepatic GPR39 and preproghrelin mRNA expression was greater in T1DM, T2DM, and obese rats than in lean controls, whereas pancreatic GPR39 mRNA and protein and preproghrelin mRNA expression was decreased in T1DM, T2DM, and DIO rats. Higher GPR39 and preproghrelin protein and mRNA levels were found in adipose tissues of T1DM compared with control. In adipose tissues of T2DM and DIO rats, GPR39 protein levels were lower than in lean or T1DM rats. Preproghrelin mRNA was higher in adipose tissues of T1DM, T2DM, and DIO than lean rats. We hypothesize that changes in obestatin, GPR39, and ghrelin may contribute to metabolic abnormalities in T1DM, T2DM, and obesity. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/GPR41 as new potential therapeutic targets

PubMed Central

Ulven, Trond

2012-01-01

The deorphanization of the free fatty acid (FFA) receptors FFA1 (GPR40), FFA2 (GPR43), FFA3 (GPR41), GPR84, and GPR120 has made clear that the body is capable of recognizing and responding directly to nonesterified fatty acid of virtually any chain length. Colonic fermentation of dietary fiber produces high concentrations of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, a process which is important to health. The phylogenetically related 7-transmembrane (7TM) receptors free fatty acid receptor 2 (FFA2) and FFA3 are activated by these SCFAs, and several lines of evidence indicate that FFA2 and FFA3 mediate beneficial effects associated with a fiber-rich diet, and that they may be of interest as targets for treatment of inflammatory and metabolic diseases. FFA2 is highly expressed on immune cells, in particular neutrophils, and several studies suggest that the receptor plays a role in diseases involving a dysfunctional neutrophil response, such as inflammatory bowel disease (IBD). Both FFA2 and FFA3 have been implicated in metabolic diseases such as type 2 diabetes and in regulation of appetite. More research is however required to clarify the potential of the receptors as drug targets and establish if activation or inhibition would be the preferred mode of action. The availability of potent and selective receptor modulators is a prerequisite for these studies. The few modulators of FFA2 or FFA3 that have been published hitherto in the peer-reviewed literature in general have properties that make them less than ideal as such tools, but published patent applications indicate that better tool compounds might soon become available which should enable studies critical to validate the receptors as new drug targets. PMID:23060857

Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling*

PubMed Central

Moreno, Estefanía; Andradas, Clara; Medrano, Mireia; Caffarel, María M.; Pérez-Gómez, Eduardo; Blasco-Benito, Sandra; Gómez-Cañas, María; Pazos, M. Ruth; Irving, Andrew J.; Lluís, Carme; Canela, Enric I.; Fernández-Ruiz, Javier; Guzmán, Manuel; McCormick, Peter J.; Sánchez, Cristina

2014-01-01

The G protein-coupled receptors CB2 (CB2R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB2R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology. PMID:24942731

The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon

PubMed Central

Cohen, L; Sekler, I; Hershfinkel, M

2014-01-01

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn2+ are not well understood. Here, we determined a role of the colonocytic Zn2+ sensing receptor, ZnR/GPR39, in mediating Zn2+-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn2+-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn2+ by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases. PMID:24967969

The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon.

PubMed

Cohen, L; Sekler, I; Hershfinkel, M

2014-06-26

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn(2+) are not well understood. Here, we determined a role of the colonocytic Zn(2+) sensing receptor, ZnR/GPR39, in mediating Zn(2+)-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn(2+)-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn(2+) by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases.

A medium-chain fatty acid receptor Gpr84 in zebrafish: expression pattern and roles in immune regulation.

PubMed

Huang, Qiaoyan; Feng, Dong; Liu, Kai; Wang, Peng; Xiao, Hongyan; Wang, Ying; Zhang, Shicui; Liu, Zhenhui

2014-08-01

Gpr84 was recently identified as a receptor for medium-chain fatty acids, but its functions remain to be clarified. We reported the identification of a zebrafish Gpr84 homologue (zGpr84), which has a higher gene expression in the tissues of intestine, heart and liver. During embryogenesis, zGpr84 is maternally expressed and a significant increase is observed at segmentation period, and it is mainly restricted to the head region, pectoral fins, branchial arches, intestine and lateral line neuromast. Fasting or treatment with lipopolysaccharide (LPS) can induce significant up-regulation of zGpr84. We further demonstrated that zGpr84 is involved in the accumulation of lipid droplets in cells. Moreover, undecanoic acid (UA) can amplify LPS induced production of the proinflammatory cytokine IL-12 p40 through zGpr84, supporting the proposal that Gpr84 may play a role in directly linking fatty acid metabolism to immunological regulation. The resulting data in fish lay a foundation for a comprehensive exploration of the functions and evolution of Gpr84. Copyright © 2014 Elsevier Ltd. All rights reserved.

Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.

PubMed

Justus, Calvin R; Dong, Lixue; Yang, Li V

2013-12-05

The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis, and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs) in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A), regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention.

Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

PubMed Central

De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

2016-01-01

Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties. PMID:27716822

Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

PubMed

De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

2016-01-01

Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.

Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells.

PubMed

Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla; Hansson, Stefan; Casslén, Bertil

2009-02-01

Epidermal growth factor (EGF) stimulates proliferation and migration in ovarian cancer cells, and high tumor expression of the EGF system correlates with poor prognosis. Epidermal growth factor upregulates urokinase plasminogen activator receptor (uPAR) on the cell surface via 3 distinct mechanisms: rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression and cell migration in ovarian cancer cells and further to identify the ER involved.We used 7 ovarian cancer cell lines, cell migration assay, cellular binding of (125)I-uPA, cellular degradation of (125)I-uPA/PAI-1 complex, enzyme-linked immunosorbent assay for uPAR, solid-phase enzyme immunoassay for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents mobilization of uPAR from detergent-resistant domains such as lipid rafts. Estradiol influenced neither the amount of uPAR mRNA nor the rate of uPAR degradation or solubilization. The nuclear ER antagonists ICI 182780 and tamoxifen, which are GPR30 agonists, as well as the specifically constructed GPR30 agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates that this effect is mediated via the membrane ER GPR30.

G protein-coupled receptor 84 controls osteoclastogenesis through inhibition of NF-κB and MAPK signaling pathways.

PubMed

Park, Ji-Wan; Yoon, Hye-Jin; Kang, Woo Youl; Cho, Seungil; Seong, Sook Jin; Lee, Hae Won; Yoon, Young-Ran; Kim, Hyun-Ju

2018-02-01

GPR84, a member of the G protein-coupled receptor family, is found predominantly in immune cells, such as macrophages, and functions as a pivotal modulator of inflammatory responses. In this study, we investigated the role of GPR84 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. Our microarray data showed that GPR84 was significantly downregulated in osteoclasts compared to in their precursors, macrophages. The overexpression of GPR84 in bone marrow-derived macrophages suppressed the formation of multinucleated osteoclasts without affecting precursor proliferation. In addition, GPR84 overexpression attenuated the induction of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which are transcription factors that are critical for osteoclastogenesis. Furthermore, knockdown of GPR84 using a small hairpin RNA promoted RANKL-mediated osteoclast differentiation and gene expression of osteoclastogenic markers. Mechanistically, GPR84 overexpression blocked RANKL-stimulated phosphorylation of IκBα and three MAPKs, JNK, ERK, and p38. GPR84 also suppressed NF-κB transcriptional activity mediated by RANKL. Conversely, GPR84 knockdown enhanced RANKL-induced activation of IκBα and the three MAPKs. Collectively, our results revealed that GPR84 functions as a negative regulator of osteoclastogenesis, suggesting that it may be a potential therapeutic target for osteoclast-mediated bone-destructive diseases. © 2017 Wiley Periodicals, Inc.

Estrogen regulates hepcidin expression via GPR30-BMP6-dependent signaling in hepatocytes.

PubMed

Ikeda, Yasumasa; Tajima, Soichiro; Izawa-Ishizawa, Yuki; Kihira, Yoshitaka; Ishizawa, Keisuke; Tomita, Shuhei; Tsuchiya, Koichiro; Tamaki, Toshiaki

2012-01-01

Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E(2)) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E(2)-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E(2) and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E(2)-induced hepcidin expression. BMP6 expression induced by E(2) was abolished by GPR30 silencing. Finally, both E(2) and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E(2) and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism.

Targeting GPR30 in Abiraterone and MDV3100 Resistant Prostate Cancer

DTIC Science & Technology

2016-10-01

environment in vivo. We had previously demonstrated that G-1 inhibited growth in cell culture experiments and a hormone -independent PC-3Published by...coupled receptor 30 (GPR30) is a seven-transmembrane estrogen receptor and activation by its specific agonist G-1 inhibited growth in multiple...significantly inhibited the growth and extended the progression-free survival of patient-derived xenograft models that are sensitive (LuCaP 136CR, P

Discovery and Characterization of a Novel Small-Molecule Agonist for Medium-Chain Free Fatty Acid Receptor G Protein-Coupled Receptor 84.

PubMed

Zhang, Qing; Yang, Hui; Li, Jing; Xie, Xin

2016-05-01

G protein-coupled receptor 84 (GPR84) is a free fatty acid receptor activated by medium-chain free fatty acids with 9-14 carbons. It is expressed mainly in the immune-related tissues, such as spleen, bone marrow, and peripheral blood leukocytes. GPR84 plays significant roles in inflammatory processes and may represent a novel drug target for the treatment of immune-mediated diseases. However, the lack of potent and specific ligands for GPR84 hindered the study of its functions and the development of potential clinical applications. Here, we report the screen of 160,000 small-molecule compounds with a calcium mobilization assay using a human embryonic kidney 293 cell line stably expressing GPR84 and Gα16, and the identification of 2-(hexylthio)pyrimidine-4,6-diol (ZQ-16) as a potent and selective agonist of GPR84 with a novel structure. ZQ-16 activates several GPR84-mediated signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, phosphorylation of extracellular signal-regulated protein kinase 1/2, receptor desensitization and internalization, and receptor-β-arrestin interaction. This compound may be a useful tool to study the functions of GPR84 and a potential candidate for further structural optimization. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

GPR142 Controls Tryptophan-Induced Insulin and Incretin Hormone Secretion to Improve Glucose Metabolism

PubMed Central

Efanov, Alexander M.; Fang, Xiankang; Beavers, Lisa S.; Wang, Xuesong; Wang, Jingru; Gonzalez Valcarcel, Isabel C.; Ma, Tianwei

2016-01-01

GPR142, a putative amino acid receptor, is expressed in pancreatic islets and the gastrointestinal tract, but the ligand affinity and physiological role of this receptor remain obscure. In this study, we show that in addition to L-Tryptophan, GPR142 signaling is also activated by L-Phenylalanine but not by other naturally occurring amino acids. Furthermore, we show that Tryptophan and a synthetic GPR142 agonist increase insulin and incretin hormones and improve glucose disposal in mice in a GPR142-dependent manner. In contrast, Phenylalanine improves in vivo glucose disposal independently of GPR142. Noteworthy, refeeding-induced elevations in insulin and glucose-dependent insulinotropic polypeptide are blunted in Gpr142 null mice. In conclusion, these findings demonstrate GPR142 is a Tryptophan receptor critically required for insulin and incretin hormone regulation and suggest GPR142 agonists may be effective therapies that leverage amino acid sensing pathways for the treatment of type 2 diabetes. PMID:27322810

"Host tissue damage" signal ATP promotes non-directional migration and negatively regulates toll-like receptor signaling in human monocytes.

PubMed

Kaufmann, Andreas; Musset, Boris; Limberg, Sven H; Renigunta, Vijay; Sus, Rainer; Dalpke, Alexander H; Heeg, Klaus M; Robaye, Bernard; Hanley, Peter J

2005-09-16

The activation of Toll-like receptors (TLRs) by lipopolysaccharide or other ligands evokes a proinflammatory immune response, which is not only capable of clearing invading pathogens but can also inflict damage to host tissues. It is therefore important to prevent an overshoot of the TLR-induced response where necessary, and here we show that extracellular ATP is capable of doing this in human monocytes. Using reverse transcription-PCR, we showed that monocytes express P2Y(1), P2Y(2), P2Y(4), P2Y(11), and P2Y(13) receptors, as well as several P2X receptors. To elucidate the function of these receptors, we first studied Ca(2+) signaling in single cells. ATP or UTP induced a biphasic increase in cytosolic Ca(2+), which corresponded to internal Ca(2+) release followed by activation of store-operated Ca(2+) entry. The evoked Ca(2+) signals stimulated Ca(2+)-activated K(+) channels, producing transient membrane hyperpolarization. In addition, ATP promoted cytoskeleton reorganization and cell migration; however, unlike chemoattractants, the migration was non-directional and further analysis showed that ATP did not activate Akt, essential for sensing gradients. When TLR2, TLR4, or TLR2/6 were stimulated with their respective ligands, ATPgammaS profoundly inhibited secretion of proinflammatory cytokines (tumor necrosis factor-alpha and monocyte chemoattractant protein-1) but increased the production of interleukin-10, an anti-inflammatory cytokine. In radioimmune assays, we found that ATP (or ATPgammaS) strongly increased cAMP levels, and, moreover, the TLR-response was inhibited by forskolin, whereas UTP neither increased cAMP nor inhibited the TLR-response. Thus, our data suggest that ATP promotes non-directional migration and, importantly, acts as a "host tissue damage" signal via the G(s) protein-coupled P2Y(11) receptor and increased cAMP to negatively regulate TLR signaling.

Central Agonism of GPR120 Acutely Inhibits Food Intake and Food Reward and Chronically Suppresses Anxiety-Like Behavior in Mice

PubMed Central

Fisette, Alexandre; Fernandes, Maria F.; Hryhorczuk, Cécile; Poitout, Vincent; Alquier, Thierry; Fulton, Stephanie

2016-01-01

Background: GPR120 (FFAR4) is a G-protein coupled receptor implicated in the development of obesity and the antiinflammatory and insulin-sensitizing effects of omega-3 (ω-3) polyunsaturated fatty acids. Increasing central ω-3 polyunsaturated fatty acid levels has been shown to have both anorectic and anxiolytic actions. Despite the strong clinical interest in GPR120, its role in the brain is largely unknown, and thus we sought to determine the impact of central GPR120 pharmacological activation on energy balance, food reward, and anxiety-like behavior. Methods: Male C57Bl/6 mice with intracerebroventricular cannulae received a single injection (0.1 or 1 µM) or continuous 2-week infusion (1 µM/d; mini-pump) of a GPR120 agonist or vehicle. Free-feeding intake, operant lever-pressing for palatable food, energy expenditure (indirect calorimetry), and body weight were measured. GPR120 mRNA expression was measured in pertinent brain areas. Anxiety-like behavior was assessed in the elevated-plus maze and open field test. Results: GPR120 agonist injections substantially reduced chow intake during 4 hours postinjection, suppressed the rewarding effects of high-fat/-sugar food, and blunted approach-avoidance behavior in the open field. Conversely, prolonged central GPR120 agonist infusions reduced anxiety-like behavior in the elevated-plus maze and open field, yet failed to affect free-feeding intake, energy expenditure, and body weight on a high-fat diet. Conclusion: Acute reductions in food intake and food reward suggest that GPR120 could mediate the effects of central ω-3 polyunsaturated fatty acids to inhibit appetite. The anxiolytic effect elicited by GPR120 agonist infusions favors the testing of compounds that can enter the brain to activate GPR120 for the mitigation of anxiety. PMID:26888796

GPR40 agonists for the treatment of type 2 diabetes: life after 'TAKing' a hit.

PubMed

Mancini, A D; Poitout, V

2015-07-01

The free fatty acid receptor GPR40 has been proposed as a potential target for type 2 diabetes (T2D) pharmacotherapy. This idea has been validated in both preclinical and clinical studies, in which activation of GPR40 was shown to improve glycaemic control by stimulating glucose-dependent insulin secretion; however, the recent termination of phase III clinical trials using the GPR40 agonist TAK-875 (fasiglifam) has raised important questions regarding the long-term safety and viability of targeting GPR40 and, more specifically, about our understanding of this receptor's basic biology. In the present review, we provide a summary of established and novel concepts related to GPR40's pharmacobiology and discuss the current status and future outlook for GPR40-based drug development for the treatment of T2D. © 2015 John Wiley & Sons Ltd.

Lactate Transport and Receptor Actions in Retina: Potential Roles in Retinal Function and Disease.

PubMed

Kolko, Miriam; Vosborg, Fia; Henriksen, Ulrik L; Hasan-Olive, Md Mahdi; Diget, Elisabeth Holm; Vohra, Rupali; Gurubaran, Iswariya Raja Sridevi; Gjedde, Albert; Mariga, Shelton Tendai; Skytt, Dorte M; Utheim, Tor Paaske; Storm-Mathisen, Jon; Bergersen, Linda H

2016-06-01

In retina, like in brain, lactate equilibrates across cell membranes via monocarboxylate transporters and in the extracellular space by diffusion, forming a basis for the action of lactate as a transmitter of metabolic signals. In the present paper, we argue that the lactate receptor GPR81, also known as HCAR1, may contribute importantly to the control of retinal cell functions in health and disease. GPR81, a G-protein coupled receptor, is known to downregulate cAMP both in adipose and nervous tissue. The receptor also acts through other down-stream mechanisms to control functions, such as excitability, metabolism and inflammation. Recent publications predict effects of the lactate receptor on neurodegeneration. Neurodegenerative diseases in retina, where the retinal ganglion cells die, notably glaucoma and diabetic retinopathy, may be linked to disturbed lactate homeostasis. Pilot studies reveal high GPR81 mRNA in retina and indicate GPR81 localization in Müller cells and retinal ganglion cells. Moreover, monocarboxylate transporters are expressed in retinal cells. We envision that lactate receptors and transporters could be useful future targets of novel therapeutic strategies to protect neurons and prevent or counteract glaucoma as well as other retinal diseases.

P2X and P2Y nucleotide receptors as targets in cardiovascular disease.

PubMed

Kennedy, Charles; Chootip, Krongkarn; Mitchell, Callum; Syed, Nawazish-i-Husain; Tengah, Asrin

2013-03-01

Endogenous nucleotides have widespread actions in the cardiovascular system, but it is only recently that the P2X and P2Y receptor subtypes, at which they act, have been identified and subtype-selective agonists and antagonists developed. These advances have greatly increased our understanding of the physiological and pathophysiological functions of P2X and P2Y receptors, but investigation of the clinical usefulness of selective ligands is at an early stage. Nonetheless, the evidence considered in this review demonstrates clearly that various cardiovascular disorders, including vasospasm, hypertension, congestive heart failure and cardiac damage during ischemic episodes, may be viable targets. With further development of novel, selective agonists and antagonists, our understanding will continue to improve and further therapeutic applications are likely to be discovered.

Application of GPCR Structures for Modelling of Free Fatty Acid Receptors.

PubMed

Tikhonova, Irina G

2017-01-01

Five G protein-coupled receptors (GPCRs) have been identified to be activated by free fatty acids (FFA). Among them, FFA1 (GPR40) and FFA4 (GPR120) bind long-chain fatty acids, FFA2 (GPR43) and FFA3 (GPR41) bind short-chain fatty acids and GPR84 binds medium-chain fatty acids. Free fatty acid receptors have now emerged as potential targets for the treatment of diabetes, obesity and immune diseases. The recent progress in crystallography of GPCRs has now enabled the elucidation of the structure of FFA1 and provided reliable templates for homology modelling of other FFA receptors. Analysis of the crystal structure and improved homology models, along with mutagenesis data and structure activity, highlighted an unusual arginine charge-pairing interaction in FFA1-3 for receptor modulation, distinct structural features for ligand binding to FFA1 and FFA4 and an arginine of the second extracellular loop as a possible anchoring point for FFA at GPR84. Structural data will be helpful for searching novel small-molecule modulators at the FFA receptors.

Functional expression of ionotropic purinergic receptors on mouse taste bud cells.

PubMed

Hayato, Ryotaro; Ohtubo, Yoshitaka; Yoshii, Kiyonori

2007-10-15

Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 microm ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca(2+) imaging showed that similarly applied 1 mum ATP, 30 microm BzATP (a P2X(7) agonist), or 1 microm 2MeSATP (a P2Y(1) and P2Y(11) agonist) increased intracellular Ca(2+) concentration, but 100 microm UTP (a P2Y(2) and P2Y(4) agonist) and alpha,beta-meATP (a P2X agonist except for P2X(2), P2X(4) and P2X(7)) did not. RT-PCR suggested the expression of P2X(2), P2X(4), P2X(7), P2Y(1), P2Y(13) and P2Y(14) among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X(2). The exposure of the basolateral membranes to 3 mm ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 microm PPADS (a non-selective P2 blocker) and 1 microm KN-62 (a P2X(7) blocker). These results showed for the first time the functional expression of P2X(2) and P2X(7) on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.

CB2 Receptor Activation Inhibits Melanoma Cell Transmigration through the Blood-Brain Barrier

PubMed Central

Haskó, János; Fazakas, Csilla; Molnár, Judit; Nyúl-Tóth, Ádám; Herman, Hildegard; Hermenean, Anca; Wilhelm, Imola; Persidsky, Yuri; Krizbai, István A.

2014-01-01

During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB). The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2); therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A), GPR18 (transcriptional variant 1) and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A), GPR18 (transcriptional variants 1 and 2), GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma. PMID:24815068

G protein-coupled estrogen receptor 1 (GPER 1) mediates estrogen-induced, proliferation of leiomyoma cells.

PubMed

Jiang, Xiuxiu; Ye, Xiaolei; Ma, Junyan; Li, Wen; Wu, Ruijin; Jun, Lin

2015-01-01

G protein-coupled estrogen receptor 1 (GPER-1, formerly known as GPR30) has been proposed as the receptor for estrogen-induced, growth of leiomyomas though its precise mechanisms of action are not clear. We obtained leiomyoma cells (LC) and normal smooth muscle cells from 28 women (n = 28, median age 38 years, median parity 1.0). We incubated them with 17-β estradiol (E(2)), after blocking, or upregulating, expression of GPER-1 with ICI182,780 (a GPER-1 agonist) and siGPR30, respectively. We evaluated the role of GPER-1 in the mitogen-activated protein kinase (MAPK) signaling pathway using Western blot analysis. We studied cell proliferation with 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, and, mitotic activity with phosphohistone H3 (PPH3) expression in leiomyoma, and, matched, normal, smooth muscle tissues using standard immunohistochemistry. Downregulation of GPER-1 expression with siGPR30 partially attenuated the E(2)-activated MAPK signaling pathway (p < 0.01). Upregulation of GPER-1 with ICI182,780 enhanced the E(2)-activated MAPK signaling pathway (p < 0.01). ICI182,780 enhanced E(2)-induced proliferation of LC (p < 0.01), while knock down of the GPER-1 gene with GPER-1 small interfering RNA partially inhibited E(2)-induced cell proliferation (p < 0.01). There were no significant differences in PPH3 expression between LCs and normal smooth muscle tissues (p > 0.05). Neither ICI182,780 nor siGPR30 increased mitosis in LCs (p > 0.05). Our results indicate that GPER-1 mediates proliferation of estrogen-induced, LC by activating the MAPK pathway, and, not by promoting mitosis.

GPR30 Signaling and Regulation in Breast Cancer

DTIC Science & Technology

2011-04-01

binds to and activates the orphan G protein-coupled receptor, GPR30, which has been renamed G Protein Coupled Estrogen Receptor ( GPER ). Since the...that inhibition of GPER reduces estrogen-mediated tumor growth. 15. SUBJECT TERMS Breast Cancer, GPR30, Proliferation, Metastasis 16. SECURITY...receptor ( GPER ) [5,  6]. E2 plays a central role in the progression of breast cancer (BrCa), and enhances the proliferation, migration, and invasion of

Palmitoylethanolamide Modulates GPR55 Receptor Signaling in the Ventral Hippocampus to Regulate Mesolimbic Dopamine Activity, Social Interaction, and Memory Processing.

PubMed

Kramar, Cecilia; Loureiro, Michael; Renard, Justine; Laviolette, Steven R

2017-01-01

Introduction: The GPR55 receptor has been identified as an atypical cannabinoid receptor and is implicated in various physiological processes. However, its functional role in the central nervous system is not currently understood. The presence of GPR55 receptor in neural regions such as the ventral hippocampus (vHipp), which is critical for cognition, recognition memory, and affective processing, led us to hypothesize that intra-vHipp GPR55 transmission may modulate mesolimbic activity states and related behavioral phenomena. The vHipp is involved in contextual memory and affective regulation through functional interactions with the mesolimbic dopamine system. Materials and Methods: Using a combination of in vivo electrophysiology and behavioral pharmacological assays in rats, we tested whether intra-vHipp activation of GPR55 receptor transmission with the fatty acid amide, palmitoylethanolamide (PEA), a lipid neuromodulator with agonist actions at the GPR55 receptor, may modulate mesolimbic dopaminergic activity states. We further examined the potential effects of intra-vHipp PEA in affective, cognitive and contextual memory tasks. Discussion: We report that intra-vHipp PEA produces a hyper-dopaminergic state in the mesolimbic system characterized by increased firing and bursting activity of ventral tegmental area dopaminergic neuron populations. Furthermore, while PEA-induced activation of GPR55 transmission had no effects on opiate-related reward-related memory formation, we observed strong disruptions in social interaction and recognition memory, spatial location memory, and context-independent associative fear memory formation. Finally, the effects of intra-vHipp PEA were blocked by a selective GPR55 receptor antagonist, CID160 and were dependent upon NMDA receptor transmission, directly in the vHipp. Conclusions: The present results add to a growing body of evidence demonstrating important functional roles for GPR55 signaling in cannabinoid-related neuronal and behavioral phenomena and underscore the potential for GPR55 signaling in the mediation of cannabinoid-related effects independently of the CB1/CB2 receptor systems.

An Orphan G Protein-Coupled Receptor, GPR1, Acts as a Coreceptor To Allow Replication of Human Immunodeficiency Virus Types 1 and 2 in Brain-Derived Cells

PubMed Central

Shimizu, Nobuaki; Soda, Yasushi; Kanbe, Katsuaki; Liu, Hui-Yu; Jinno, Atsushi; Kitamura, Toshio; Hoshino, Hiroo

1999-01-01

Twelve G protein-coupled receptors, including chemokine receptors, act as coreceptors and determinants for the cell tropisms of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We isolated HIV-1 variants from T-cell-line (T)- and macrophage (M)-tropic (i.e., dualtropic) (R5-R3-X4) HIV-1 strains and also produced six HIV-1 mutants carrying single-point amino acid substitutions at the tip of the V3 region of the Env protein of HIV-1. These variants and three mutants infected brain-derived CD4-positive cells that are resistant to M-, T-, or dualtropic (R5, X4, or R5-X4) HIV-1 strains. However, a factor that determines this cell tropism has not been identified. This study shows that primary brain-derived fibroblast-like cell strains, BT-3 and BT-20/N, as well as a CD4-transduced glioma cell line, U87/CD4, which were susceptible to these HIV-1 variants and mutants and the HIV-2ROD strain, expressed mRNA of an orphan G protein-coupled receptor (GPCR), GPR1. When a CD4-positive cell line which was strictly resistant to infection with diverse HIV-1 and HIV-2 strains was transduced with GPR1, the cell line became susceptible to these HIV-1 variants and mutants and to an HIV-2 strain but not to T- or dualtropic HIV-1 strains, and numerous syncytia formed after infection. These results indicate that GPR1 functions as a coreceptor for the HIV-1 variants and mutants and for the HIV-2ROD strain in vitro. PMID:10233994

G protein-coupled receptor 91 signaling in diabetic retinopathy and hypoxic retinal diseases.

PubMed

Hu, Jianyan; Li, Tingting; Du, Xinhua; Wu, Qiang; Le, Yun-Zheng

2017-10-01

G protein-coupled receptor 91 (GPR91) is a succinate-specific receptor and activation of GPR91 could initiate a complex signal transduction cascade and upregulate inflammatory and pro-angiogenic cytokines. In the retina, GPR91 is predominately expressed in ganglion cells, a major cellular entity involved in the pathogenesis of diabetic retinopathy (DR) and other hypoxic retinal diseases. During the development of DR and retinopathy of prematurity (ROP), chronic hypoxia causes an increase in the levels of local succinate. Succinate-mediated GPR91 activation upregulates vascular endothelial growth factor (VEGF) through ERK1/2-C/EBP β (c-Fos) and/or ERK1/2-COX-2/PGE2 signaling pathways, which in turn, leads to the breakdown of blood-retina barriers in these disorders. In this review, we will have a brief introduction of GPR91 and its biological functions and a more detailed discussion about the role and mechanisms of GPR91 in DR and ROP. A better understanding of GPR91 regulation may be of great significance in identifying new biomarkers and drug targets for the prediction and treatment of DR, ROP, and hypoxic retinal diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

GPR81, a Cell-Surface Receptor for Lactate, Regulates Intestinal Homeostasis and Protects Mice from Experimental Colitis.

PubMed

Ranganathan, Punithavathi; Shanmugam, Arulkumaran; Swafford, Daniel; Suryawanshi, Amol; Bhattacharjee, Pushpak; Hussein, Mohamed S; Koni, Pandelakis A; Prasad, Puttur D; Kurago, Zoya B; Thangaraju, Muthusamy; Ganapathy, Vadivel; Manicassamy, Santhakumar

2018-03-01

At mucosal sites such as the intestine, the immune system launches robust immunity against invading pathogens while maintaining a state of tolerance to commensal flora and ingested food Ags. The molecular mechanisms underlying this phenomenon remain poorly understood. In this study, we report that signaling by GPR81, a receptor for lactate, in colonic dendritic cells and macrophages plays an important role in suppressing colonic inflammation and restoring colonic homeostasis. Genetic deletion of GPR81 in mice led to increased Th1/Th17 cell differentiation and reduced regulatory T cell differentiation, resulting in enhanced susceptibility to colonic inflammation. This was due to increased production of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) and decreased expression of immune regulatory factors (IL-10, retinoic acid, and IDO) by intestinal APCs lacking GPR81. Consistent with these findings, pharmacological activation of GPR81 decreased inflammatory cytokine expression and ameliorated colonic inflammation. Taken together, these findings identify a new and important role for the GPR81 signaling pathway in regulating immune tolerance and colonic inflammation. Thus, manipulation of the GPR81 pathway could provide novel opportunities for enhancing regulatory responses and treating colonic inflammation. Copyright © 2018 by The American Association of Immunologists, Inc.

Genetic variants of GPER/GPR30, a novel estrogen-related G protein receptor, are associated with human seminoma.

PubMed

Chevalier, Nicolas; Paul-Bellon, Rachel; Camparo, Philippe; Michiels, Jean-François; Chevallier, Daniel; Fénichel, Patrick

2014-01-21

Testicular germ cell tumors (TGCTs) are the most common solid cancers in young men, with an increasing incidence over several years. However, their pathogenesis remains a matter of debate. Some epidemiological data suggest the involvement of both environmental and genetic factors. We reported two distinct effects of estrogens and/or xeno-estrogens on in vitro human seminoma-derived cells proliferation: (1) an antiproliferative effect via a classical estrogen receptor beta-dependent pathway, and (2) a promotive effect via a non-classical membrane G-protein-coupled receptor, GPR30/GPER, which is only overexpressed in seminomas, the most common TGCT. In order to explain this overexpression, we investigated the possible association of polymorphisms in the GPER gene by using allele-specific tetra-primer polymerase chain reaction performed on tissue samples from 150 paraffin-embedded TGCT specimens (131 seminomas, 19 non seminomas). Compared to control population, loss of homozygous ancestral genotype GG in two polymorphisms located in the promoter region of GPER (rs3808350 and rs3808351) was more frequent in seminomas but not in non-seminomas (respectively, OR = 1.960 (1.172-3.277) and 7.000 (2.747-17.840); p < 0.01). These polymorphisms may explain GPER overexpression and represent a genetic factor of susceptibility supporting the contribution of environmental GPER ligands in testicular carcinogenesis.

Medium-chain fatty acid-sensing receptor, GPR84, is a proinflammatory receptor.

PubMed

Suzuki, Masakatsu; Takaishi, Sachiko; Nagasaki, Miyuki; Onozawa, Yoshiko; Iino, Ikue; Maeda, Hiroaki; Komai, Tomoaki; Oda, Tomiichiro

2013-04-12

G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiological roles have not yet been clarified. Here, we show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. We also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [(35)S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated production of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the intravenous injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. Our results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation associated-disease.

Medium-chain Fatty Acid-sensing Receptor, GPR84, Is a Proinflammatory Receptor

PubMed Central

Suzuki, Masakatsu; Takaishi, Sachiko; Nagasaki, Miyuki; Onozawa, Yoshiko; Iino, Ikue; Maeda, Hiroaki; Komai, Tomoaki; Oda, Tomiichiro

2013-01-01

G protein-coupled receptor 84 (GPR84) is a putative receptor for medium-chain fatty acids (MCFAs), whose pathophysiological roles have not yet been clarified. Here, we show that GPR84 was activated by MCFAs with the hydroxyl group at the 2- or 3-position more effectively than nonhydroxylated MCFAs. We also identified a surrogate agonist, 6-n-octylaminouracil (6-OAU), for GPR84. These potential ligands and the surrogate agonist, 6-OAU, stimulated [35S]GTP binding and accumulated phosphoinositides in a GPR84-dependent manner. The surrogate agonist, 6-OAU, internalized GPR84-EGFP from the cell surface. Both the potential ligands and 6-OAU elicited chemotaxis of human polymorphonuclear leukocytes (PMNs) and macrophages and amplified LPS-stimulated production of the proinflammatory cytokine IL-8 from PMNs and TNFα from macrophages. Furthermore, the intravenous injection of 6-OAU raised the blood CXCL1 level in rats, and the inoculation of 6-OAU into the rat air pouch accumulated PMNs and macrophages in the site. Our results indicate a proinflammatory role of GPR84, suggesting that the receptor may be a novel target to treat chronic low grade inflammation associated-disease. PMID:23449982

CYP2C19*17 increases clopidogrel-mediated platelet inhibition but does not alter the pharmacokinetics of the active metabolite of clopidogrel.

PubMed

Pedersen, Rasmus Steen; Nielsen, Flemming; Stage, Tore Bjerregaard; Vinholt, Pernille Just; el Achwah, Alaa Bilal; Damkier, Per; Brosen, Kim

2014-11-01

The aim of the present study was to determine the impact of CYP2C19*17 on the pharmacokinetics and pharmacodynamics of the active metabolite of clopidogrel and the pharmacokinetics of proguanil. Thus, we conducted an open-label two-phase cross-over study in 31 healthy male volunteers (11 CYP2C19*1/*1, 11 CYP2C19*1/*17 and nine CYP2C19*17/*17). In Phase A, the pharmacokinetics of the derivatized active metabolite of clopidogrel (CAMD) and platelet function were determined after administration of a single oral dose of 600 mg clopidogrel (Plavix; Sanofi-Avensis, Horsholm, Denmark). In Phase B, the pharmacokinetics of proguanil and its metabolites cycloguanil and 4-chlorphenylbiguanide (4-CPB) were determined in 29 of 31 subjects after a single oral dose of 200 mg proguanil given as the combination drug Malarone (GlaxoSmithKline Pharma, Brondby, Denmark). Significant correlations were found between the area under the time-concentration curve (AUC0-∞ ) of CAMD and both the absolute ADP-induced P2Y12 receptor-activated platelet aggregation (r = -0.60, P = 0.0007) and the percentage inhibition of aggregation (r = 0.59, P = 0.0009). In addition, the CYP2C19*17/*17 and CYP2C19*1/*17 genotype groups had significantly higher percentage inhibition of platelet aggregation compared with the CYP2C19*1/*1 subjects (geometric mean percentage inhibition of 84%, 73% and 63%, respectively; P = 0.014). Neither the absolute ADP-induced P2Y12 receptor-activated platelet aggregation, exposure to CAMD nor the pharmacokinetic parameters of proguanil, cycloguanil and 4-CPB exhibited any significant differences among the genotype groups. In conclusion, carriers of CYP2C19*17 exhibit higher percentage inhibition of platelet aggregation, but do not have significantly lower absolute P2Y12 receptor-activated platelet aggregation or higher exposure to the active metabolite after a single oral administration of 600 mg clopidogrel. © 2014 Wiley Publishing Asia Pty Ltd.

Fatty acid receptor GPR120: a novel marker for human melanoma.

PubMed

Kleemann, Johannes; Hrgovic, Igor; Ter-Nedden, Jan; Kleimann, Pia; Steinhorst, Katja; Härle, Katja; Müller, Jutta; Kaufmann, Roland; Meissner, Markus; Kippenberger, Stefan

2018-03-21

The correlation between ultraviolet radiation of the skin and melanoma incidence in humans is well established. Interestingly, epidemiologic data suggest also a correlation to an increased BMI pointing to metabolic trigger factors in melanoma pathogenesis. To substantiate this connection, we studied the expression of G-protein-coupled receptor 120 (GPR120), a receptor sensitive to unsaturated long-chain free fatty acids in melanoma tissues. One-hundred fourteen tissue sections histologically confirmed as nevi (n=32), primary melanoma (n=39), and melanoma metastasis (n=43) were immunohistochemically stained against GPR120. The staining was evaluated by three trained dermatopathologists and independently scored. Compared with nevi, primary melanoma and melanoma metastasis showed significantly higher levels of GPR120 staining. Only three out of 32 nevi showed strong GPR120 expression [median immunoreactivity-scoring system (IRS) score: 1, range: 0-10], whereas in primary melanomas 14 out of 39 were highly GPR120-positive (median IRS score: 7, range: 0-12) and in melanoma metastasis 27 out of 43 were highly GPR120-positive (median IRS score: 9, range: 0-12). GPR120 expression and tumor thickness (mm) show a statistically significant correlation in primary melanoma (P=0.011). Moreover, GPR120-positive staining was found throughout the epidermis and in sebaceous and sweat glands, which is yet not described. This study identified GPR120 as a novel marker for melanoma, indicating that melanoma cells are sensitive to free fatty acids. It is tempting to speculate that pharmacologically interfering with GPR120 signaling might improve melanoma therapy.

Functional analysis of free fatty acid receptor GPR120 in human eosinophils: implications in metabolic homeostasis.

PubMed

Konno, Yasunori; Ueki, Shigeharu; Takeda, Masahide; Kobayashi, Yoshiki; Tamaki, Mami; Moritoki, Yuki; Oyamada, Hajime; Itoga, Masamichi; Kayaba, Hiroyuki; Omokawa, Ayumi; Hirokawa, Makoto

2015-01-01

Recent evidence has shown that eosinophils play an important role in metabolic homeostasis through Th2 cytokine production. GPR120 (FFA4) is a G protein-coupled receptor (GPCR) for long-chain fatty acids that functions as a regulator of physiological energy metabolism. In the present study, we aimed to investigate whether human eosinophils express GPR120 and, if present, whether it possesses a functional capacity on eosinophils. Eosinophils isolated from peripheral venous blood expressed GPR120 at both the mRNA and protein levels. Stimulation with a synthetic GPR120 agonist, GW9508, induced rapid down-regulation of cell surface expression of GPR120, suggesting ligand-dependent receptor internalization. Although GPR120 activation did not induce eosinophil chemotactic response and degranulation, we found that GW9508 inhibited eosinophil spontaneous apoptosis and Fas receptor expression. The anti-apoptotic effect was attenuated by phosphoinositide 3-kinase (PI3K) inhibitors and was associated with inhibition of caspase-3 activity. Eosinophil response investigated using ELISpot assay indicated that stimulation with a GPR120 agonist induced IL-4 secretion. These findings demonstrate the novel functional properties of fatty acid sensor GPR120 on human eosinophils and indicate the previously unrecognized link between nutrient metabolism and the immune system.

The platelet P2Y(12) receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [(3)H]PSB-0413.

PubMed

Ohlmann, Philippe; Lecchi, Anna; El-Tayeb, Ali; Müller, Christa E; Cattaneo, Marco; Gachet, Christian

2013-03-01

Various radioligands have been used to characterize and quantify the platelet P2Y(12) receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y(1) and P2Y(12). We used the [(3)H]PSB-0413 selective P2Y(12) receptor antagonist radioligand to reevaluate the number of P2Y(12) receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [(3)H]PSB-0413 bound to 425 ± 50 sites/platelet (K (D) = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 6.5 ± 3.6 nM), and (3) competition studies confirmed the known features of P2Y(12), with the expected rank order of potency: AR-C69931MX > 2MeSADP ≫ ADPβS > ADP, while the P2Y(1) ligand MRS2179 and the P2X(1) ligand α,β-Met-ATP did not displace [(3)H]PSB-0413 binding. Patients with severe P2Y(12) deficiency displayed virtually no binding of [(3)H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y(12) receptor had normal binding. Studies in mice showed that: (1) [(3)H]PSB-0413 bound to 634 ± 87 sites/platelet (K (D) = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [(3)H]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 9.1 ± 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [(3)H]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y(12) receptors, to identify patients with P2Y(12) deficiencies or quantify the effect of P2Y(12) targeting drugs.

Regulating the effects of GPR21, a novel target for type 2 diabetes

NASA Astrophysics Data System (ADS)

Leonard, Siobhán; Kinsella, Gemma K.; Benetti, Elisa; Findlay, John B. C.

2016-05-01

Type 2 diabetes is a chronic metabolic disorder primarily caused by insulin resistance to which obesity is a major contributor. Expression levels of an orphan G protein-coupled receptor (GPCR), GPR21, demonstrated a trend towards a significant increase in the epididymal fat pads of wild type high fat high sugar (HFHS)-fed mice. To gain further insight into the potential role this novel target may play in the development of obesity-associated type 2 diabetes, the signalling capabilities of the receptor were investigated. Overexpression studies in HEK293T cells revealed GPR21 to be a constitutively active receptor, which couples to Gαq type G proteins leading to the activation of mitogen activated protein kinases (MAPKs). Overexpression of GPR21 in vitro also markedly attenuated insulin signalling. Interestingly, the effect of GPR21 on the MAPKs and insulin signalling was reduced in the presence of serum, inferring the possibility of a native inhibitory ligand. Homology modelling and ligand docking studies led to the identification of a novel compound that inhibited GPR21 activity. Its effects offer potential as an anti-diabetic pharmacological strategy as it was found to counteract the influence of GPR21 on the insulin signalling pathway.

SNARE-dependent upregulation of KCC2 activity following metabotropic zinc receptor (mZnR/GPR39) activation in rat cortical neurons in vitro

PubMed Central

Saadi, Robert A.; He, Kai; Hartnett, Karen A.; Kandler, Karl; Hershfinkel, Michal; Aizenman, Elias

2012-01-01

The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABAA receptor channels. In a recent study, we showed that Zn2+ regulates GABAA reversal potentials in the hippocampus by enhancing the activity of KCC2 via an increase in its surface expression. Zn2+ initiates this process by activating the Gq-coupled metabotropic Zn2+ receptor mZnR/GPR39. Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture and then tested the hypothesis that the increase in KCC2 activity is mediated through a SNARE-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca2+ indicator and exposing cells to Zn2+, which triggered consistent Ca2+ responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist MCPG. Importantly, Zn2+ treatment under these conditions did not increase the intracellular concentrations of Zn2+ itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH4+ influx via the co-transporter using an intracellular pH sensitive fluorescent indicator. We observed that Zn2+ pretreatment induced a Ca2+-dependent increase in KCC2 activity. The effects of Zn2+ on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39−/− mice, suggesting that Zn2+ acts via mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and SNAP-25. Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn2+-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn2+. These results suggest that SNARE proteins are necessary for the increased activity of KCC2 following Zn2+ stimulation of mZnR/GPR39. PMID:22441041

Expression and immunoaffinity purification of recombinant soluble human GPR56 protein for the analysis of GPR56 receptor shedding by ELISA.

PubMed

Yang, Tai-Yun; Chiang, Nien-Yi; Tseng, Wen-Yi; Pan, Hsiao-Lin; Peng, Yen-Ming; Shen, Jiann-Jong; Wu, Kuo-An; Kuo, Ming-Ling; Chang, Gin-Wen; Lin, Hsi-Hsien

2015-05-01

GPR56 is a multi-functional adhesion-class G protein-coupled receptor involved in biological systems as diverse as brain development, male gonad development, myoblast fusion, hematopoietic stem cell maintenance, tumor growth and metastasis, and immune-regulation. Ectodomain shedding of human GPR56 receptor has been demonstrated previously, however the quantitative detection of GPR56 receptor shedding has not been investigated fully due to the lack of appropriate assays. Herein, an efficient system of expression and immune-affinity purification of the recombinant soluble extracellular domain of human GPR56 (sGPR56) protein from a stably transduced human melanoma cell line was established. The identity and functionality of the recombinant human sGPR56 protein were verified by Western blotting and mass spectrometry, and ligand-binding assays, respectively. Combined with the use of two recently generated anti-GPR56 monoclonal antibodies, a sensitive sandwich ELISA assay was successfully developed for the quantitative detection of human sGPR56 molecule. We found that GPR56 receptor shedding occurred constitutively and was further increased in activated human melanoma cells expressing endogenous GPR56. In conclusion, we report herein an efficient system for the production and purification of human sGPR56 protein for the establishment of a quantitative ELISA analysis of GPR56 receptor shedding. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of free fatty acid receptors in the regulation of energy metabolism.

PubMed

Hara, Takafumi; Kashihara, Daiji; Ichimura, Atsuhiko; Kimura, Ikuo; Tsujimoto, Gozoh; Hirasawa, Akira

2014-09-01

Free fatty acids (FFAs) are energy-generating nutrients that act as signaling molecules in various cellular processes. Several orphan G protein-coupled receptors (GPCRs) that act as FFA receptors (FFARs) have been identified and play important physiological roles in various diseases. FFA ligands are obtained from food sources and metabolites produced during digestion and lipase degradation of triglyceride stores. FFARs can be grouped according to ligand profiles, depending on the length of carbon chains of the FFAs. Medium- and long-chain FFAs activate FFA1/GPR40 and FFA4/GPR120. Short-chain FFAs activate FFA2/GPR43 and FFA3/GPR41. However, only medium-chain FFAs, and not long-chain FFAs, activate GPR84 receptor. A number of pharmacological and physiological studies have shown that these receptors are expressed in various tissues and are primarily involved in energy metabolism. Because an impairment of these processes is a part of the pathology of obesity and type 2 diabetes, FFARs are considered as key therapeutic targets. Here, we reviewed recently published studies on the physiological functions of these receptors, primarily focusing on energy homeostasis. Copyright © 2014 Elsevier B.V. All rights reserved.

GPR30 Gene Polymorphisms Are Associated with Gynecomastia Risk in Adolescents.

PubMed

Korkmaz, Hüseyin Anıl; Edgünlü, Tuba; Eren, Erdal; Demir, Korcan; Çakir, Esra Deniz Papatya; Çelik, Sevim Karakaș; Özkan, Behzat

2015-01-01

The G protein-coupled receptor, GPR30, which is a third estrogen receptor, has been shown to mediate estrogenic effects on the essential features of human breast cancer cells. The aim of this study was to evaluate the association between GPR30 single nucleotide polymorphisms and gynecomastia in males. This study included 109 male adolescents with gynecomastia and 104 controls. Follicle stimulating hormone, luteinizing hormone, total testosterone, estradiol (E2), dehydroepiandrosterone sulfate (DHEAS), and prolactin levels were measured. DNA was extracted from whole blood using a GeneJET Genomic DNA purification kit. The genotypes of the GPR30 gene (rs3808350, rs3808351 and rs11544331) were studied using a tetra-primer ARMS (amplification refractory mutation system) PCR approach. The median E2 (11.80 vs. 16.86 IU/l, p < 0.001) and DHEAS levels (116.8 vs. 146.5 μg/dl, p = 0.044) were higher in the gynecomastia group. The G allele of rs3808350 and the A allele of rs3808351 were frequently observed in patients with gynecomastia. Gynecomastia was more common in patients with the GG genotype of rs3808350 and in patients with the AA genotype of rs3808351. Our results suggest that increased E2 levels, the G allele of rs3808350 and the A allele of rs3808351 might explain why certain adolescents are affected by gynecomastia. © 2014 S. Karger AG, Basel.

A novel loss-of-function mutation in GPR54/KISS1R leads to hypogonadotropic hypogonadism in a highly consanguineous family.

PubMed

Nimri, Revital; Lebenthal, Yael; Lazar, Liora; Chevrier, Lucie; Phillip, Moshe; Bar, Meytal; Hernandez-Mora, Eva; de Roux, Nicolas; Gat-Yablonski, Galia

2011-03-01

The G protein-coupled receptor 54 (GPR54), the kisspeptin receptor, is essential for stimulation of GnRH secretion and induction of puberty. Recently loss-of-function mutations of the GPR54 have been implicated as a cause of isolated idiopathic hypogonadotropic hypogonadism (IHH). The objective of the study was to identify the genetic cause of IHH in a consanguineous pedigree and to characterize the phenotypic features from infancy through early adulthood. In six patients with normosmic IHH belonging to two families of Israeli Muslim-Arab origin highly related to one another, DNA was analyzed for mutations in the GnRHR and GPR54 genes, with functional analysis of the mutation found. The five males underwent comprehensive endocrine evaluation and were under longitudinal follow-up; the one female presented in early adulthood. A new homozygous mutation (c.T815C) in GPR54 leading to a phenylalanine substitution by serine (p.F272S) was detected in all patients. Functional analysis showed an almost complete inhibition of kisspeptin-induced GPR54 signaling and a dramatic decrease of the mutated receptor expression at the cell surface. The males exhibited the same clinical features from infancy to adulthood, characterized by cryptorchidism, a relatively short penis, and no spontaneous pubertal development. The female patient presented at 18 yr with impuberism and primary amenorrhea. Repeated stimulation tests demonstrated complete gonadotropin deficiency throughout follow-up. A novel loss-of-function mutation (p.F272S) in the GPR54 gene is associated with familial normosmic IHH. Underdeveloped external genitalia and impuberism point to the major role of GPR54 in the activation of the gonadotropic axis from intrauterine life to adulthood.

Pharmacological profiles of cloned mammalian P2Y-receptor subtypes.

PubMed

von Kügelgen, Ivar

2006-06-01

Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signalling. P2X-receptors are ligand-gated ion channels, whereas P2Y-receptors belong to the superfamily of G-protein-coupled receptors (GPCRs). So far, the P2Y family is composed out of 8 human subtypes that have been cloned and functionally defined; species orthologues have been found in many vertebrates. P2Y1-, P2Y2-, P2Y4-, P2Y6-, and P2Y11-receptors all couple to stimulation of phospholipase C. The P2Y11-receptor mediates in addition a stimulation of adenylate cyclase. In contrast, activation of the P2Y12-, P2Y13-, and P2Y14-receptors causes an inhibition of adenylate cyclase activity. The expression of P2Y1-receptors is widespread. The receptor is involved in blood platelet aggregation, vasodilatation and neuromodulation. It is activated by ADP and ADP analogues including 2-methylthio-ADP (2-MeSADP). 2'-Deoxy-N6-methyladenosine-3',5'-bisphosphate (MRS2179) and 2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate (MRS2279) are potent and selective antagonists. P2Y2 transcripts are abundantly distributed. One important example for its functional role is the control of chloride ion fluxes in airway epithelia. The P2Y2-receptor is activated by UTP and ATP and blocked by suramin. The P2Y2-agonist diquafosol is used for the treatment of the dry eye disease. P2Y4-receptors are expressed in the placenta and in epithelia. The human P2Y4-receptor has a strong preference for UTP as agonist, whereas the rat P2Y4-receptor is activated about equally by UTP and ATP. The P2Y4-receptor is not blocked by suramin. The P2Y6-receptor has a widespread distribution including heart, blood vessels, and brain. The receptor prefers UDP as agonist and is selectively blocked by 1,2-di-(4-isothiocyanatophenyl)ethane (MRS2567). The P2Y11-receptor may play a role in the differentiation of immunocytes. The human P2Y11-receptor is activated by ATP as naturally occurring agonist and it is blocked by suramin and reactive blue 2 (RB2). The P2Y12-receptor plays a crucial role in platelet aggregation as well as in inhibition of neuronal cells. It is activated by ADP and very potently by 2-methylthio-ADP. Nucleotide antagonists including N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (=cangrelor; AR-C69931MX), the nucleoside analogue AZD6140, as well as active metabolites of the thienopyridine compounds clopidogrel and prasugrel block the receptor. These P2Y12-antagonists are used in pharmacotherapy to inhibit platelet aggregation. The P2Y13-receptor is expressed in immunocytes and neuronal cells and is again activated by ADP and 2-methylthio-ADP. The 2-chloro-5-nitro pyridoxal-phosphate analogue 6-(2'-chloro-5'-nitro-azophenyl)-pyridoxal-alpha5-phosphate (MRS2211) is a selective antagonist. mRNA encoding for the human P2Y14-receptor is found in many tissues. However, a physiological role of the receptor has not yet been established. UDP-glucose and related analogues act as agonists; antagonists are not known. Finally, UDP has been reported to act on receptors for cysteinyl leukotrienes as an additional agonist--indicating a dual agonist specificity of these receptors.

A selective antagonist reveals a potential role of G protein-coupled receptor 55 in platelet and endothelial cell function.

PubMed

Kargl, Julia; Brown, Andrew J; Andersen, Liisa; Dorn, Georg; Schicho, Rudolf; Waldhoer, Maria; Heinemann, Akos

2013-07-01

The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²⁺ release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB₁ or CB₂). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.

Modeling ligand recognition at the P2Y12 receptor in light of X-ray structural information

NASA Astrophysics Data System (ADS)

Paoletta, Silvia; Sabbadin, Davide; von Kügelgen, Ivar; Hinz, Sonja; Katritch, Vsevolod; Hoffmann, Kristina; Abdelrahman, Aliaa; Straßburger, Jens; Baqi, Younis; Zhao, Qiang; Stevens, Raymond C.; Moro, Stefano; Müller, Christa E.; Jacobson, Kenneth A.

2015-08-01

The G protein-coupled P2Y12 receptor (P2Y12R) is an important antithrombotic target and of great interest for pharmaceutical discovery. Its recently solved, highly divergent crystallographic structures in complex either with nucleotides (full or partial agonist) or with a nonnucleotide antagonist raise the question of which structure is more useful to understand ligand recognition. Therefore, we performed extensive molecular modeling studies based on these structures and mutagenesis, to predict the binding modes of major classes of P2Y12R ligands previously reported. Various nucleotide derivatives docked readily to the agonist-bound P2Y12R, but uncharged nucleotide-like antagonist ticagrelor required a hybrid receptor resembling the agonist-bound P2Y12R except for the top portion of TM6. Supervised molecular dynamics (SuMD) of ticagrelor binding indicated interactions with the extracellular regions of P2Y12R, defining possible meta-binding sites. Ureas, sulfonylureas, sulfonamides, anthraquinones and glutamic acid piperazines docked readily to the antagonist-bound P2Y12R. Docking dinucleotides at both agonist- and antagonist-bound structures suggested interactions with two P2Y12R pockets. Thus, our structure-based approach consistently rationalized the main structure-activity relationships within each ligand class, giving useful information for designing improved ligands.

G protein-coupled estrogen receptor 1 (GPER1)/GPR30 increases ERK1/2 activity through PDZ motif-dependent and -independent mechanisms.

PubMed

Gonzalez de Valdivia, Ernesto; Broselid, Stefan; Kahn, Robin; Olde, Björn; Leeb-Lundberg, L M Fredrik

2017-06-16

G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of G i/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that G i/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two G i/o -mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Two alternatively spliced GPR39 transcripts in seabream: molecular cloning, genomic organization, and regulation of gene expression by metabolic signals.

PubMed

Zhang, Yong; Liu, Yun; Huang, Xigui; Liu, Xiaochun; Jiao, Baowei; Meng, Zining; Zhu, Pei; Li, Shuisheng; Lin, Haoran; Cheng, Christopher H K

2008-12-01

Two GPR39 transcripts, designated as sbGPR39-1a and sbGPR39-1b, were identified in black seabream (Acanthopagrus schlegeli). The deduced amino acid (aa) sequence of sbGPR39-1a contains 423 residues with seven putative transmembrane (TM) domains. On the other hand, sbGPR39-1b contains 284 aa residues with only five putative TM domains. Northern blot analysis confirmed the presence of two GPR39 transcripts in the seabream intestine, stomach, and liver. Apart from seabream, the presence of two GPR39 transcripts was also found to exist in a number of teleosts (zebrafish and pufferfish) and mammals (human and mouse). Analysis of the GPR39 gene structure in different species suggests that the two GPR39 transcripts are generated by alternative splicing. When the seabream receptors were expressed in cultured HEK293 cells, Zn(2)(+) could trigger sbGPR39-1a signaling through the serum response element pathway, but no such functionality could be detected for the sbGPR39-1b receptor. The two receptors were found to be differentially expressed in seabream tissues. sbGPR39-1a is predominantly expressed in the gastrointestinal tract. On the other hand, sbGPR39-1b is widely expressed in most central and peripheral tissues except muscle and ovary. The expression of sbGPR39-1a in the intestine and the expression of sbGPR39-1b in the hypothalamus were decreased significantly during food deprivation in seabream. On the contrary, the expression of the GH secretagogue receptors (sbGHSR-1a and sbGHSR-1b) was significantly increased in the hypothalamus of the food-deprived seabream. The reciprocal regulatory patterns of expression of these two genes suggest that both of them are involved in controlling the physiological response of the organism during starvation.

GPR139, an Orphan Receptor Highly Enriched in the Habenula and Septum, Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.

PubMed

Liu, Changlu; Bonaventure, Pascal; Lee, Grace; Nepomuceno, Diane; Kuei, Chester; Wu, Jiejun; Li, Qingqin; Joseph, Victory; Sutton, Steven W; Eckert, William; Yao, Xiang; Yieh, Lynn; Dvorak, Curt; Carruthers, Nicholas; Coate, Heather; Yun, Sujin; Dugovic, Christine; Harrington, Anthony; Lovenberg, Timothy W

2015-11-01

GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-μM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Low concentration of BPA induces mice spermatocytes apoptosis via GPR30.

PubMed

Wang, Chaoliang; Zhang, Jianxiang; Li, Qi; Zhang, Tianbiao; Deng, Zishi; Lian, Jing; Jia, Donghui; Li, Rui; Zheng, Tao; Ding, Xiaoju; Yang, Fan; Ma, Chao; Wang, Rui; Zhang, Weixing; Guo Wen, Jian

2017-07-25

Bisphenol A (BPA) acts as xenoestrogen and has a great impact on disorders of human reproductive system. However, the mechanism through which BPA can affect human testicular function remains to be identified. GPR30 is a novel membrane estrogen receptor with high-affinity and low-capacity binding to estrogens. We demonstrated that estrogen receptor α (ERα), estrogen receptor β (ERβ) as well as GPR30 are expressed in mouse spermatocyte-derived GC-2 cells using Real-time PCR. We treated the cells with different doses of BPA and found that even low doses of BPA can inhibit GC-2 cell growth using MTT assay. To make sure which receptor is responsible for the biological function of BPA, we used ER down-regulator ICI and indicated that BPA could bind to GPR30. We also observed that BPA was able to induce Erk1/2 phosphorylation in GC-2 cells and proved that this process was mediated by GPR30-related EGFR-MAPK pathway using western blot. By Real-time PCR, we found that the expression of c-Fos was up-regulated and Cyclin D1 gene was down-regulated, in the presence of BPA and ICI. The results of MTT assay, comet assay and flow cytometry indicated that the activation of GPR30 induced by BPA inhibited the cell growth and induced cell apoptosis and ICI, GPR30 siRNA, EGFR inhibitor (AG), and MAPK (PD) inhibitor could partially reverse this effect. Immunohistochemistry on the testis of BPA -damaged mice showed that BPA induced spermatocyte apoptosis without affecting the seminiferous tubules and spermatocyte. In conclusion, BPA triggered spermatocyte apoptosis via GPR30.

Retrograde transport of the transmembrane estrogen receptor, G-protein-coupled-receptor-30 (GPR30/GPER) from the plasma membrane towards the nucleus.

PubMed

Cheng, Shi-Bin; Graeber, Carl T; Quinn, Jeffrey A; Filardo, Edward J

2011-08-01

G-protein-coupled receptor 30 (GPR30/GPER) belongs to the seven transmembrane receptor (7TMR) superfamily, the most common class of surface receptor with approximately 800 known members. GPER promotes estrogen binding and rapid signaling via membrane-associated enzymes resulting in increased cAMP and release of heparan bound epidermal growth factor (proHB-EGF) from breast cancer cells. However, GPER is predominately localized intracellularly in breast cancer cells with minor amounts of receptor on the cell surface, an observation that has caused some controversy regarding its potential role as a plasma membrane estrogen receptor. Using the widely employed approach of tracking recombinant 7TMRs by surface labeling live cells, we have begun to characterize and compare the endocytic fate of GPER to other similarly labeled 7TMRs. Upon ectopic expression in human embryonic kidney HEK-293 cells, functional GPER is generated as these cells acquire the capacity to stimulate cAMP and activate cyclic AMP responsive binding protein in response to estradiol-17 beta stimulation. GPER is detectable on the cell surface by immunofluorescent analysis using HA-specific antibodies, albeit the bulk of the receptor is located intracellularly. Like β1AR (beta 1 adrenergic receptor) and CXCR4 (C-X-C chemokine receptor 4), GPER exits the plasma membrane via clathrin-coated pits and enters early endosomes. Interestingly, GPER has a destination that is uncommon among 7TMRs, as it accumulates in a perinuclear compartment. Like many 7TMRs (approximately one-third), GPER trafficking from the plasma membrane is constitutive (occurs in the absence of agonist). However, its route of intracellular trafficking is highly unusual, as 7TMRs typically recycle to the plasma membrane (e.g. β1AR) or are degraded in lysosomes (e.g. CXCR4). The accumulation of GPER in the perinuclear space and its possible significance for attenuating estrogen action via this newly recognized membrane estrogen receptor is discussed herein. Published by Elsevier Inc.

Cell adhesion controlled by adhesion G protein-coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo-Dock.

PubMed

Hernández-Vásquez, Magda Nohemí; Adame-García, Sendi Rafael; Hamoud, Noumeira; Chidiac, Rony; Reyes-Cruz, Guadalupe; Gratton, Jean Philippe; Côté, Jean-François; Vázquez-Prado, José

2017-07-21

Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Proresolving lipid mediators resolvin D1, resolvin D2, and maresin 1 are critical in modulating T cell responses.

PubMed

Chiurchiù, Valerio; Leuti, Alessandro; Dalli, Jesmond; Jacobsson, Anders; Battistini, Luca; Maccarrone, Mauro; Serhan, Charles N

2016-08-24

Resolution of inflammation is a finely regulated process mediated by specialized proresolving lipid mediators (SPMs), including docosahexaenoic acid (DHA)-derived resolvins and maresins. The immunomodulatory role of SPMs in adaptive immune cells is of interest. We report that D-series resolvins (resolvin D1 and resolvin D2) and maresin 1 modulate adaptive immune responses in human peripheral blood lymphocytes. These lipid mediators reduce cytokine production by activated CD8(+) T cells and CD4(+) T helper 1 (TH1) and TH17 cells but do not modulate T cell inhibitory receptors or abrogate their capacity to proliferate. Moreover, these SPMs prevented naïve CD4(+) T cell differentiation into TH1 and TH17 by down-regulating their signature transcription factors, T-bet and Rorc, in a mechanism mediated by the GPR32 and ALX/FPR2 receptors; they concomitantly enhanced de novo generation and function of Foxp3(+) regulatory T (Treg) cells via the GPR32 receptor. These results were also supported in vivo in a mouse deficient for DHA synthesis (Elovl2(-/-)) that showed an increase in TH1/TH17 cells and a decrease in Treg cells compared to wild-type mice. Additionally, either DHA supplementation in Elovl2(-/-) mice or in vivo administration of resolvin D1 significantly reduced cytokine production upon specific stimulation of T cells. These findings demonstrate actions of specific SPMs on adaptive immunity and provide a new avenue for SPM-based approaches to modulate chronic inflammation. Copyright © 2016, American Association for the Advancement of Science.

The G protein-coupled receptor GPR34 - The past 20 years of a grownup.

PubMed

Schöneberg, Torsten; Meister, Jaroslawna; Knierim, Alexander Bernd; Schulz, Angela

2018-04-22

Research on GPR34, which was discovered in 1999 as an orphan G protein-coupled receptor of the rhodopsin-like class, disclosed its physiologic relevance only piece by piece. Being present in all recent vertebrate genomes analyzed so far it seems to improve the fitness of species although it is not essential for life and reproduction as GPR34-deficient mice demonstrate. However, closer inspection of macrophages and microglia, where it is mainly expressed, revealed its relevance in immune cell function. Recent data clearly demonstrate that GPR34 function is required to arrest microglia in the M0 homeostatic non-phagocytic phenotype. Herein, we summarize the current knowledge on its evolution, genomic and structural organization, physiology, pharmacology and relevance in human diseases including neurodegenerative diseases and cancer, which accumulated over the last 20 years. Copyright © 2018 Elsevier Inc. All rights reserved.

HOMEOSTATIC REGULATION OF KCC2 ACTIVITY BY THE ZINC RECEPTOR mZnR/GPR39 DURING SEIZURES

PubMed Central

Gilad, David; Shorer, Sharon; Ketzef, Maya; Friedman, Alon; Sekler, Israel; Aizenman, Elias; Hershfinkel, Michal

2015-01-01

The aim of this study was to investigate the role of the synaptic metabotropic zinc receptor mZnR/GPR39 in physiological adaptation to epileptic seizures. We previously demonstrated that synaptic activation of mZnR/GPR39 enhances inhibitory drive in the hippocampus by upregulating neuronal K+/Cl− co-transporter 2 (KCC2) activity. Here, we first show that mZnR/GPR39 knockout (KO) adult mice have dramatically enhanced susceptibility to seizures triggered by a single intraperitoneal injection of kainic acid, when compared to wild type (WT) littermates. Kainate also substantially enhances seizure-associated gamma oscillatory activity in juvenile mZnR/GPR39 KO hippocampal slices, a phenomenon that can be reproduced in WT tissue by extracellular Zn2+ chelation. Importantly, kainate-induced synaptic Zn2+ release enhances surface expression and transport activity of KCC2 in WT, but not mZnR/GPR39 KO hippocampal neurons. Kainate-dependent upregulation of KCC2 requires mZnR/GPR39 activation of the Gαq/phospholipase C/extracellular regulated kinase (ERK1/2) signaling cascade. We suggest that mZnR/GPR39-dependent upregulation of KCC2 activity provides homeostatic adaptation to an excitotoxic stimulus by increasing inhibition. As such, mZnR/GPR39 may provide a novel pharmacological target for dampening epileptic seizure activity. PMID:25562657

Diindolylmethane Derivatives: Potent Agonists of the Immunostimulatory Orphan G Protein-Coupled Receptor GPR84.

PubMed

Pillaiyar, Thanigaimalai; Köse, Meryem; Sylvester, Katharina; Weighardt, Heike; Thimm, Dominik; Borges, Gleice; Förster, Irmgard; von Kügelgen, Ivar; Müller, Christa E

2017-05-11

The G i protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently, 3,3'-diindolylmethane was identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and β-arrestin assays. Structure-activity relationships (SARs) were steep. 3,3'-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane (38, PSB-15160, EC 50 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC 50 41.3 nM). In β-arrestin assays, SARs were different, indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further investigated and found to display an ago-allosteric mechanism of action and increased stability in comparison to the lead structure.

GPR3 Receptor, a Novel Actor in the Emotional-Like Responses

PubMed Central

Valverde, Olga; Célérier, Evelyne; Baranyi, Mária; Vanderhaeghen, Pierre; Maldonado, Rafael; Sperlagh, Beata; Vassart, Gilbert; Ledent, Catherine

2009-01-01

GPR3 is an orphan G protein-coupled receptor endowed with constitutive Gs signaling activity, which is expressed broadly in the central nervous system, with maximal expression in the habenula. We investigated the consequences of its genetic deletion in several behavioral paradigms and on neurotransmission. Compared to wild-type, hippocampal neurons from Gpr3−/− mice displayed lower basal intracellular cAMP levels, consistent with the strong constitutive activity of GPR3 in transiently transfected cells. Behavioral analyses revealed that Gpr3−/− mice exhibited a high level of avoidance of novel and unfamiliar environment, associated with increased stress reactivity in behavioral despair paradigms and aggressive behavior in the resident-intruder test. On the contrary, no deficit was found in the learning ability to avoid an aversive event in active avoidance task. The reduced ability of Gpr3 −/− mice to cope with stress was unrelated to dysfunction of the hypothalamic-pituitary-adrenal axis, with Gpr3−/− mice showing normal corticosterone production under basal or stressful conditions. In contrast, dramatic alterations of monoamine contents were found in hippocampus, hypothalamus and frontal cortex of Gpr3−/− mice. Our results establish a link between tonic stimulation of the cAMP signaling pathway by GPR3 and control of neurotransmission by monoamines throughout the forebrain. GPR3 qualifies as a new player in the modulation of behavioral responses to stress and constitutes a novel promising pharmacological target for treatment of emotional disorders. PMID:19259266

G-protein-coupled receptor 91 and succinate are key contributors in neonatal postcerebral hypoxia-ischemia recovery.

PubMed

Hamel, David; Sanchez, Melanie; Duhamel, François; Roy, Olivier; Honoré, Jean-Claude; Noueihed, Baraa; Zhou, Tianwei; Nadeau-Vallée, Mathieu; Hou, Xin; Lavoie, Jean-Claude; Mitchell, Grant; Mamer, Orval A; Chemtob, Sylvain

2014-02-01

Prompt post-hypoxia-ischemia (HI) revascularization has been suggested to improve outcome in adults and newborn subjects. Other than hypoxia-inducible factor, sensors of metabolic demand remain largely unknown. During HI, anaerobic respiration is arrested resulting in accumulation of carbohydrate metabolic intermediates. As such succinate readily increases, exerting its biological effects via a specific receptor, G-protein-coupled receptor (GPR) 91. We postulate that succinate/GPR91 enhances post-HI vascularization and reduces infarct size in a model of newborn HI brain injury. The Rice-Vannucci model of neonatal HI was used. Succinate was measured by mass spectrometry, and microvascular density was evaluated by quantification of lectin-stained cryosection. Gene expression was evaluated by real-time polymerase chain reaction. Succinate levels rapidly increased in the penumbral region of brain infarcts. GPR91 was foremost localized not only in neurons but also in astrocytes. Microvascular density increased at 96 hours after injury in wild-type animals; it was diminished in GPR91-null mice leading to an increased infarct size. Stimulation with succinate led to an increase in growth factors implicated in angiogenesis only in wild-type mice. To explain the mode of action of succinate/GPR91, we investigated the role of prostaglandin E2-prostaglandin E receptor 4, previously proposed in neural angiogenesis. Succinate-induced vascular endothelial growth factor expression was abrogated by a cyclooxygenase inhibitor and a selective prostaglandin E receptor 4 antagonist. This antagonist also abolished succinate-induced neovascularization. We uncover a dominant metabolic sensor responsible for post-HI neurovascular adaptation, notably succinate/GPR91, acting via prostaglandin E2-prostaglandin E receptor 4 to govern expression of major angiogenic factors. We propose that pharmacological intervention targeting GPR91 could improve post-HI brain recovery.

Expression and function of the metabotropic purinergic P2Y receptor family in experimental seizure models and patients with drug-refractory epilepsy.

PubMed

Alves, Mariana; Gomez-Villafuertes, Rosa; Delanty, Norman; Farrell, Michael A; O'Brien, Donncha F; Miras-Portugal, Maria Teresa; Hernandez, Miguel Diaz; Henshall, David C; Engel, Tobias

2017-09-01

ATP is released into the extracellular space during pathologic processes including increased neuronal firing. Once released, ATP acts on P2 receptors including ionotropic P2X and metabotropic P2Y receptors, resulting in changes to glial function and neuronal network excitability. Evidence suggests an involvement of P2Y receptors in the pathogenesis of epilepsy, but there has been no systematic effort to characterize the expression and function of the P2Y receptor family during seizures and in experimental and human epilepsy. Status epilepticus was induced using either intra-amygdala kainic acid or pilocarpine to characterize the acute- and long-term changes in hippocampal P2Y expression. P2Y expression was also investigated in brain tissue from patients with temporal lobe epilepsy. Finally, we analyzed the effects of two specific P2Y agonists, ADP and UTP, on seizure severity and seizure-induced cell death. Both intra-amygdala kainic acid and pilocarpine-induced status epilepticus increased the transcription of the uracil-sensitive P2Y receptors P2ry 2 , P2ry 4 , and P2ry 6 and decreased the transcription of the adenine-sensitive P2Y receptors P2ry 1 , P2ry 12 , P2ry 13 . Protein levels of P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 6 were increased after status epilepticus, whereas P2Y 12 expression was decreased. In the chronic phase, P2ry 1 , P2ry 2 , and P2ry 6 transcription and P2Y 1 , P2Y 2 , and P2Y 12 protein levels were increased with no changes for the other P2Y receptors. In hippocampal samples from patients with temporal lobe epilepsy, P2Y 1 and P2Y 2 protein expression was increased, whereas P2Y 13 levels were lower. Demonstrating a functional contribution of P2Y receptors to seizures, central injection of ADP exacerbated seizure severity, whereas treatment with UTP decreased seizure severity during status epilepticus in mice. The present study is the first to establish the specific hippocampal expression profile and function of the P2Y receptor family after experimental status epilepticus and in human temporal lobe epilepsy and offers potential new targets for seizure control and disease modification. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

Aromatic D-amino acids act as chemoattractant factors for human leukocytes through a G protein-coupled receptor, GPR109B.

PubMed

Irukayama-Tomobe, Yoko; Tanaka, Hirokazu; Yokomizo, Takehiko; Hashidate-Yoshida, Tomomi; Yanagisawa, Masashi; Sakurai, Takeshi

2009-03-10

GPR109B (HM74) is a putative G protein-coupled receptor (GPCR) whose cognate ligands have yet to be characterized. GPR109B shows a high degree of sequence similarity to GPR109A, another GPCR that was identified as a high-affinity nicotinic acid (niacin) receptor. However, the affinity of nicotinic acid to GPR109B is very low. In this study, we found that certain aromatic D-amino acids, including D-phenylalanine, D-tryptophan, and the metabolite of the latter, D-kynurenine, decreased the activity of adenylate cyclase in cells transfected with GPR109B cDNA through activation of pertussis toxin (PTX)-sensitive G proteins. These D-amino acids also elicited a transient rise of intracellular Ca(2+) level in cells expressing GPR109B in a PTX-sensitive manner. In contrast, these D-amino acids did not show any effects on cells expressing GPR109A. We found that the GPR109B mRNA is abundantly expressed in human neutrophils. D-phenylalanine and D-tryptophan induced a transient increase of intracellular Ca(2+) level and a reduction of cAMP levels in human neutrophils. Furthermore, knockdown of GPR109B by RNA interference inhibited the D-amino acids-induced decrease of cellular cAMP levels in human neutrophils. These D-amino acids induced chemotactic activity of freshly prepared human neutrophils. We also found that D-phenylalanine and D-tryptophan induced chemotactic responses in Jurkat cells transfected with the GPR109B cDNA but not in mock-transfected Jurkat cells. These results suggest that these aromatic D-amino acids elicit a chemotactic response in human neutrophils via activation of GPR109B.

GPR41/FFAR3 and GPR43/FFAR2 as cosensors for short-chain fatty acids in enteroendocrine cells vs FFAR3 in enteric neurons and FFAR2 in enteric leukocytes.

PubMed

Nøhr, Mark K; Pedersen, Maria H; Gille, Andreas; Egerod, Kristoffer L; Engelstoft, Maja S; Husted, Anna Sofie; Sichlau, Rasmus M; Grunddal, Kaare V; Poulsen, Steen Seier; Han, Sangdon; Jones, Robert M; Offermanns, Stefan; Schwartz, Thue W

2013-10-01

The expression of short-chain fatty acid receptors GPR41/FFAR3 and GPR43/ free fatty acid receptor 2 (FFAR2) was studied in the gastrointestinal tract of transgenic monomeric red fluorescent protein (mRFP) reporter mice. In the stomach free fatty acid receptor 3 (FFAR3)-mRFP was expressed in a subpopulation of ghrelin and gastrin cells. In contrast, strong expression of FFAR3-mRFP was observed in all cholecystokinin, glucose-dependent insulinotropic peptide (GIP), and secretin cells of the proximal small intestine and in all glucagon-like peptide-1 (GLP-1), peptide YY, and neurotensin cells of the distal small intestine. Throughout the colon and rectum, FFAR3-mRFP was strongly expressed in the large population of peptide YY and GLP-1 cells and in the neurotensin cells of the proximal colon. A gradient of expression of FFAR3-mRFP was observed in the somatostatin cells from less than 5% in the stomach to more than 95% in the rectum. Substance P-containing enterochromaffin cells displayed a similar gradient of FFAR3-mRFP expression throughout the small intestine. Surprisingly, FFAR3-mRFP was also expressed in the neuronal cells of the submucosal and myenteric ganglia. Quantitative PCR analysis of fluorescence-activated cell sorting (FACS) purified FFAR3-mRFP positive cells confirmed the coexpression with the various peptide hormones as well as key neuronal marker proteins. The FFAR2-mRFP reporter was strongly expressed in a large population of leukocytes in the lamina propria of in particular the small intestine but surprisingly only weakly in a subpopulation of enteroendocrine cells. Nevertheless, synthetic ligands specific for either FFAR3 or FFAR2 each released GLP-1 from colonic crypt cultures and the FFAR2 agonist mobilized intracellular Ca²⁺ in FFAR2 positive enteroendocrine cells. It is concluded that FFAR3-mRFP serves as a useful marker for the majority of enteroendocrine cells of the small and large intestine and that FFAR3 and FFAR2 both act as sensors for short-chain fatty acids in enteroendocrine cells, whereas FFAR3 apparently has this role alone in enteric neurons and FFAR2 in enteric leukocytes.

GPER/GPR30, a membrane estrogen receptor, is expressed in the brain and retina of a social fish (Carassius auratus) and colocalizes with isotocin.

PubMed

Mangiamele, Lisa A; Gomez, Julia R; Curtis, Nancy J; Thompson, Richmond R

2017-02-01

Estradiol rapidly (within 30 minutes) influences a variety of sociosexual behaviors in both mammalian and nonmammalian vertebrates, including goldfish, in which it rapidly stimulates approach responses to the visual cues of females. Such rapid neuromodulatory effects are likely mediated via membrane-associated estrogen receptors; however, the localization and distribution of such receptors within the nervous system is not well described. To begin to address this gap, we identified GPER/GPR30, a G-protein-coupled estrogen receptor, in goldfish (Carassius auratus) neural tissue and used reverse-transcription polymerase chain reaction (RT-PCR) and in situ hybridization to test if GPR30 is expressed in the brain regions that might mediate visually guided social behaviors in males. We then used immunohistochemistry to determine whether GPR30 colocalizes with isotocin-producing cells in the preoptic area, a critical node in the highly conserved vertebrate social behavior network. We used quantitative (q)PCR to test whether GPR30 mRNA levels differ in males in breeding vs. nonbreeding condition and in males that were socially interacting with a female vs. a rival male. Our results show that GPR30 is expressed in the retina and in many brain regions that receive input from the retina and/or optic tectum, as well as in a few nodes in the social behavior network, including cell populations that produce isotocin. J. Comp. Neurol. 525:252-270, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

PubMed Central

ABBRACCHIO, MARIA P.; BURNSTOCK, GEOFFREY; BOEYNAEMS, JEAN-MARIE; BARNARD, ERIC A.; BOYER, JOSÉ L.; KENNEDY, CHARLES; KNIGHT, GILLIAN E.; FUMAGALLI, MARTA; GACHET, CHRISTIAN; JACOBSON, KENNETH A.; WEISMAN, GARY A.

2012-01-01

There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes have been cloned and characterized and most orphan receptors deorphanized, so that it is now possible to provide a basis for a future subdivision of P2Y receptor subtypes. More is known about the functional elements of the P2Y receptor molecules and the signaling pathways involved, including interactions with ion channels. There have been substantial developments in the design of selective agonists and antagonists to some of the P2Y receptor subtypes. There are new findings about the mechanisms underlying nucleotide release and ectoenzymatic nucleotide breakdown. Interactions between P2Y receptors and receptors to other signaling molecules have been explored as well as P2Y-mediated control of gene transcription. The distribution and roles of P2Y receptor subtypes in many different cell types are better understood and P2Y receptor-related compounds are being explored for therapeutic purposes. These and other advances are discussed in the present review. PMID:16968944

Genome-wide association analysis in primary sclerosing cholangitis and ulcerative colitis identifies risk loci at GPR35 and TCF4.

PubMed

Ellinghaus, David; Folseraas, Trine; Holm, Kristian; Ellinghaus, Eva; Melum, Espen; Balschun, Tobias; Laerdahl, Jon K; Shiryaev, Alexey; Gotthardt, Daniel N; Weismüller, Tobias J; Schramm, Christoph; Wittig, Michael; Bergquist, Annika; Björnsson, Einar; Marschall, Hanns-Ulrich; Vatn, Morten; Teufel, Andreas; Rust, Christian; Gieger, Christian; Wichmann, H-Erich; Runz, Heiko; Sterneck, Martina; Rupp, Christian; Braun, Felix; Weersma, Rinse K; Wijmenga, Cisca; Ponsioen, Cyriel Y; Mathew, Christopher G; Rutgeerts, Paul; Vermeire, Séverine; Schrumpf, Erik; Hov, Johannes R; Manns, Michael P; Boberg, Kirsten M; Schreiber, Stefan; Franke, Andre; Karlsen, Tom H

2013-09-01

Approximately 60%-80% of patients with primary sclerosing cholangitis (PSC) have concurrent ulcerative colitis (UC). Previous genome-wide association studies (GWAS) in PSC have detected a number of susceptibility loci that also show associations in UC and other immune-mediated diseases. We aimed to systematically compare genetic associations in PSC with genotype data in UC patients with the aim of detecting new susceptibility loci for PSC. We performed combined analyses of GWAS for PSC and UC comprising 392 PSC cases, 987 UC cases, and 2,977 controls and followed up top association signals in an additional 1,012 PSC cases, 4,444 UC cases, and 11,659 controls. We discovered novel genome-wide significant associations with PSC at 2q37 [rs3749171 at G-protein-coupled receptor 35 (GPR35); P = 3.0 × 10(-9) in the overall study population, combined odds ratio [OR] and 95% confidence interval [CI] of 1.39 (1.24-1.55)] and at 18q21 [rs1452787 at transcription factor 4 (TCF4); P = 2.61 × 10(-8) , OR (95% CI) = 0.75 (0.68-0.83)]. In addition, several suggestive PSC associations were detected. The GPR35 rs3749171 is a missense single nucleotide polymorphism resulting in a shift from threonine to methionine. Structural modeling showed that rs3749171 is located in the third transmembrane helix of GPR35 and could possibly alter efficiency of signaling through the GPR35 receptor. By refining the analysis of a PSC GWAS by parallel assessments in a UC GWAS, we were able to detect two novel risk loci at genome-wide significance levels. GPR35 shows associations in both UC and PSC, whereas TCF4 represents a PSC risk locus not associated with UC. Both loci may represent previously unexplored aspects of PSC pathogenesis. Copyright © 2012 American Association for the Study of Liver Diseases.

Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer.

PubMed

Aihara, Masamune; Yamamoto, Shigeru; Nishioka, Hiroko; Inoue, Yutaro; Hamano, Kimikazu; Oka, Masaaki; Mizukami, Yoichi

2012-06-15

G protein-coupled receptor 30/G protein estrogen receptor-1 (GPR30/GPER-1) is a novel membrane receptor for estrogen whose mRNA is expressed at high levels in estrogen-dependent cells such as breast cancer cell lines. However, mutations in GRP30 related to diseases remain unreported. To detect unknown mutations in the GPR30 open reading frame (ORF) quickly, the experimental conditions for high-resolution melting (HRM) analysis were examined for PCR primers, Taq polymerases, saturation DNA binding dyes, Mg(2+) concentration, and normalized temperatures. Nine known SNPs and 13 artificial point mutations within the GPR30 ORF, as well as single nucleotide variants in DNA extracted from subjects with breast cancers were tested under the optimal experimental conditions. The combination of Expand High Fidelity(PLUS) and SYTO9 in the presence of 2.0 mM MgCl(2) produced the best separation in melting curves of mutations in all regions of the GPR30 ORF. Under these experimental conditions, the mutations were clearly detected in both heterozygotes and homozygotes. HRM analysis of GPR30 using genomic DNA from subjects with breast cancers showed a novel single nucleotide variant, 111C>T in GPR30 and 4 known SNPs. The experimental conditions determined in this study for HRM analysis are useful for high throughput assays to detect unknown mutations within the GPR30 ORF. Copyright © 2012 Elsevier B.V. All rights reserved.

G protein-coupled receptor 30 expression is up-regulated by EGF and TGF alpha in estrogen receptor alpha-positive cancer cells.

PubMed

Vivacqua, Adele; Lappano, Rosamaria; De Marco, Paola; Sisci, Diego; Aquila, Saveria; De Amicis, Francesca; Fuqua, Suzanne A W; Andò, Sebastiano; Maggiolini, Marcello

2009-11-01

In the present study, we evaluated the regulation of G protein-coupled receptor (GPR)30 expression in estrogen receptor (ER)-positive endometrial, ovarian, and estrogen-sensitive, as well as tamoxifen-resistant breast cancer cells. We demonstrate that epidermal growth factor (EGF) and TGF alpha transactivate the GPR30 promoter and accordingly up-regulate GPR30 mRNA and protein levels only in endometrial and tamoxifen-resistant breast cancer cells. These effects exerted by EGF and TGF alpha were dependent on EGF receptor (EGFR) expression and activation and involved phosphorylation of the Tyr(1045) and Tyr(1173) EGFR sites. Using gene-silencing experiments and specific pharmacological inhibitors, we have ascertained that EGF and TGF alpha induce GPR30 expression through the EGFR/ERK transduction pathway, and the recruitment of c-fos to the activator protein-1 site located within GPR30 promoter sequence. Interestingly, we show that functional cross talk of GPR30 with both activated EGFR and ER alpha relies on a physical interaction among these receptors, further extending the potential of estrogen to trigger a complex stimulatory signaling network in hormone-sensitive tumors. Given that EGFR/HER2 overexpression is associated with tamoxifen resistance, our data may suggest that ligand-activated EGFR could contribute to the failure of tamoxifen therapy also by up-regulating GPR30, which in turn could facilitates the action of estrogen. In addition, important for resistance is the ability of tamoxifen to bind to and activate GPR30, the expression of which is up-regulated by EGFR activation. Our results emphasize the need for new endocrine agents able to block widespread actions of estrogen without exerting any stimulatory activity on transduction pathways shared by the steroid and growth factor-signaling networks.

The G-protein coupled receptor, GPR84 regulates IL-4 production by T lymphocytes in response to CD3 crosslinking.

PubMed

Venkataraman, Chandrasekar; Kuo, Frederick

2005-11-15

The orphan G-protein coupled receptor, GPR84 is highly expressed in the bone marrow, and in splenic T cells and B cells. In this study, GPR84-deficient mice were generated to understand the biological function of this orphan receptor. The proliferation of T and B cells in response to various mitogens was normal in GPR84-deficient mice. Interestingly, primary stimulation of T cells with anti-CD3 resulted in increased IL-4 but not IL-2 or IFN-gamma production in GPR84(-/-) mice compared to wild-type mice. Augmented IL-4 production in GPR84-deficient T cells was not related to increased frequency of IL-4-secreting cells in response to anti-CD3 stimulation. In fact, stimulation with anti-CD3 and anti-CD28 resulted in increased levels of IL-4 but not IFN-gamma steady-state mRNA in GPR84(-/-) T cells. In addition, Th2 effector cells generated in vitro from GPR84(-/-) mice produced higher levels of IL-4, IL-5 and IL-13 compared to wild-type mice. However, there was no detectable difference in the extent of IL-4 and IL-5 production between the two groups of mice in response to antigen stimulation of spleen cells, isolated from mice previously immunized with OVA in alum. These studies reveal a novel role for GPR84 in regulating early IL-4 gene expression in activated T cells.

An Update on Non-CB1, Non-CB2 Cannabinoid Related G-Protein-Coupled Receptors

PubMed Central

Morales, Paula; Reggio, Patricia H.

2017-01-01

Abstract The endocannabinoid system (ECS) has been shown to be of great importance in the regulation of numerous physiological and pathological processes. To date, two Class A G-protein-coupled receptors (GPCRs) have been discovered and validated as the main therapeutic targets of this system: the cannabinoid receptor type 1 (CB1), which is the most abundant neuromodulatory receptor in the brain, and the cannabinoid receptor type 2 (CB2), predominantly found in the immune system among other organs and tissues. Endogenous cannabinoid receptor ligands (endocannabinoids) and the enzymes involved in their synthesis, cell uptake, and degradation have also been identified as part of the ECS. However, its complex pharmacology suggests that other GPCRs may also play physiologically relevant roles in this therapeutically promising system. In the last years, GPCRs such as GPR18 and GPR55 have emerged as possible missing members of the cannabinoid family. This categorization still stimulates strong debate due to the lack of pharmacological tools to validate it. Because of their close phylogenetic relationship, the Class A orphan GPCRs, GPR3, GPR6, and GPR12, have also been associated with the cannabinoids. Moreover, certain endo-, phyto-, and synthetic cannabinoid ligands have displayed activity at other well-established GPCRs, including the opioid, adenosine, serotonin, and dopamine receptor families. In addition, the cannabinoid receptors have also been shown to form dimers with other GPCRs triggering cross-talk signaling under specific conditions. In this mini review, we aim to provide insight into the non-CB1, non-CB2 cannabinoid-related GPCRs that have been reported thus far. We consider the physiological relevance of these molecular targets in modulating the ECS. PMID:29098189

Agonists for G-protein-coupled receptor 84 (GPR84) alter cellular morphology and motility but do not induce pro-inflammatory responses in microglia.

PubMed

Wei, Li; Tokizane, Kyohei; Konishi, Hiroyuki; Yu, Hua-Rong; Kiyama, Hiroshi

2017-10-03

Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood. We used endogenous, natural, and surrogate agonists for GPR84 (capric acid, embelin, and 6-OAU, respectively) and examined their effect on mouse primary cultured microglia in vitro. 6-n-Octylaminouracil (6-OAU), embelin, and capric acid rapidly induced membrane ruffling and motility in cultured microglia obtained from C57BL/6 mice, although these agonists failed to promote microglial pro-inflammatory cytokine expression. Concomitantly, 6-OAU suppressed forskolin-induced increase of cAMP in cultured microglia. Pertussis toxin, an inhibitor of Gi-coupled signaling, completely suppressed 6-OAU-induced microglial membrane ruffling and motility. In contrast, no 6-OAU-induced microglial membrane ruffling and motility was observed in microglia from DBA/2 mice, a mouse strain that does not express functional GPR84 protein due to endogenous nonsense mutation of the GPR84 gene. GPR84 mediated signaling causes microglial motility and membrane ruffling but does not promote pro-inflammatory responses. As GPR84 is a known receptor for medium-chain fatty acids, those released from damaged brain cells may be involved in the enhancement of microglial motility through GPR84 after neuronal injury.

Adenosine triphosphate induces P2Y2 activation and interleukin-8 release in human esophageal epithelial cells.

PubMed

Wu, Liping; Oshima, Tadayuki; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

2017-07-01

Immune-mediated mucosal inflammation characterized by the release of interleukin (IL)-8 is associated with gastroesophageal reflux disease. ATP released by human esophageal epithelial cells (HEECs) mediates the release of cytokines through P2 nucleotide receptors that are present on various cells, including HEECs. This study characterized and identified human esophageal epithelial P2 receptors that are responsible for ATP-mediated release of IL-8 by using a human esophageal stratified squamous epithelial model. Primary HEECs were cultured with the use of an air-liquid interface (ALI) system. The ATP analogue adenosine 5'-O-3-thiotriphosphate (ATP-γ-S) was added to the basolateral compartment, and IL-8 release was measured. Involvement of the P2Y2 receptor was assessed with the use of selective and non-selective receptor antagonists and a P2Y2 receptor agonist. Expression of the P2Y2 receptor was assessed using western blotting and immunohistochemistry. Adenosine triphosphate-γ-S induced IL-8 release through the P2Y2 receptor. A P2Y2 receptor antagonist but not a P2X3 receptor antagonist or a P2Y1 receptor antagonist blocked ATP-γ-S-mediated IL-8 release. Conversely, a P2Y2 receptor agonist induced IL-8 release. Western blotting and immunohistochemistry of the P2Y2 receptor showed strong expression of the P2Y2 receptor on ALI-cultured HEECs and in human esophagus. Inhibition of extracellular signal-regulated kinase but not of protein kinase C blocked the ATP-mediated release of IL-8. ATP-γ-S induced phosphorylation of extracellular signal-regulated kinase, and a P2Y2 receptor antagonist blocked this phosphorylation. Interleukin-8 release after purinergic stimulation in ALI-cultured HEECs is mediated through P2Y2 receptor activation. ATP-induced IL-8 release maybe involved in the pathogenesis of refractory gastroesophageal reflux disease. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

In vivo functions of GPR30/GPER-1, a membrane receptor for estrogen: from discovery to functions in vivo.

PubMed

Mizukami, Yoichi

2010-01-01

G protein-coupled receptor 30/G protein-coupled estrogen receptor-1 (GPR30/GPER-1) was reported as a novel membrane receptor for estrogen in 2005. However, the research on GPR30 has produced conflicting reports with regard to its intracellular localization, the tissue distribution of its expression, and some its functions. Recently, in addition to the finding of G-1, a GPR30 agonist, GPR30 KO mice have been produced in laboratories, and this has significantly increased the confidence in the data. In this review, the intrinsic appearance of GPR30 is approached based mainly on data obtained in vivo.

G protein-coupled receptor 30 is critical for a progestin-induced growth inhibition in MCF-7 breast cancer cells.

PubMed

Ahola, Tytti M; Manninen, Tommi; Alkio, Niina; Ylikomi, Timo

2002-09-01

The issue of how progesterone affects mammary gland growth is controversial, and the mechanism governing the effects of the hormone remains mostly unknown. We have previously shown that G protein-coupled receptor 30 (GPR30) is a progestin target gene whose expression correlates with progestin-induced growth inhibition in breast cancer cells. In this study, we investigate the role of GPR30 in regulating cell proliferation and mediating progestin-induced growth inhibition. When progestin failed to inhibit the growth of MCF-7 cells and instead stimulated growth, GPR30 was down-regulated. In this way, the inhibitory or stimulatory affects that progestin has on proliferation correlated with the level of expression of GPR30. Transient expression of GPR30 resulted in a marked inhibition of cell proliferation independent of estrogen treatment. GPR30 antisense was used to evaluate the role of GPR30 expression in progestin-induced growth inhibition. A diminished GPR30 mRNA expression by the antisense stimulated growth. Interestingly, GPR30 antisense abrogated the growth inhibitory effect of progestin and progesterone. Indeed, progestin induced 1) a reduction in cell proliferation, 2) G1-phase arrest, and 3) down-regulation of cyclin D1 was diminished. These data suggest that the orphan receptor, GPR30, is important for the inhibitory effect of progestin on growth.

The 2-monoacylglycerol moiety of dietary fat appears to be responsible for the fat-induced release of GLP-1 in humans.

PubMed

Mandøe, Mette J; Hansen, Katrine B; Hartmann, Bolette; Rehfeld, Jens F; Holst, Jens J; Hansen, Harald S

2015-09-01

Dietary triglycerides can, after digestion, stimulate the intestinal release of incretin hormones through activation of G protein-coupled receptor (GPR) 119 by 2-monoacylglycerol and by the activation of fatty acid receptors for long- and short-chain fatty acids. Medium-chain fatty acids do not stimulate the release of intestinal hormones. To dissect the mechanism of fat-induced glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) release in humans, we compared the effects of tributyrin (containing short-chain fatty acids; i.e., butyric acid), olive oil [containing long-chain fatty acids; e.g., oleic acid plus 2-oleoyl glycerol (2-OG)], and 1,3-dioctanoyl-2-oleoyl glycerol (C8-dietary oil), which is digested to form medium-chain fatty acids : i.e., octanoic acid : and 2-OG. In a randomized, single-blinded crossover study, 12 healthy white men [mean age: 24 y; BMI (in kg/m(2)): 22] were given the following 4 meals on 4 different days: 200 g carrots + 6.53 g tributyrin, 200 g carrots + 13.15 g C8-dietary oil, 200 g carrots + 19 g olive oil, or 200 g carrots. All of the lipids totaled 0.0216 mol. Main outcome measures were incremental areas under the curve for total GLP-1, GIP, and cholecystokinin (CCK) in plasma. C8-dietary oil and olive oil showed the same GLP-1 response [583 ± 101 and 538 ± 71 (pmol/L) × 120 min; P = 0.733], whereas the GIP response was higher for olive oil than for C8-dietary oil [3293 ± 404 and 1674 ± 270 (pmol/L) × 120 min; P = 0.002]. Tributyrin and carrots alone resulted in no increase in any of the measured hormones. Peptide YY (PYY) and neurotensin responses resembled those of GLP-1. Only olive oil stimulated CCK release. Under our study conditions, 2-OG and GPR119 activation can fully explain the olive oil-induced secretion of GLP-1, PYY, and neurotensin. In contrast, both oleic acid and 2-OG contributed to the GIP response. Dietary butyrate did not stimulate gut hormone secretion. Olive oil-derived oleic acid seems to be fully responsible for olive oil-induced CCK secretion. This trial was registered at clinicaltrials.gov as NCT02264951. © 2015 American Society for Nutrition.

GPR30 activation decreases anxiety in the open field test but not in the elevated plus maze test in female mice.

PubMed

Anchan, Divya; Clark, Sara; Pollard, Kevin; Vasudevan, Nandini

2014-01-01

The GPR30 is a novel estrogen receptor (ER) that is a candidate membrane ER based on its binding to 17β estradiol and its rapid signaling properties such as activation of the extracellular-regulated kinase (ERK) pathway. Its distribution in the mouse limbic system predicts a role for this receptor in the estrogenic modulation of anxiety behaviors in the mouse. A previous study showed that chronic administration of a selective agonist to the GPR30 receptor, G-1, in the female rat can improve spatial memory, suggesting that GPR30 plays a role in hippocampal-dependent cognition. In this study, we investigated the effect of a similar chronic administration of G-1 on behaviors that denote anxiety in adult ovariectomized female mice, using the elevated plus maze (EPM) and the open field test as well as the activation of the ERK pathway in the hippocampus. Although estradiol benzoate had no effect on behaviors in the EPM or the open field, G-1 had an anxiolytic effect solely in the open field that was independent of ERK signaling in either the ventral or dorsal hippocampus. Such an anxiolytic effect may underlie the ability of G-1 to increase spatial memory, by acting on the hippocampus.

Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk.

PubMed

Lassen, Kara G; McKenzie, Craig I; Mari, Muriel; Murano, Tatsuro; Begun, Jakob; Baxt, Leigh A; Goel, Gautam; Villablanca, Eduardo J; Kuo, Szu-Yu; Huang, Hailiang; Macia, Laurence; Bhan, Atul K; Batten, Marcel; Daly, Mark J; Reggiori, Fulvio; Mackay, Charles R; Xavier, Ramnik J

2016-06-21

Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense. Copyright © 2016 Elsevier Inc. All rights reserved.

Structural basis for regulation of GPR56/ADGRG1 by its alternatively spliced extracellular domains

PubMed Central

Salzman, Gabriel S.; Ackerman, Sarah D.; Ding, Chen; Koide, Akiko; Leon, Katherine; Luo, Rong; Stoveken, Hannah M.; Fernandez, Celia G.; Tall, Gregory G.; Piao, Xianhua; Monk, Kelly R.; Koide, Shohei; Araç, Demet

2016-01-01

Summary Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we term Pentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development in vivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology. PMID:27657451

Novel effects of diosgenin on skin aging.

PubMed

Tada, Yayoi; Kanda, Naoko; Haratake, Akinori; Tobiishi, Megumi; Uchiwa, Hideyo; Watanabe, Shinichi

2009-06-01

Extracts of Dioscorea coomposita or Dioscorea villosa are consumed as supplemental health foods at the time of climacteric. The extracts contain large amounts of the plant steroid, diosgenin. Here, we studied the safety and efficacy of diosgenin against skin aging at the time of climacteric. In vitro, diosgenin enhanced DNA synthesis in a human 3D skin equivalent model, and increased bromodeoxyuridine uptake and intracellular cAMP level in adult human keratinocytes. The increase of bromodeoxyuridine uptake by diosgenin was blocked by an adenylate cyclase inhibitor, but not by antisense oligonucleotides against estrogen receptor alpha, estrogen receptor beta or an orphan G-protein-coupled receptor, GPR30, indicating the involvement of cAMP but not estrogen receptor alpha, estrogen receptor beta or GPR30. In vivo, administration of diosgenin improved the epidermal thickness in the ovariectomized mice, a climacteric model, without altering the degree of fat accumulation. In order to examine the safety of diosgenin, diosgenin and 17beta-estradiol were administered to breast cancer-burdened mice. The results revealed that while 17beta-estradiol accelerated the tumor growth, diosgenin did not show this effect. Our finding, a restoration of keratinocyte proliferation in aged skin, suggests that diosgenin may have potential as a safe health food for climacteric.

Activation of the omega-3 fatty acid receptor GPR120 mediates anti-inflammatory actions in immortalized hypothalamic neurons.

PubMed

Wellhauser, Leigh; Belsham, Denise D

2014-03-27

Overnutrition and the ensuing hypothalamic inflammation is a major perpetuating factor in the development of metabolic diseases, such as obesity and diabetes. Inflamed neurons of the CNS fail to properly regulate energy homeostasis leading to pathogenic changes in glucose handling, feeding, and body weight. Hypothalamic neurons are particularly sensitive to pro-inflammatory signals derived locally and peripherally, and it is these neurons that become inflamed first upon high fat feeding. Given the prevalence of metabolic disease, efforts are underway to identify therapeutic targets for this inflammatory state. At least in the periphery, omega-3 fatty acids and their receptor, G-protein coupled receptor 120 (GPR120), have emerged as putative targets. The role for GPR120 in the hypothalamus or CNS in general is poorly understood. Here we introduce a novel, immortalized cell model derived from the rat hypothalamus, rHypoE-7, to study GPR120 activation at the level of the individual neuron. Gene expression levels of pro-inflammatory cytokines were studied by quantitative reverse transcriptase-PCR (qRT-PCR) upon exposure to tumor necrosis factor α (TNFα) treatment in the presence or absence of the polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA). Signal transduction pathway involvement was also studied using phospho-specific antibodies to key proteins by western blot analysis. Importantly, rHypoE-7 cells exhibit a transcriptional and translational inflammatory response upon exposure to TNFα and express abundant levels of GPR120, which is functionally responsive to DHA. DHA pretreatment prevents the inflammatory state and this effect was inhibited by the reduction of endogenous GPR120 levels. GPR120 activates both AKT (protein kinase b) and ERK (extracellular signal-regulated kinase); however, the anti-inflammatory action of this omega-3 fatty acid (FA) receptor is AKT- and ERK-independent and likely involves the GPR120-transforming growth factor-β-activated kinase 1 binding protein (TAB1) interaction as identified in the periphery. Taken together, GPR120 is functionally active in the hypothalamic neuronal line, rHypoE-7, wherein it mediates the anti-inflammatory actions of DHA to reduce the inflammatory response to TNFα.

GPR30 activation improves memory and facilitates DHPG-induced LTD in the hippocampal CA3 of middle-aged mice.

PubMed

Xu, Wen; Cao, Jian; Zhou, Yan; Wang, Lina; Zhu, Guoqi

2018-03-01

Reduced estrogen levels and decreased expression of related receptors are typical cerebral features of aging. The G protein-coupled estrogen receptor 1 (GPER1, also known as GPR30) is considered a novel therapeutic target for neurodegenerative diseases. In this study, we demonstrated that hippocampal GPR30 expression was reduced in middle-aged mice compared with young adult mice. GPR30 agonist G1 improved both fear and spatial memory in both male and female middle-aged mice, but not in young adult mice, which were blocked by the GPR30 antagonist G15. Interestingly, a group I metabotropic glutamate receptor (mGluR) agonist, 3,5-dihydroxyphenylglycine (DHPG)-induced long-term depression (LTD) in mossy fiber-cornu ammonis 3 (MF-CA3) synapses but not Schaffer collateral-CA1 (SC-CA1) synapses was facilitated in brain slices from G1-treated middle-aged mice. Long-term potentiation (LTP) in SC-CA1 synapses was not affected in slices from G1-treated mice. The effects of GPR30 activation on memory and DHPG-LTD in MF-CA3 synapses were further confirmed by viral expression of GPR30 in the CA3. The regulation of hippocampal synaptic plasticity by G1 treatment might be related to brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signaling, as G15 also blocked G1-induced activation of the BDNF-TrkB pathway. Moreover, we found that DHPG triggered GluA internalization in slices from G1-treated mice but not control mice. Pharmacological experiments showed that G1-mediated facilitation of DHPG-induced LTD in MF-CA3 synapses was dependent on protein kinase B (Akt), mammalian target of rapamycin (mTor), and TrkB signaling. In conclusion, our results indicate that GPR30 activation improves memory in middle-aged mice, likely through facilitating synaptic plasticity in the CA3. This study provides novel evidence that GPR30 activation can improve memory in middle-aged animals. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular Recognition at Purine and Pyrimidine Nucleotide (P2) Receptors

PubMed Central

Jacobson, Kenneth A.; Constanzi, Stefano; Ohno, Michihiro; Joshi, Bhalchandra V.; Besada, Pedro; Xu, Bin; Tchilibon, Susanna

2015-01-01

In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X1 (derivatives of pyridoxal phosphates and suramin), P2X3 (A-317491), P2X7 (derivatives of the isoquinoline KN-62), P2Y1 (nucleotide analogues MRS 2179 and MRS 2279), P2Y2 (thiouracil derivatives such as AR-C126313), and P2Y12 (nucleotide/nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y1 receptors. The dinucleotide INS 37217 potently activates the P2Y2 receptor. UTP-γ-S and UDP-β-S are selective agonists for P2Y2/P2Y4 and P2Y6 receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y1 receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7. PMID:15078212

IFNγ Induces DNA Methylation-Silenced GPR109A Expression via pSTAT1/p300 and H3K18 Acetylation in Colon Cancer.

PubMed

Bardhan, Kankana; Paschall, Amy V; Yang, Dafeng; Chen, May R; Simon, Priscilla S; Bhutia, Yangzom D; Martin, Pamela M; Thangaraju, Muthusamy; Browning, Darren D; Ganapathy, Vadivel; Heaton, Christopher M; Gu, Keni; Lee, Jeffrey R; Liu, Kebin

2015-07-01

Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A have been the subject of extensive studies; however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wild-type mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoter to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoter to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer, and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. ©2015 American Association for Cancer Research.

IFNγ induces DNA methylation-silenced GPR109A expression via pSTAT1/p300 and H3K18 acetylation in colon cancer

PubMed Central

Bardhan, Kankana; Paschall, Amy V.; Yang, Dafeng; Chen, May R.; Simon, Priscilla S.; Bhutia, Yangzom; Martin, Pamela M.; Thangaraju, Muthusamy; Browning, Darren D.; Ganapathy, Vadivel; Heaton, Christopher M.; Gu, Keni; Lee, Jeffrey R.; Liu, Kebin

2015-01-01

Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A has been the subject of extensive studies, however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wildtype mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoters to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoters to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. PMID:25735954

Microbial short chain fatty acid metabolites lower blood pressure via endothelial G protein-coupled receptor 41.

PubMed

Natarajan, Niranjana; Hori, Daijiro; Flavahan, Sheila; Steppan, Jochen; Flavahan, Nicholas A; Berkowitz, Dan E; Pluznick, Jennifer L

2016-11-01

Short chain fatty acid (SCFA) metabolites are byproducts of gut microbial metabolism that are known to affect host physiology via host G protein-coupled receptor (GPCRs). We previously showed that an acute SCFA bolus decreases blood pressure (BP) in anesthetized mice, an effect mediated primarily via Gpr41. In this study, our aims were to identify the cellular localization of Gpr41 and to determine its role in BP regulation. We localized Gpr41 to the vascular endothelium using RT-PCR: Gpr41 is detected in intact vessels (with endothelium) but is absent from denuded vessels (without endothelium). Furthermore, using pressure myography we confirmed that SCFAs dilate resistance vessels in an endothelium-dependent manner. Since we previously found that Gpr41 mediates a hypotensive response to acute SCFA administration, we hypothesized that Gpr41 knockout (KO) mice would be hypertensive. Here, we report that Gpr41 KO mice have isolated systolic hypertension compared with wild-type (WT) mice; diastolic BP was not different between WT and KO. Older Gpr41 KO mice also exhibited elevated pulse wave velocity, consistent with a phenotype of systolic hypertension; however, there was no increase in ex vivo aorta stiffness (measured by mechanical tensile testing). Plasma renin concentrations were also similar in KO and WT mice. The systolic hypertension in Gpr41 KO is not salt sensitive, as it is not significantly altered on either a high- or low-salt diet. In sum, these studies suggest that endothelial Gpr41 lowers baseline BP, likely by decreasing active vascular tone without altering passive characteristics of the blood vessels, and that Gpr41 KO mice have hypertension of a vascular origin. Copyright © 2016 the American Physiological Society.

Germline or somatic GPR101 duplication leads to X-linked acrogigantism: a clinico-pathological and genetic study.

PubMed

Iacovazzo, Donato; Caswell, Richard; Bunce, Benjamin; Jose, Sian; Yuan, Bo; Hernández-Ramírez, Laura C; Kapur, Sonal; Caimari, Francisca; Evanson, Jane; Ferraù, Francesco; Dang, Mary N; Gabrovska, Plamena; Larkin, Sarah J; Ansorge, Olaf; Rodd, Celia; Vance, Mary L; Ramírez-Renteria, Claudia; Mercado, Moisés; Goldstone, Anthony P; Buchfelder, Michael; Burren, Christine P; Gurlek, Alper; Dutta, Pinaki; Choong, Catherine S; Cheetham, Timothy; Trivellin, Giampaolo; Stratakis, Constantine A; Lopes, Maria-Beatriz; Grossman, Ashley B; Trouillas, Jacqueline; Lupski, James R; Ellard, Sian; Sampson, Julian R; Roncaroli, Federico; Korbonits, Márta

2016-06-01

Non-syndromic pituitary gigantism can result from AIP mutations or the recently identified Xq26.3 microduplication causing X-linked acrogigantism (XLAG). Within Xq26.3, GPR101 is believed to be the causative gene, and the c.924G > C (p.E308D) variant in this orphan G protein-coupled receptor has been suggested to play a role in the pathogenesis of acromegaly.We studied 153 patients (58 females and 95 males) with pituitary gigantism. AIP mutation-negative cases were screened for GPR101 duplication through copy number variation droplet digital PCR and high-density aCGH. The genetic, clinical and histopathological features of XLAG patients were studied in detail. 395 peripheral blood and 193 pituitary tumor DNA samples from acromegaly patients were tested for GPR101 variants.We identified 12 patients (10 females and 2 males; 7.8 %) with XLAG. In one subject, the duplicated region only contained GPR101, but not the other three genes in found to be duplicated in the previously reported patients, defining a new smallest region of overlap of duplications. While females presented with germline mutations, the two male patients harbored the mutation in a mosaic state. Nine patients had pituitary adenomas, while three had hyperplasia. The comparison of the features of XLAG, AIP-positive and GPR101&AIP-negative patients revealed significant differences in sex distribution, age at onset, height, prolactin co-secretion and histological features. The pathological features of XLAG-related adenomas were remarkably similar. These tumors had a sinusoidal and lobular architecture. Sparsely and densely granulated somatotrophs were admixed with lactotrophs; follicle-like structures and calcifications were commonly observed. Patients with sporadic of familial acromegaly did not have an increased prevalence of the c.924G > C (p.E308D) GPR101 variant compared to public databases.In conclusion, XLAG can result from germline or somatic duplication of GPR101. Duplication of GPR101 alone is sufficient for the development of XLAG, implicating it as the causative gene within the Xq26.3 region. The pathological features of XLAG-associated pituitary adenomas are typical and, together with the clinical phenotype, should prompt genetic testing.

Role of the X-linked gene GPR174 in autoimmune Addison's disease.

PubMed

Napier, C; Mitchell, A L; Gan, E; Wilson, I; Pearce, S H S

2015-01-01

Autoimmune endocrinopathies demonstrate a profound gender bias, but the reasons for this remain obscure. The 1000 genes on the X chromosome are likely to be implicated in this inherent susceptibility; various theories, including skewed X chromosome inactivation and fetal microchimerism, have been proposed. GPR174 is an Xq21 putative purinergic receptor that is widely expressed in lymphoid tissues. A single-nucleotide polymorphism, rs3827440, encoding Ser162Pro, has recently been associated with Graves' disease in Chinese and Polish populations, suggesting a role of this X chromosome gene in autoimmune disease. We investigated the role of rs3827440 in a UK cohort of patients with autoimmune Addison's disease (AAD). Samples from 286 AAD cases and 288 healthy controls were genotyped using TaqMan single-nucleotide polymorphism genotyping assays (C_25954273_10) on the Applied Biosystems 7900HT Fast real-time PCR system. Using a dominant (present/absent) model, the serine-encoding T allele of rs3827440 was present in 189 of 286 AAD patients (66%) compared with 132 of 288 unaffected controls (46%) [P = .010, odds ratio 1.80 (5%-95% confidence interval 1.22-2.67)]. An allele dosage model found a significant excess of the T allele in AAD patients compared with controls [P = .03, odds ratio 1.34 (5%-95% confidence interval 1.07-1.67)]. We have demonstrated a significant association of this X chromosome-encoded immunoreceptor with AAD for the first time. This X-linked gene could have a more generalized role in autoimmunity pathogenesis: G protein-coupled receptors are promising drugable targets, and further work to elucidate the functional role of GPR174 is now warranted.

Adenine Nucleotide Analogues Locked in a Northern Methanocarba Conformation: Enhanced Stability and Potency as P2Y1 Receptor Agonists

PubMed Central

Ravi, R. Gnana; Kim, Hak Sung; Servos, Jörg; Zimmermann, Herbert; Lee, Kyeong; Maddileti, Savitri; Boyer, José L.; Harden, T. Kendall; Jacobson, Kenneth A.

2016-01-01

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5′-triphosphate agonists at P2Y1, P2Y2, P2Y4, and P2Y11 receptors, but not P2Y6 receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208–218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y1 or human P2Y1 and P2Y2 receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC50 at the hP2Y1 receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5′-diphosphate. 2-Cl–(N)-methanocarba-ATP and its N6-Me analogue were also highly selective, full agonists at P2Y1 receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y1 receptors, while the corresponding ribosides were inactive. Although β,γ-methylene-ATP was inactive at P2Y receptors, β,γ-methylene-(N)-methanocarba-ATP was a potent hP2Y1 receptor agonist with an EC50 of 160 nM and was selective versus hP2Y2 and hP2Y4 receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5′-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of the (N)-conformation, which greatly enhanced potency at P2Y1 receptors. PMID:11985476

Molecular mechanisms of platelet P2Y(12) receptor regulation.

PubMed

Cunningham, Margaret R; Nisar, Shaista P; Mundell, Stuart J

2013-02-01

Platelets are critical for haemostasis, however inappropriate activation can lead to the development of arterial thrombosis, which can result in heart attack and stroke. ADP is a key platelet agonist that exerts its actions via stimulation of two surface GPCRs (G-protein-coupled receptors), P2Y(1) and P2Y(12). Similar to most GPCRs, P2Y receptor activity is tightly regulated by a number of complex mechanisms including receptor desensitization, internalization and recycling. In the present article, we review the molecular mechanisms that underlie P2Y(1) and P2Y(12) receptor regulation, with particular emphasis on the structural motifs within the P2Y(12) receptor, which are required to maintain regulatory protein interaction. The implications of these findings for platelet responsiveness are also discussed.

Effects of a combined treatment with GPR30 agonist G-1 and herceptin on growth and gene expression of human breast cancer cell lines.

PubMed

Lubig, Julia; Lattrich, Claus; Springwald, Anette; Häring, Julia; Schüler, Susanne; Ortmann, Olaf; Treeck, Oliver

2012-06-01

Expression of G-protein-coupled receptor 30 (GPR30) is present in HER2-overexpressing breast cancer. In this study, we examined to what extent GPR30-agonist G-1 would affect the antitumoral action of trastuzumab (Herceptin). Combined treatment with both drugs exerted an additive growth-inhibitory effect on breast cancer cells which was accompanied by a significant decline of cyclin A2 expression both on the protein and the mRNA level. Combined treatment also resulted in expression changes of c-fos, cyclin D1, or p21/WAF-1. The results of our study encourage further attempts to test the relevance of these in vitro data in the clinical setting.

Identification of endogenous surrogate ligands for human P2Y receptors through an in silico search.

PubMed

Hiramoto, Takeshi; Nonaka, Yosuke; Inoue, Kazuko; Yamamoto, Takefumi; Omatsu-Kanbe, Mariko; Matsuura, Hiroshi; Gohda, Keigo; Fujita, Norihisa

2004-05-01

G protein-coupled receptors (GPCRs) are distributed widely throughout the human body, and nearly 50% of current medicines act on a GPCR. GPCRs are considered to consist of seven transmembrane alpha-helices that form an alpha-helical bundle in which agonists and antagonists bind. A 3D structure of the target GPCR is indispensable for designing novel medicines acting on a GPCR. We have previously constructed the 3D structure of human P2Y(1) (hP2Y(1)) receptor, a GPCR, by homology modeling with the 3D structure of bovine rhodopsin as a template. In the present study, we have employed an in silico screening for compounds that could bind to the hP2Y(1)-receptor model using AutoDock 3.0. We selected 21 of the 30 top-ranked compounds, and by measuring intracellular Ca(2+) concentration, we identified 12 compounds that activated or blocked the hP2Y(1) receptor stably expressed in recombinant CHO cells. 5-Phosphoribosyl-1-pyrophosphate (PRPP) was found to activate the hP2Y(1) receptor with a low ED(50) value of 15 nM. The Ca(2+) assays showed it had no significant effect on P2Y(2), P2Y(6), or P2X(2) receptors, but acted as a weak agonist on the P2Y(12) receptor. This is the first study to rationally identify surrogate ligands for the P2Y-receptor family.

Effect of Substitution on the Aniline Moiety of the GPR88 Agonist 2-PCCA: Synthesis, Structure-Activity Relationships, and Molecular Modeling Studies.

PubMed

Jin, Chunyang; Decker, Ann M; Harris, Danni L; Blough, Bruce E

2016-10-19

GPR88, an orphan receptor richly expressed in the striatum, is implicated in a number of basal ganglia-associated disorders. In order to elucidate the functions of GPR88, an in vivo probe appropriate for CNS investigation is required. We previously reported that 2-PCCA was able to modulate GPR88-mediated cAMP production through a Gα i -coupled pathway. Early structure-activity relationship (SAR) studies suggested that the aniline moiety of 2-PCCA is a suitable site for diverse modifications. Aimed at elucidating structural requirements in this region, we have designed and synthesized a series of analogues bearing a variety of substituents at the phenyl ring of the aniline moiety. Several compounds (e.g., 5j, 5o) showed improved or comparable potency, but have lower lipophilicity than 2-PCCA (clogP 6.19). These compounds provide the basis for further optimization to probe GPR88 in vivo functions. Computational studies confirmed the SAR trends and supported the notion that 4'-substituents on the biphenyl ring exit through a largely hydrophobic binding site to the extracellular loop.

Association analysis of the chromosome 4p-located G protein-coupled receptor 78 (GPR78) gene in bipolar affective disorder and schizophrenia.

PubMed

Underwood, S L; Christoforou, A; Thomson, P A; Wray, N R; Tenesa, A; Whittaker, J; Adams, R A; Le Hellard, S; Morris, S W; Blackwood, D H R; Muir, W J; Porteous, D J; Evans, K L

2006-04-01

The orphan G protein-coupled receptor 78 (GPR78) gene lies within a region of chromosome 4p where we have previously shown linkage to bipolar affective disorder (BPAD) in a large Scottish family. GPR78 was screened for single-nucleotide polymorphisms (SNPs) and a linkage disequilibrium map was constructed. Six tagging SNPs were selected and tested for association on a sample of 377 BPAD, 392 schizophrenia (SCZ) and 470 control individuals. Using standard chi(2) statistics and a backwards logistic regression approach to adjust for the effect of sex, SNP rs1282, located approximately 3 kb upstream of the coding region, was identified as a potentially important variant in SCZ (chi(2) P=0.044; LRT P=0.065). When the analysis was restricted to females, the strength of association increased to an uncorrected allele P-value of 0.015 (odds ratios (OR)=1.688, 95% confidence intervals (CI): 1.104-2.581) and uncorrected genotype P-value of 0.015 (OR=5.991, 95% CI: 1.545-23.232). Under the recessive model, the genotype P-value improved further to 0.005 (OR=5.618, 95% CI: 1.460-21.617) and remained significant after correcting for multiple testing (P=0.017). No single-marker association was detected in the SCZ males, in the BPAD individuals or with any other SNP. Haplotype analysis of the case-control samples revealed several global and individual haplotypes, with P-values <0.05, all but one of which contained SNP rs1282. After correcting for multiple testing, two haplotypes remained significant in both the female BPAD individuals (P=0.038 and 0.032) and in the full sample of affected female individuals (P=0.044 and 0.033). Our results provide preliminary evidence for the involvement of GPR78 in susceptibility to BPAD and SCZ in the Scottish population. Molecular Psychiatry (2006) 11, 384-394. doi:10.1038/sj.mp.4001786; published online 3 January 2006.

Functional distribution of Ca2+-coupled P2 purinergic receptors among adrenergic and noradrenergic bovine adrenal chromaffin cells.

PubMed

Tomé, Angelo R; Castro, Enrique; Santos, Rosa M; Rosário, Luís M

2007-06-14

Adrenal chromaffin cells mediate acute responses to stress through the release of epinephrine. Chromaffin cell function is regulated by several receptors, present both in adrenergic (AD) and noradrenergic (NA) cells. Extracellular ATP exerts excitatory and inhibitory actions on chromaffin cells via ionotropic (P2X) and metabotropic (P2Y) receptors. We have taken advantage of the actions of the purinergic agonists ATP and UTP on cytosolic free Ca2+ concentration ([Ca2+]i) to determine whether P2X and P2Y receptors might be asymmetrically distributed among AD and NA chromaffin cells. The [Ca2+]i and the [Na+]i were recorded from immunolabeled bovine chromaffin cells by single-cell fluorescence imaging. Among the ATP-sensitive cells ~40% did not yield [Ca2+]i responses to ATP in the absence of extracellular Ca2+ (Ca2+o), indicating that they expressed P2X receptors and did not express Ca2+- mobilizing P2Y receptors; the remainder expressed Ca2+-mobilizing P2Y receptors. Relative to AD-cells approximately twice as many NA-cells expressed P2X receptors while not expressing Ca2+- mobilizing P2Y receptors, as indicated by the proportion of cells lacking [Ca2+]i responses and exhibiting [Na+]i responses to ATP in the absence and presence of Ca2+o, respectively. The density of P2X receptors in NA-cells appeared to be 30-50% larger, as suggested by comparing the average size of the [Na+]i and [Ca2+]i responses to ATP. Conversely, approximately twice as many AD-cells expressed Ca2+-mobilizing P2Y receptors, and they appeared to exhibit a higher (~20%) receptor density. UTP raised the [Ca2+]i in a fraction of the cells and did not raise the [Na+]i in any of the cells tested, confirming its specificity as a P2Y agonist. The cell density of UTP-sensitive P2Y receptors did not appear to vary among AD- and NA-cells. Although neither of the major purinoceptor types can be ascribed to a particular cell phenotype, P2X and Ca2+-mobilizing P2Y receptors are preferentially located to noradrenergic and adrenergic chromaffin cells, respectively. ATP might, in addition to an UTP-sensitive P2Y receptor, activate an UTP-insensitive P2Y receptor subtype. A model for a short-loop feedback interaction is presented whereby locally released ATP acts upon P2Y receptors in adrenergic cells, inhibiting Ca2+ influx and contributing to terminate evoked epinephrine secretion.

Subtype specific internalization of P2Y1 and P2Y2 receptors induced by novel adenosine 5′-O-(1-boranotriphosphate) derivatives

PubMed Central

Tulapurkar, M E; Laubinger, W; Nahum, V; Fischer, B; Reiser, G

2004-01-01

P2Y-nucleotide receptors represent important targets for drug development. The lack of stable and receptor specific agonists, however, has prevented successful therapeutic applications. A novel series of P-boronated ATP derivatives (ATP-α-B) were synthesized by substitution of a nonbridging O at Pα with a BH3 group. This introduces a chiral center, thus resulting in diastereoisomers. In addition, at C2 of the adenine ring a further substitution was made (Cl- or methylthio-). The pairs of diastereoisomers were denoted here as A and B isomers. Here, we tested the receptor subtype specificity of these analogs on HEK 293 cells stably expressing rat P2Y1 and rat P2Y2 receptors, respectively, both attached to the fluorescent marker protein GFP (rP2Y1-GFP, rP2Y2-GFP). We investigated agonist-induced receptor endocytosis, [Ca2+]i rise and arachidonic acid (AA) release. Agonist-induced endocytosis of rP2Y1-GFP was more pronounced for the A isomers than the corresponding B counterparts for all ATP-α-B analogs. Both 2-MeS-substituted diastereoisomers induced a greater degree of agonist-induced receptor endocytosis as compared to the 2-Cl-substituted derivatives. Endocytosis results are in accordance with the potency to induce Ca2+ release by these compounds in HEK 293 cells stably transfected with rP2Y1. In case of rP2Y2-GFP, the borano-nucleotides were very weak agonists in comparison to UTP and ATP in terms of Ca2+ release, AA release and in inducing receptor endocytosis. The different ATP-α-B derivatives and also the diastereoisomers were equally ineffective. Thus, the new agonists may be considered as potent and highly specific agonist drug candidates for P2Y1 receptors. The difference in activity of the ATP analogs at P2Y receptors could be used as a tool to investigate structural differences between P2Y receptor subtypes. PMID:15197109

The role of G-protein receptor 84 in experimental neuropathic pain.

PubMed

Nicol, Louise S C; Dawes, John M; La Russa, Federica; Didangelos, Athanasios; Clark, Anna K; Gentry, Clive; Grist, John; Davies, John B; Malcangio, Marzia; McMahon, Stephen B

2015-06-10

G-protein receptor 84 (GPR84) is an orphan receptor that is induced markedly in monocytes/macrophages and microglia during inflammation, but its pathophysiological function is unknown. Here, we investigate the role of GPR84 in a murine model of traumatic nerve injury. Naive GPR84 knock-out (KO) mice exhibited normal behavioral responses to acute noxious stimuli, but subsequent to partial sciatic nerve ligation (PNL), KOs did not develop mechanical or thermal hypersensitivity, in contrast to wild-type (WT) littermates. Nerve injury increased ionized calcium binding adapter molecule 1 (Iba1) and phosphorylated p38 MAPK immunoreactivity in the dorsal horn and Iba1 and cluster of differentiation 45 expression in the sciatic nerve, with no difference between genotypes. PCR array analysis revealed that Gpr84 expression was upregulated in the spinal cord and sciatic nerve of WT mice. In addition, the expression of arginase-1, a marker for anti-inflammatory macrophages, was upregulated in KO sciatic nerve. Based on this evidence, we investigated whether peripheral macrophages behave differently in the absence of GPR84. We found that lipopolysaccharide-stimulated KO macrophages exhibited attenuated expression of several proinflammatory mediators, including IL-1β, IL-6, and TNF-α. Forskolin-stimulated KO macrophages also showed greater cAMP induction, a second messenger associated with immunosuppression. In summary, our results demonstrate that GPR84 is a proinflammatory receptor that contributes to nociceptive signaling via the modulation of macrophages, whereas in its absence the response of these cells to an inflammatory insult is impaired. Copyright © 2015 Nicol et al.

The Role of G-Protein Receptor 84 in Experimental Neuropathic Pain

PubMed Central

Nicol, Louise S.C.; Dawes, John M.; La Russa, Federica; Didangelos, Athanasios; Clark, Anna K.; Gentry, Clive; Grist, John; Davies, John B.; Malcangio, Marzia

2015-01-01

G-protein receptor 84 (GPR84) is an orphan receptor that is induced markedly in monocytes/macrophages and microglia during inflammation, but its pathophysiological function is unknown. Here, we investigate the role of GPR84 in a murine model of traumatic nerve injury. Naive GPR84 knock-out (KO) mice exhibited normal behavioral responses to acute noxious stimuli, but subsequent to partial sciatic nerve ligation (PNL), KOs did not develop mechanical or thermal hypersensitivity, in contrast to wild-type (WT) littermates. Nerve injury increased ionized calcium binding adapter molecule 1 (Iba1) and phosphorylated p38 MAPK immunoreactivity in the dorsal horn and Iba1 and cluster of differentiation 45 expression in the sciatic nerve, with no difference between genotypes. PCR array analysis revealed that Gpr84 expression was upregulated in the spinal cord and sciatic nerve of WT mice. In addition, the expression of arginase-1, a marker for anti-inflammatory macrophages, was upregulated in KO sciatic nerve. Based on this evidence, we investigated whether peripheral macrophages behave differently in the absence of GPR84. We found that lipopolysaccharide-stimulated KO macrophages exhibited attenuated expression of several proinflammatory mediators, including IL-1β, IL-6, and TNF-α. Forskolin-stimulated KO macrophages also showed greater cAMP induction, a second messenger associated with immunosuppression. In summary, our results demonstrate that GPR84 is a proinflammatory receptor that contributes to nociceptive signaling via the modulation of macrophages, whereas in its absence the response of these cells to an inflammatory insult is impaired. PMID:26063927

The oral lipid sensor GPR120 is not indispensable for the orosensory detection of dietary lipids in mice

PubMed Central

Ancel, Déborah; Bernard, Arnaud; Subramaniam, Selvakumar; Hirasawa, Akira; Tsujimoto, Gozoh; Hashimoto, Toshihiro; Passilly-Degrace, Patricia; Khan, Naim-Akhtar; Besnard, Philippe

2015-01-01

Implication of the long-chain fatty acid (LCFA) receptor GPR120, also termed free fatty acid receptor 4, in the taste-guided preference for lipids is a matter of debate. To further unravel the role of GPR120 in the “taste of fat”, the present study was conducted on GPR120-null mice and their wild-type littermates. Using a combination of morphological [i.e., immunohistochemical staining of circumvallate papillae (CVP)], behavioral (i.e., two-bottle preference tests, licking tests and conditioned taste aversion) and functional studies [i.e., calcium imaging in freshly isolated taste bud cells (TBCs)], we show that absence of GPR120 in the oral cavity was not associated with changes in i) gross anatomy of CVP, ii) LCFA-mediated increases in intracellular calcium levels ([Ca2+]i), iii) preference for oily and LCFA solutions and iv) conditioned avoidance of LCFA solutions. In contrast, the rise in [Ca2+]i triggered by grifolic acid, a specific GPR120 agonist, was dramatically curtailed when the GPR120 gene was lacking. Taken together, these data demonstrate that activation of lingual GPR120 and preference for fat are not connected, suggesting that GPR120 expressed in TBCs is not absolutely required for oral fat detection in mice PMID:25489006

P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice

PubMed Central

Guns, Pieter-Jan DF; Hendrickx, Jan; Van Assche, Tim; Fransen, Paul; Bult, Hidde

2010-01-01

Background and purpose: P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis. Experimental approach: mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE–/–) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE–/– mice. Key results: P2Y6 receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y2 receptor expression remained unchanged. Expression of P2Y1 or P2Y4 receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y6 mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y6-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y6 receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y6-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100–300 µM) or pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS, 10–30 µM). Finally, 4-week treatment of cholesterol-fed apoE–/– mice with suramin or PPADS (50 and 25 mg·kg−1·day−1 respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication. Conclusions and implications: These results suggest involvement of nucleotide receptors, particularly P2Y6 receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y6 receptor-deficient mice. PMID:20050854

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium.

PubMed

Carneiro, Inês; Timóteo, M Alexandrina; Silva, Isabel; Vieira, Cátia; Baldaia, Catarina; Ferreirinha, Fátima; Silva-Ramos, Miguel; Correia-de-Sá, Paulo

2014-07-01

Despite the abundant expression of the UDP-sensitive P2Y6 receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood. We compared the effects of UDP and of the selective P2Y6 receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [(3) H]-ACh overflow experiments. Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y6 receptor antagonist, MRS2578. Effects mediated by urothelial P2Y6 receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y1 receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [(3) H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y1 receptor antagonist, MRS2179. The P2Y6 receptor agonist increased threefold ATP levels in the voided fluid. Activation of P2Y6 receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y1 receptors. © 2014 The British Pharmacological Society.

Unique transcriptome signatures and GM-CSF expression in lymphocytes from patients with spondyloarthritis.

PubMed

Al-Mossawi, M H; Chen, L; Fang, H; Ridley, A; de Wit, J; Yager, N; Hammitzsch, A; Pulyakhina, I; Fairfax, B P; Simone, D; Yi, Yao; Bandyopadhyay, S; Doig, K; Gundle, R; Kendrick, B; Powrie, F; Knight, J C; Bowness, P

2017-11-15

Spondyloarthritis encompasses a group of common inflammatory diseases thought to be driven by IL-17A-secreting type-17 lymphocytes. Here we show increased numbers of GM-CSF-producing CD4 and CD8 lymphocytes in the blood and joints of patients with spondyloarthritis, and increased numbers of IL-17A + GM-CSF + double-producing CD4, CD8, γδ and NK cells. GM-CSF production in CD4 T cells occurs both independently and in combination with classical Th1 and Th17 cytokines. Type 3 innate lymphoid cells producing predominantly GM-CSF are expanded in synovial tissues from patients with spondyloarthritis. GM-CSF + CD4 + cells, isolated using a triple cytokine capture approach, have a specific transcriptional signature. Both GM-CSF + and IL-17A + GM-CSF + double-producing CD4 T cells express increased levels of GPR65, a proton-sensing receptor associated with spondyloarthritis in genome-wide association studies and pathogenicity in murine inflammatory disease models. Silencing GPR65 in primary CD4 T cells reduces GM-CSF production. GM-CSF and GPR65 may thus serve as targets for therapeutic intervention of spondyloarthritis.

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65

PubMed Central

Huang, Xi-Ping; Karpiak, Joel; Kroeze, Wesley K.; Zhu, Hu; Chen, Xin; Moy, Sheryl S.; Saddoris, Kara A.; Nikolova, Viktoriya; Farrell, Martilias S.; Wang, Sheng; Mangano, Thomas J.; Deshpande, Deepak A.; Jiang, Alice; Penn, Raymond B.; Jin, Jian; Koller, Beverly H.; Kenakin, Terry; Shoichet, Brian K.; Roth, Bryan L.

2016-01-01

At least 120 non-olfactory G protein-coupled receptors in the human genome are ”orphans” for which endogenous ligands are unknown, and many have no selective ligands, hindering elucidation of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Yeast-based screens against GPR68 identified the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. Over 3000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators many of which were confirmed in functional assays. One potent GPR68 modulator—ogerin– suppressed recall in fear conditioning in wild-type, but not in GPR68 knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs. PMID:26550826

GPR120 in adipocytes has differential roles in the production of pro-inflammatory adipocytokines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasan, Arif Ul, E-mail: ahasan@med.kagawa-u.ac.jp; Department of Pharmacology, Faculty of Medicine, Kagawa University, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793; Ohmori, Koji

How nutritional excess leads to inflammatory responses in metabolic syndrome is not well characterized. Here, we evaluated the effects of ω-3 polyunsaturated fatty acid specific G-protein coupled receptor 120 (GPR120) activation on inflammatory pathways in adipocytes, and the influence of this process on macrophage migration. Using 3T3-L1 adipocytes, we found that agonizing GPR120 using its synthetic ligand, GSK137647, attenuated both basal and lipopolysaccharide-induced production of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2). Moreover, the intervention reduced the phosphorylation of nuclear factor kappa B inhibitor alpha (IκBα) and nuclear translocation of nuclear factor kappa-B p65 subunit (p65). Furthermore, themore » silencing of GPR120 itself reduced IL-6 and CCL2 mRNA expression. Inhibition of protein kinase C (PKC) augmented the down-regulatory effect of GSK137647 on IL-6 and CCL2 mRNA. Using a luciferase assay to measure promoter activity of the IL-6 gene in mouse embryonic fibroblasts, we demonstrated that exogenous transfection of GPR120 alone reduced the promoter activity, which was augmented by GSK137647. Inhibition of PKC further reduced the promoter activity. Nevertheless, RAW 264.7 macrophages grown in conditioned medium collected from GSK137647-treated adipocytes attenuated the expressions of matrix metalloproteinases-9 and -3, and tissue inhibitor of metalloproteinase-1. Conditioned medium also inhibited the lipopolysaccharide-induced migration of these macrophages. Taken together, these findings provide critical evidence that although GPR120 is associated with a PKC-mediated pro-inflammatory pathway, the direct inhibitory effects of GPR120 on the nuclear factor kappa B pathway are anti-inflammatory. Moreover, GPR120 activity can attenuate the adipocyte-mediated enhanced production of extracellular matrix-modulating factors in macrophages and can reduce their migration by a paracrine mechanism. - Highlights: • Agonizing GPR120 differentially regulates the pro-inflammatory adipocytokines. • Agonizing GPR120 in adipocytes attenuates NF-κB mediated IL-6 and CCL2 production. • Agonizing GPR120 concomitantly triggers a PKC mediated pro-inflammatory pathway. • However, the resulted effect in adipocytes remains anti-inflammatory. • Agonizing GPR120 in adipocytes reduces macrophage migration in a paracrine manner.« less

Rapid resensitization of purinergic receptor function in human platelets.

PubMed

Mundell, S J; Barton, J F; Mayo-Martin, M B; Hardy, A R; Poole, A W

2008-08-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), the P2Y(1) and P2Y(12) purinergic receptors. Recently, we demonstrated that both receptors desensitize and internalize in human platelets by differential kinase-dependent mechanisms. To demonstrate whether responses to P2Y(1) and P2Y(12) purinergic receptors resensitize in human platelets and determine the role of receptor traffic in this process. These studies were undertaken either in human platelets or in cells stably expressing epitope-tagged P2Y(1) and P2Y(12) purinergic receptor constructs. In this study we show for the first time that responses to both of these receptors can rapidly resensitize following agonist-dependent desensitization in human platelets. Further, we show that in human platelets or in 1321N1 cells stably expressing receptor constructs, the disruption of receptor internalization, dephosphorylation or subsequent receptor recycling is sufficient to block resensitization of purinergic receptor responses. We also show that, in platelets, internalization of both these receptors is dependent upon dynamin, and that this process is required for resensitization of responses. This study is therefore the first to show that both P2Y(1) and P2Y(12) receptor activities are rapidly and reversibly modulated in human platelets, and it reveals that the underlying mechanism requires receptor trafficking as an essential part of this process.

Important roles of P2Y receptors in the inflammation and cancer of digestive system.

PubMed

Wan, Han-Xing; Hu, Jian-Hong; Xie, Rei; Yang, Shi-Ming; Dong, Hui

2016-05-10

Purinergic signaling is important for many biological processes in humans. Purinoceptors P2Y are widely distributed in human digestive system and different subtypes of P2Y receptors mediate different physiological functions from metabolism, proliferation, differentiation to apoptosis etc. The P2Y receptors are essential in many gastrointestinal functions and also involve in the occurrence of some digestive diseases. Since different subtypes of P2Y receptors are present on the same cell of digestive organs, varying subtypes of P2Y receptors may have opposite or synergetic functions on the same cell. Recently, growing lines of evidence strongly suggest the involvement of P2Y receptors in the pathogenesis of several digestive diseases. In this review, we will focus on their important roles in the development of digestive inflammation and cancer. We anticipate that as the special subtypes of P2Y receptors are studied in depth, specific modulators for them will have good potentials to become promising new drugs to treat human digestive diseases in the near future.

Molecular recognition at adenine nucleotide (P2) receptors in platelets.

PubMed

Jacobson, Kenneth A; Mamedova, Liaman; Joshi, Bhalchandra V; Besada, Pedro; Costanzi, Stefano

2005-04-01

Transmembrane signaling through P2Y receptors for extracellular nucleotides controls a diverse array of cellular processes, including thrombosis. Selective agonists and antagonists of the two P2Y receptors present on the platelet surface-the G (q)-coupled P2Y (1) subtype and the G (i)-coupled P2Y (12) subtype-are now known. High-affinity antagonists of each have been developed from nucleotide structures. The (N)-methanocarba bisphosphate derivatives MRS2279 and MRS2500 are potent and selective P2Y (1) receptor antagonists. The carbocyclic nucleoside AZD6140 is an uncharged, orally active P2Y (12) receptor antagonist of nM affinity. Another nucleotide receptor on the platelet surface, the P2X (1) receptor, the activation of which may also be proaggregatory, especially under conditions of high shear stress, has high-affinity ligands, although high selectivity has not yet been achieved. Although alpha,beta-methylene-adenosine triphosphate (ATP) is the classic agonist for the P2X (1) receptor, where it causes rapid desensitization, the agonist BzATP is among the most potent in activating this subtype. The aromatic sulfonates NF279 and NF449 are potent antagonists of the P2X (1) receptor. The structures of the two platelet P2Y receptors have been modeled, based on a rhodopsin template, to explain the basis for nucleotide recognition within the putative transmembrane binding sites. The P2Y (1) receptor model, especially, has been exploited in the design and optimization of antagonists targeted to interact selectively with that subtype.

G protein-coupled receptor 30 localizes to the endoplasmic reticulum and is not activated by estradiol.

PubMed

Otto, Christiane; Rohde-Schulz, Beate; Schwarz, Gilda; Fuchs, Iris; Klewer, Mario; Brittain, Dominic; Langer, Gernot; Bader, Benjamin; Prelle, Katja; Nubbemeyer, Reinhard; Fritzemeier, Karl-Heinrich

2008-10-01

The classical estrogen receptor (ER) mediates genomic as well as rapid nongenomic estradiol responses. In case of genomic responses, the ER acts as a ligand-dependent transcription factor that regulates gene expression in estrogen target tissues. In contrast, nongenomic effects are initiated at the plasma membrane and lead to rapid activation of cytoplasmic signal transduction pathways. Recently, an orphan G protein-coupled receptor, GPR30, has been claimed to bind to and to signal in response to estradiol. GPR30 therefore might mediate some of the nongenomic estradiol effects. The present study was performed to clarify the controversy about the subcellular localization of GPR30 and to gain insight into the in vivo function of this receptor. In transiently transfected cells as well as cells endogenously expressing GPR30, we confirmed that the receptor localized to the endoplasmic reticulum. However, using radioactive estradiol, we observed only saturable, specific binding to the classical ER but not to GPR30. Estradiol stimulation of cells expressing GPR30 had no impact on intracellular cAMP or calcium levels. To elucidate the physiological role of GPR30, we performed in vivo experiments with estradiol and G1, a compound that has been claimed to act as selective GPR30 agonist. In two classical estrogen target organs, the uterus and the mammary gland, G1 did not show any estrogenic effect. Taken together, we draw the conclusion that GPR30 is still an orphan receptor.

P2Y receptor-mediated transient relaxation of rat longitudinal ileum preparations involves phospholipase C activation, intracellular Ca(2+) release and SK channel activation.

PubMed

Mader, Felix; Krause, Ludwig; Tokay, Tursonjan; Hakenberg, Oliver W; Köhling, Rüdiger; Kirschstein, Timo

2016-05-01

Purinergic signaling plays a major role in the enteric nervous system, where it governs gut motility through a number of P2X and P2Y receptors. The aim of this study was to investigate the P2Y receptor-mediated motility in rat longitudinal ileum preparations. Ileum smooth muscle strips were prepared from rats, and fixed in an organ bath. Isometric contraction and relaxation responses of the muscle strips were measured with force transducers. Drugs were applied by adding of stock solutions to the organ bath to yield the individual final concentrations. Application of the non-hydrolyzable P2 receptor agonists α,β-Me-ATP or 2-Me-S-ADP (10, 100 μmol/L) dose-dependently elicited a transient relaxation response followed by a sustained contraction. The relaxation response was largely blocked by SK channel blockers apamin (500 nmol/L) and UCL1684 (10 μmol/L), PLC inhibitor U73122 (100 μmol/L), IP3 receptor blocker 2-APB (100 μmol/L) or sarcoendoplasmic Ca(2+) ATPase inhibitor thapsigargin (1 μmol/L), but not affected by atropine, NO synthase blocker L-NAME or tetrodotoxin. Furthermore, α,β-Me-ATP-induced relaxation was suppressed by P2Y1 receptor antagonist MRS2179 (50 μmol/L) or P2Y13 receptor antagonist MRS2211 (100 μmol/L), and was abolished by co-application of the two antagonists, whereas 2-Me-S-ADP-induced relaxation was abolished by P2Y6 receptor antagonist MRS2578 (50 μmol/L). In addition, P2Y1 receptor antagonist MRS2500 (1 μmol/L) not only abolished α,β-Me-ATP-induced relaxation, but also suppressed 2-Me-S-ADP-induced relaxation. P2Y receptor agonist-induced transient relaxation of rat ileum smooth muscle strips is mediated predominantly by P2Y1 receptor, but also by P2Y6 and P2Y13 receptors, and involves PLC, IP3, Ca(2+) release and SK channel activation, but is independent of acetylcholine and NO release.

A critical review of fundamental controversies in the field of GPR30 research.

PubMed

Langer, Gernot; Bader, Benjamin; Meoli, Luca; Isensee, Jörg; Delbeck, Martina; Noppinger, Patricia Ruiz; Otto, Christiane

2010-01-01

The female sex hormone estradiol plays an important role in reproduction, mammary gland development, bone turnover, metabolism, and cardiovascular function. The effects of estradiol are mediated by two classical nuclear receptors, estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). In 2005, G-protein-coupled receptor 30 (GPR30) was claimed to act as a non-classical estrogen receptor that was also activated by the ERalpha and ERbeta antagonists tamoxifen and fulvestrant (ICI 182780). Despite many conflicting results regarding the potential role of GPR30 as an estrogen receptor, the official nomenclature was changed to GPER (G-protein-coupled estrogen receptor). This review revisits the inconsistencies that still exist in the literature and focuses on selected publications that basically address the following two questions: what is the evidence for and against the hypothesis that GPR30 acts as an estrogen receptor? What is the potential in vivo role of GPR30? Thus, in the first part we focus on conflicting results from in vitro studies analysing the subcellular localization of GPR30, its ability to bind (or not to bind) estradiol and to signal (or not to signal) in response to estradiol. In the second part, we discuss the strengths and limitations of four available GPR30 mouse models. We elucidate the potential impact of different targeting strategies on phenotypic diversity. Copyright 2010 Elsevier Inc. All rights reserved.

Small interfering RNA-mediated silencing of G-protein-coupled receptor 137 inhibits growth of osteosarcoma cells.

PubMed

Li, Hao; Fu, Xiaodong; Gao, Yingjian; Li, Xiaomiao; Shen, Yi; Wang, Weili

2018-06-01

Osteosarcoma is the most widespread primary carcinoma in bones. Osteosarcoma cells are highly metastatic and frequently develop resistance to chemotherapy making this disease harder to treat. This identifies an urgent need of novel therapeutic strategies for osteosarcoma. G-Protein-coupled receptor 137 (GPR137) is involved in several human cancers and may be a novel therapeutic target. The expression of GPR137 was assessed in one osteoblast and three human osteosarcoma cell lines via the quantitative real-time polymerase chain reaction and western blot assays. Stable GPR137 knockdown cell lines were established using an RNA interference lentivirus system. Viability, colony formation, and flow cytometry assays were performed to measure the effects of GPR137 depletion on cell growth. The underlying molecular mechanism was determined using signaling array analysis and western blot assays. GPR137 expression was higher in the three human osteosarcoma cell lines, Saos-2, U2OS, and SW1353, than in osteoblast hFOB 1.19 cells. Lentivirus-mediated small interfering RNA targeting GPR137 successfully knocked down GPR137 mRNA and protein expression in both Saos-2 and U2OS cells. In the absence of GPR137, cell viability and colony formation ability were seriously impaired. The extent of apoptosis was also increased in both cell lines. Moreover, AMP-activated protein kinase α, proline-rich AKT substrate of 40 kDa, AKT, and extracellular signal-regulated kinase phosphorylation levels were down-regulated in GPR137 knockdown cells. The results of this study highlight the crucial role of GPR137 in promoting osteosarcoma cell growth in vitro . GPR137 could serve as a potential therapeutic target against osteosarcoma.

Differential eosinophil and mast cell regulation: Mast cell viability and accumulation in inflammatory tissue are independent of proton-sensing receptor GPR65

PubMed Central

Zhu, Xiang; Mose, Eucabeth; Hogan, Simon P.

2014-01-01

Extracellular acidification has been observed in allergic inflammatory diseases. Recently, we demonstrated that the proton-sensing receptor G protein-coupled receptor 65 (GPR65) regulates eosinophil survival in an acidic environment in vitro and eosinophil accumulation in an allergic lung inflammation model. For mast cells, another inflammatory cell type critical for allergic responses, it remains unknown whether GPR65 is expressed and/or regulates mast cell viability. Thus, in the present study, we employed in vitro experiments and an intestinal anaphylaxis model in which both mastocytosis and eosinophilia can be observed, particularly in the gastrointestinal tract, to enable us to directly compare the effect of GPR65 expression on these two cell types. We identified GPR65 expression on mast cells; however, unlike eosinophil viability, mast cell viability in vitro is not affected by acidification or GPR65 expression. Mechanistically, we determined that mast cells do not respond to extracellular acidification with increased cAMP levels. Furthermore, in the intestinal anaphylaxis model, we observed a significant reduction of eosinophils (59.1 ± 9.2% decrease) in the jejunum of allergen-challenged GPR65-deficient mice compared with allergen-challenged wild-type mice, despite the degree of antigen sensitization and the expression levels of Th2 cytokines (Il4, Il13) and eosinophil chemokines (Ccl11, Ccl24) in the jejunum being comparable. In contrast, the accumulation of mast cells in allergen-challenged mice was not affected by GPR65 deficiency. In conclusion, our study demonstrates differential regulation of eosinophils and mast cells in inflammatory tissue, with mast cell viability and accumulation being independent of GPR65. PMID:24742990

P2Y2 Receptor and EGFR Cooperate to Promote Prostate Cancer Cell Invasion via ERK1/2 Pathway.

PubMed

Li, Wei-Hua; Qiu, Ying; Zhang, Hong-Quan; Tian, Xin-Xia; Fang, Wei-Gang

2015-01-01

As one member of G protein-coupled P2Y receptors, P2Y2 receptor can be equally activated by extracellular ATP and UTP. Our previous studies have proved that activation of P2Y2 receptor by extracellular ATP could promote prostate cancer cell invasion and metastasis in vitro and in vivo via regulating the expressions of some epithelial-mesenchymal transition/invasion-related genes (including IL-8, E-cadherin, Snail and Claudin-1), and the most significant change in expression of IL-8 was observed after P2Y2 receptor activation. However, the signaling pathway downstream of P2Y2 receptor and the role of IL-8 in P2Y2-mediated prostate cancer cell invasion remain unclear. Here, we found that extracellular ATP/UTP induced activation of EGFR and ERK1/2. After knockdown of P2Y2 receptor, the ATP -stimulated phosphorylation of EGFR and ERK1/2 was significantly suppressed. Further experiments showed that inactivation of EGFR and ERK1/2 attenuated ATP-induced invasion and migration, and suppressed ATP-mediated IL-8 production. In addition, knockdown of IL-8 inhibited ATP-mediated invasion and migration of prostate cancer cells. These findings suggest that P2Y2 receptor and EGFR cooperate to upregulate IL-8 production via ERK1/2 pathway, thereby promoting prostate cancer cell invasion and migration. Thus blocking of the P2Y2-EGFR-ERK1/2 pathway may provide effective therapeutic interventions for prostate cancer.

Interaction between G Protein-Coupled Receptor 143 and Tyrosinase: Implications for Understanding Ocular Albinism Type 1.

PubMed

De Filippo, Elisabetta; Schiedel, Anke C; Manga, Prashiela

2017-02-01

Developmental eye defects in X-linked ocular albinism type 1 are caused by G-protein coupled receptor 143 (GPR143) mutations. Mutations result in dysfunctional melanosome biogenesis and macromelanosome formation in pigment cells, including melanocytes and retinal pigment epithelium. GPR143, primarily expressed in pigment cells, localizes exclusively to endolysosomal and melanosomal membranes unlike most G protein-coupled receptors, which localize to the plasma membrane. There is some debate regarding GPR143 function and elucidating the role of this receptor may be instrumental for understanding neurogenesis during eye development and for devising therapies for ocular albinism type I. Many G protein-coupled receptors require association with other proteins to function. These G protein-coupled receptor-interacting proteins also facilitate fine-tuning of receptor activity and tissue specificity. We therefore investigated potential GPR143 interaction partners, with a focus on the melanogenic enzyme tyrosinase. GPR143 coimmunoprecipitated with tyrosinase, while confocal microscopy demonstrated colocalization of the proteins. Furthermore, tyrosinase localized to the plasma membrane when coexpressed with a GPR143 trafficking mutant. The physical interaction between the proteins was confirmed using fluorescence resonance energy transfer. This interaction may be required in order for GPR143 to function as a monitor of melanosome maturation. Identifying tyrosinase as a potential GPR143 binding protein opens new avenues for investigating the mechanisms that regulate pigmentation and neurogenesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Expression of Cannabinoid Receptors in Human Osteoarthritic Cartilage: Implications for Future Therapies.

PubMed

Dunn, Sara L; Wilkinson, Jeremy Mark; Crawford, Aileen; Bunning, Rowena A D; Le Maitre, Christine L

2016-01-01

Introduction: Cannabinoids have shown to reduce joint damage in animal models of arthritis and reduce matrix metalloproteinase expression in primary human osteoarthritic (OA) chondrocytes. The actions of cannabinoids are mediated by a number of receptors, including cannabinoid receptors 1 and 2 (CB1 and CB2), G-protein-coupled receptors 55 and 18 (GPR55 and GPR18), transient receptor potential vanilloid-1 (TRPV1), and peroxisome proliferator-activated receptors alpha and gamma (PPARα and PPARγ). However, to date very few studies have investigated the expression and localization of these receptors in human chondrocytes, and expression during degeneration, and thus their potential in clinical applications is unknown. Methods: Human articular cartilage from patients with symptomatic OA was graded histologically and the expression and localization of cannabinoid receptors within OA cartilage and underlying bone were determined immunohistochemically. Expression levels across regions of cartilage and changes with degeneration were investigated. Results: Expression of all the cannabinoid receptors investigated was observed with no change with grade of degeneration seen in the expression of CB1, CB2, GPR55, PPARα, and PPARγ. Conversely, the number of chondrocytes within the deep zone of cartilage displaying immunopositivity for GPR18 and TRPV1 was significantly decreased in degenerate cartilage. Receptor expression was higher in chondrocytes than in osteocytes in the underlying bone. Conclusions: Chondrocytes from OA joints were shown to express a wide range of cannabinoid receptors even in degenerate tissues, demonstrating that these cells could respond to cannabinoids. Cannabinoids designed to bind to receptors inhibiting the catabolic and pain pathways within the arthritic joint, while avoiding psychoactive effects, could provide potential arthritis therapies.

GPR30: A G protein-coupled receptor for estrogen.

PubMed

Prossnitz, Eric R; Arterburn, Jeffrey B; Sklar, Larry A

2007-02-01

Estrogen is a critical steroid in human physiology exerting its effect both at the transcriptional level as well as at the level of rapid intracellular signaling through second messengers. Many of estrogen's transcriptional effects have long been known to be mediated through classical nuclear steroid receptors but recent studies also demonstrate the existence of a 7-transmembrane G protein-coupled receptor, GPR30 that responds to estrogen with rapid cellular signaling. There is currently controversy over the ability of classical estrogen receptors to recapitulate GPR30-mediated signaling mechanisms and vice versa. This article will summarize recent literature and address the relationship between GPR30 and conventional estrogen receptor signaling.

G-protein-coupled receptor 81 promotes a malignant phenotype in breast cancer through angiogenic factor secretion.

PubMed

Lee, Yu Jin; Shin, Kyeong Jin; Park, Soo-Ah; Park, Kyeong Su; Park, Seorim; Heo, Kyun; Seo, Young-Kyo; Noh, Dong-Young; Ryu, Sung Ho; Suh, Pann-Ghill

2016-10-25

G-protein-coupled receptor 81 (GPR81) functions as a receptor for lactate and plays an important role in the regulation of anti-lipolytic effects in adipocytes. However, to data, a role for GPR81 in the tumor microenvironment has not been clearly defined. Here, GPR81 expression in breast cancer patients and several breast cancer cell lines was significantly increased compared with normal mammary tissues and cells. GPR81 knockdown resulted in impaired breast cancer growth and led to apoptosis both in vitro and in vivo. Furthermore, the inhibition of GPR81 signaling suppressed angiogenesis through a phosphoinositide 3-OH kinase (PI3K)/Akt-cAMP response element binding protein (CREB) pathway, which led to decreased production of the pro-angiogenic mediator amphiregulin (AREG). Overall, these findings identify GPR81 as a tumor-promoting receptor in breast cancer progression and suggest a novel mechanism that regulates GPR81-dependent activation of the PI3K/Akt signaling axis in tumor microenvironment.

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta

PubMed Central

Guns, Pieter-Jan D F; Korda, András; Crauwels, Herta M; Van Assche, Tim; Robaye, Bernard; Boeynaems, Jean-Marie; Bult, Hidde

2005-01-01

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5′ triphosphate (ATP), uridine 5′ triphosphate (UTP), uridine 5′ diphosphate (UDP); >90%) or partial (adenosine 5′ diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP∼UTP∼ADP>adenosine 5′-[γ-thio] triphosphate (ATPγS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4-deficient mice displayed unaltered responses to ATP and UTP. The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53±0.07) was compatible with literature, but the pKb for UTP (5.19±0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y2 receptor subtype(s). Further, pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2′-deoxy-N6-methyladenosine3′,5′-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation. PMID:15997227

Virtual and biomolecular screening converge on a selective agonist for GPR30.

PubMed

Bologa, Cristian G; Revankar, Chetana M; Young, Susan M; Edwards, Bruce S; Arterburn, Jeffrey B; Kiselyov, Alexander S; Parker, Matthew A; Tkachenko, Sergey E; Savchuck, Nikolay P; Sklar, Larry A; Oprea, Tudor I; Prossnitz, Eric R

2006-04-01

Estrogen is a hormone critical in the development, normal physiology and pathophysiology of numerous human tissues. The effects of estrogen have traditionally been solely ascribed to estrogen receptor alpha (ERalpha) and more recently ERbeta, members of the soluble, nuclear ligand-activated family of transcription factors. We have recently shown that the seven-transmembrane G protein-coupled receptor GPR30 binds estrogen with high affinity and resides in the endoplasmic reticulum, where it activates multiple intracellular signaling pathways. To differentiate between the functions of ERalpha or ERbeta and GPR30, we used a combination of virtual and biomolecular screening to isolate compounds that selectively bind to GPR30. Here we describe the identification of the first GPR30-specific agonist, G-1 (1), capable of activating GPR30 in a complex environment of classical and new estrogen receptors. The development of compounds specific to estrogen receptor family members provides the opportunity to increase our understanding of these receptors and their contribution to estrogen biology.

Molecular pharmacology of promiscuous seven transmembrane receptors sensing organic nutrients.

PubMed

Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

2009-09-01

A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed in taste tissue, the gastrointestinal tract, endocrine glands, adipose tissue, and/or kidney. These receptors thus hold the potential to act as sensors of food intake, regulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue. The promiscuous tendency in ligand recognition of these receptors is in contrast to the typical specific interaction with one physiological agonist seen for most receptors, which challenges the classic "lock-and-key" concept. We here review the molecular mechanisms of nutrient sensing of the calcium-sensing receptor, the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3, which are sensing L-alpha-amino acids, the carbohydrate-sensing T1R2/T1R3 receptor, the proteolytic degradation product sensor GPR93 (also termed GPR92), and the free fatty acid (FFA) sensing receptors FFA1, FFA2, FFA3, GPR84, and GPR120. The involvement of the individual receptors in sensing of food intake has been validated to different degrees because of limited availability of specific pharmacological tools and/or receptor knockout mice. However, as a group, the receptors represent potential drug targets, to treat, for example, type II diabetes by mimicking food intake by potent agonists or positive allosteric modulators. The ligand-receptor interactions of the promiscuous receptors of organic nutrients thus remain an interesting subject of emerging functional importance.

Angelica dahurica Extracts Improve Glucose Tolerance through the Activation of GPR119

PubMed Central

Kim, Mi-Hwi; Choung, Jin-Seung; Oh, Yoon-Sin; Moon, Hong-Sub; Jun, Hee-Sook

2016-01-01

G protein-coupled receptor (GPR) 119 is expressed in pancreatic β-cells and intestinal L cells, and is involved in glucose-stimulated insulin secretion and glucagon-like peptide-1 (GLP-1) release, respectively. Therefore, the development of GPR119 agonists is a potential treatment for type 2 diabetes. We screened 1500 natural plant extracts for GPR119 agonistic actions and investigated the most promising extract, that from Angelica dahurica (AD), for hypoglycemic actions in vitro and in vivo. Human GPR119 activation was measured in GeneBLAzer T-Rex GPR119-CRE-bla CHO-K1 cells; intracellular cAMP levels and insulin secretion were measured in INS-1 cells; and GLP-1 release was measured in GLUTag cells. Glucose tolerance tests and serum plasma insulin levels were measured in normal C57BL6 mice and diabetic db/db mice. AD extract-treated cells showed significant increases in GPR119 activation, intracellular cAMP levels, GLP-1 levels and glucose-stimulated insulin secretion as compared with controls. In normal mice, a single treatment with AD extract improved glucose tolerance and increased insulin secretion. Treatment with multiple doses of AD extract or n-hexane fraction improved glucose tolerance in diabetic db/db mice. Imperatorin, phellopterin and isoimperatorin were identified in the active fraction of AD extract. Among these, phellopterin activated GPR119 and increased active GLP-1 and insulin secretion in vitro and enhanced glucose tolerance in normal and db/db mice. We suggest that phellopterin might have a therapeutic potential for the treatment of type 2 diabetes. PMID:27391814

Autocrine Regulation of UVA-Induced IL-6 Production via Release of ATP and Activation of P2Y Receptors

PubMed Central

Kawano, Ayumi; Kadomatsu, Remi; Ono, Miyu; Kojima, Shuji; Tsukimoto, Mitsutoshi; Sakamoto, Hikaru

2015-01-01

Extracellular nucleotides, such as ATP, are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. Exposure to the ultraviolet radiation A (UVA) component of sunlight causes molecular and cellular damage, and in this study, we investigated the involvement of extracellular nucleotides and P2 receptors in the UVA-induced cellular response. Human keratinocyte-derived HaCaT cells were irradiated with a single dose of UVA (2.5 J/cm2), and ATP release and interleukin (IL)-6 production were measured. ATP was released from cells in response to UVA irradiation, and the release was blocked by pretreatment with inhibitors of gap junction hemichannels or P2X7 receptor antagonist. IL-6 production was increased after UVA irradiation, and this increase was inhibited by ecto-nucleotidase or by antagonists of P2Y11 or P2Y13 receptor. These results suggest that UVA-induced IL-6 production is mediated by release of ATP through hemichannels and P2X7 receptor, followed by activation of P2Y11 and P2Y13 receptors. Interestingly, P2Y11 and P2Y13 were associated with the same pattern of IL-6 production, though they trigger different intracellular signaling cascades: Ca2+-dependent and PI3K-dependent, respectively. Thus, IL-6 production in response to UVA-induced ATP release involves at least two distinct pathways, mediated by activation of P2Y11 and P2Y13 receptors. PMID:26030257

G protein-coupled receptor 30 in tumor development.

PubMed

Wang, Dengfeng; Hu, Lina; Zhang, Guonan; Zhang, Lin; Chen, Chen

2010-08-01

Estrogen plays several important physiological and pathological functions in not only reproductive system but many other systems as well. Its transcriptional activation has been traditionally described as being mediated by classic nuclear estrogen receptors (ERs). It is however established recently that a novel functional estrogen transmembrane receptor, G protein-coupled receptor 30 (GPR30), modulates both rapid non-genomic events and genomic transcriptional events of estrogen. It has been demonstrated that GPR30 promotes the progress of estrogen-related tumors through mitogen-activated protein kinase (MAPK) signaling pathways. Effects mediated by GPR30 are maintained when classic ERs are absent or blocked. In addition, GPR30 is involved in drug resistance, which is often occurring during cancer treatments. All these new findings strongly imply that GPR30 may be an important therapeutic target for estrogen-related tumors. Simultaneously blocking both GPR30 and classic ERs may be a better strategy for the treatment of estrogen-related tumors.

Expression of the long-chain fatty acid receptor GPR120 in the gonadotropes of the mouse anterior pituitary gland.

PubMed

Moriyama, Ryutaro; Deura, Chikaya; Imoto, Shingo; Nose, Kazuhiro; Fukushima, Nobuyuki

2015-01-01

G-protein-coupled receptor 120 (GPR120) has been known to be a receptor of long-chain fatty acids. Here, we investigated GPR120 expression in the mouse pituitary gland via real-time PCR, in situ hybridization, and immunohistochemistry. GPR120 mRNA was abundantly expressed in the pituitary gland of ad-lib fed animals. In situ hybridization and immunohistochemistry revealed GPR120 expression in the gonadotropes of the anterior pituitary gland, but not in thyrotropes, somatotropes, lactotropes, corticotropes, melanotropes, and the posterior pituitary gland. Furthermore, 24 h of fasting induced an increase in GPR120 mRNA expression in the pituitary gland. These results demonstrate that GPR120 in mouse pituitary gonadotropes is upregulated by fasting and that it may play a role in controlling gonadotropin secretion.

Differential endosomal sorting of a novel P2Y12 purinoreceptor mutant.

PubMed

Cunningham, Margaret R; Nisar, Shaista P; Cooke, Alexandra E; Emery, Elizabeth D; Mundell, Stuart J

2013-05-01

P2Y12 receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y12 (P341A) whose P2Y12 receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y12 recycling and investigated P2Y12 -P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y12 . While WT P2Y12 rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y12 . Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y12 receptor traffic and explain the compromised receptor function in the platelets of the P2Y12 -P341A-expressing patient. © 2013 John Wiley & Sons A/S.

The oral lipid sensor GPR120 is not indispensable for the orosensory detection of dietary lipids in mice.

PubMed

Ancel, Déborah; Bernard, Arnaud; Subramaniam, Selvakumar; Hirasawa, Akira; Tsujimoto, Gozoh; Hashimoto, Toshihiro; Passilly-Degrace, Patricia; Khan, Naim-Akhtar; Besnard, Philippe

2015-02-01

Implication of the long-chain fatty acid (LCFA) receptor GPR120, also termed free fatty acid receptor 4, in the taste-guided preference for lipids is a matter of debate. To further unravel the role of GPR120 in the "taste of fat", the present study was conducted on GPR120-null mice and their wild-type littermates. Using a combination of morphological [i.e., immunohistochemical staining of circumvallate papillae (CVP)], behavioral (i.e., two-bottle preference tests, licking tests and conditioned taste aversion) and functional studies [i.e., calcium imaging in freshly isolated taste bud cells (TBCs)], we show that absence of GPR120 in the oral cavity was not associated with changes in i) gross anatomy of CVP, ii) LCFA-mediated increases in intracellular calcium levels ([Ca(2+)]i), iii) preference for oily and LCFA solutions and iv) conditioned avoidance of LCFA solutions. In contrast, the rise in [Ca(2+)]i triggered by grifolic acid, a specific GPR120 agonist, was dramatically curtailed when the GPR120 gene was lacking. Taken together, these data demonstrate that activation of lingual GPR120 and preference for fat are not connected, suggesting that GPR120 expressed in TBCs is not absolutely required for oral fat detection in mice. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

GPR107, a G-protein-coupled receptor essential for intoxication by Pseudomonas aeruginosa exotoxin A, localizes to the Golgi and is cleaved by furin.

PubMed

Tafesse, Fikadu G; Guimaraes, Carla P; Maruyama, Takeshi; Carette, Jan E; Lory, Stephen; Brummelkamp, Thijn R; Ploegh, Hidde L

2014-08-29

A number of toxins, including exotoxin A (PE) of Pseudomonas aeruginosa, kill cells by inhibiting protein synthesis. PE kills by ADP-ribosylation of the translation elongation factor 2, but many of the host factors required for entry, membrane translocation, and intracellular transport remain to be elucidated. A genome-wide genetic screen in human KBM7 cells was performed to uncover host factors used by PE, several of which were confirmed by CRISPR/Cas9-gene editing in a different cell type. Several proteins not previously implicated in the PE intoxication pathway were identified, including GPR107, an orphan G-protein-coupled receptor. GPR107 localizes to the trans-Golgi network and is essential for retrograde transport. It is cleaved by the endoprotease furin, and a disulfide bond connects the two cleaved fragments. Compromising this association affects the function of GPR107. The N-terminal region of GPR107 is critical for its biological function. GPR107 might be one of the long-sought receptors that associates with G-proteins to regulate intracellular vesicular transport. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Activation of P2Y6 receptors increases the voiding frequency in anaesthetized rats by releasing ATP from the bladder urothelium

PubMed Central

Carneiro, Inês; Timóteo, M Alexandrina; Silva, Isabel; Vieira, Cátia; Baldaia, Catarina; Ferreirinha, Fátima; Silva-Ramos, Miguel; Correia-de-Sá, Paulo

2014-01-01

BACKGROUND AND PURPOSE Despite the abundant expression of the UDP-sensitive P2Y6 receptor in urothelial cells and sub-urothelial myofibroblasts its role in the control of bladder function is not well understood. EXPERIMENTAL APPROACH We compared the effects of UDP and of the selective P2Y6 receptor agonist, PSB0474, on bladder urodynamics in anaesthetized rats; the voided fluid was tested for ATP bioluminescence. The isolated urinary bladder was used for in vitro myographic recordings and [3H]-ACh overflow experiments. KEY RESULTS Instillation of UDP or PSB0474 into the bladder increased the voiding frequency (VF) without affecting the amplitude (A) and the duration (Δt) of bladder contractions; an effect blocked by the P2Y6 receptor antagonist, MRS2578. Effects mediated by urothelial P2Y6 receptors required extrinsic neuronal circuitry as they were not detected in the isolated bladder. UDP-induced bladder hyperactvity was also prevented by blocking P2X3 and P2Y1 receptors, respectively, with A317491 and MRS2179 applied i.v.. UDP decreased [3H]-ACh release from stimulated bladder strips with urothelium, but not in its absence. Inhibitory effects of UDP were converted into facilitation by the P2Y1 receptor antagonist, MRS2179. The P2Y6 receptor agonist increased threefold ATP levels in the voided fluid. CONCLUSIONS AND IMPLICATIONS Activation of P2Y6 receptors increased the voiding frequency indirectly by releasing ATP from the urothelium and activation of P2X3 receptors on sub-urothelial nerve afferents. Bladder hyperactivity may be partly reversed following ATP hydrolysis to ADP by E-NTPDases, thereby decreasing ACh release from cholinergic nerves expressing P2Y1 receptors. PMID:24697602

P2Y6 receptor mediates colonic NaCl secretion via differential activation of cAMP-mediated transport

PubMed Central

Köttgen, Michael; Löffler, Thomas; Jacobi, Christoph; Nitschke, Roland; Pavenstädt, Hermann; Schreiber, Rainer; Frische, Sebastian; Nielsen, Søren; Leipziger, Jens

2003-01-01

Extracellular nucleotides are important regulators of epithelial ion transport. Here we investigated nucleotide-mediated effects on colonic NaCl secretion and the signal transduction mechanisms involved. Basolateral UDP induced a sustained activation of Cl– secretion, which was completely inhibited by 293B, a specific inhibitor of cAMP-stimulated basolateral KCNQ1/KCNE3 K+ channels. We therefore speculated that a basolateral P2Y6 receptor could increase cAMP. Indeed UDP elevated cAMP in isolated crypts. We identified an epithelial P2Y6 receptor using crypt [Ca2+]i measurements, RT-PCR, and immunohistochemistry. To investigate whether the rat P2Y6elevates cAMP, we coexpressed the P2Y1 or P2Y6 receptor together with the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel in Xenopus oocytes. A two-electrode voltage clamp was used to monitor nucleotide-induced Cl– currents. In oocytes expressing the P2Y1 receptor, ATP transiently activated the endogenous Ca2+-activated Cl– current, but not CFTR. In contrast, in oocytes expressing the P2Y6receptor, UDP transiently activated the Ca2+-activated Cl– current and subsequently CFTR. CFTR Cl– currents were identified by their halide conductance sequence. In summary we find a basolateral P2Y6 receptor in colonic epithelial cells stimulating sustained NaCl secretion by way of a synergistic increase of [Ca2+]i and cAMP. In support of these data P2Y6 receptor stimulation differentially activates CFTR in Xenopus oocytes. PMID:12569163

The emerging role of promiscuous 7TM receptors as chemosensors for food intake.

PubMed

Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

2010-01-01

In recent years, several highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized of which many are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids (FFAs) and are expressed in taste tissue, the gastrointestinal (GI) tract, endocrine glands, adipose tissue, and/or kidney. This has led to the hypothesis that these receptors may act as sensors of food intake modulating, for example, release of incretin hormones from the gut, insulin/glucagon from the pancreas, and leptin from adipose tissue. In the present review, we describe the molecular mechanisms of nutrient-sensing of the calcium-sensing receptor (CaR), the G protein-coupled receptor family C, group 6, subtype A (GPRC6A), and the taste1 receptor T1R1/T1R3-sensing L-α-amino acids; the carbohydrate-sensing T1R2/T1R3 receptor; the proteolytic degradation product sensor GPR93 (also termed GPR92); and the FFA sensing receptors FFA1, FFA2, FFA3, GPR84, and GPR120. Due to their omnipresent nature, the natural ligands have had limited usability in pharmacological/physiological studies which has hampered the elucidation of the physiological function and therapeutic prospect of their receptors. However, an increasing number of subtype-selective ligands and/or receptor knockout mice are being developed which at least for some of the receptors have validated them as promising drug targets in, for example, type II diabetes. Copyright © 2010 Elsevier Inc. All rights reserved.

SAR216471, an alternative to the use of currently available P2Y₁₂ receptor inhibitors?

PubMed

Delesque-Touchard, N; Pflieger, A M; Bonnet-Lignon, S; Millet, L; Salel, V; Boldron, C; Lassalle, G; Herbert, J M; Savi, P; Bono, F

2014-09-01

P2Y12 antagonism is a key therapeutic strategy in the management and prevention of arterial thrombosis. The objective of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of SAR216471, a novel P2Y12 receptor antagonist. SAR216471 blocks the binding of 2MeSADP to P2Y12 receptors in vitro (IC50=17 nM). This inhibition was shown to be reversible. It potently antagonized ADP-induced platelet aggregation in human and rat platelet-rich plasma (IC50=108 and 62 nM, respectively). It also inhibited platelet aggregation when blood was exposed to collagen or thromboxane A2. Its high selectivity was demonstrated against a large panel of receptors, enzymes, and ion channels. Despite its moderate bioavailability in rats, oral administration of SAR216471 resulted in a fast, potent, and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition were directly proportional to its circulating plasma levels. Pre-clinical study of SAR216471 in a rat shunt thrombosis model demonstrated a dose-dependent antithrombotic activity after oral administration (ED50=6.7 mg/kg). By comparison, ED50 values for clopidogrel, prasugrel and ticagrelor were 6.3, 0.35 and 2.6 mg/kg, respectively. Finally, the anti-hemostatic effect of SAR216471 and its competitors was investigated in a rat tail bleeding model, revealing a favorable safety profile of SAR216471. Together, these findings have established a reliable antiplatelet profile of SAR216471, and support its potential use in clinical practice as an alternative to currently available P2Y12 receptor antagonists. Copyright © 2014 Elsevier Ltd. All rights reserved.

GPR40/FFAR1 deficient mice increase noradrenaline levels in the brain and exhibit abnormal behavior.

PubMed

Aizawa, Fuka; Nishinaka, Takashi; Yamashita, Takuya; Nakamoto, Kazuo; Kurihara, Takashi; Hirasawa, Akira; Kasuya, Fumiyo; Miyata, Atsuro; Tokuyama, Shogo

2016-12-01

The free fatty acid receptor 1 (GPR40/FFAR1) is a G protein-coupled receptor, which is activated by long chain fatty acids. We have previously demonstrated that activation of brain GPR40/FFAR1 exerts an antinociceptive effect that is mediated by the modulation of the descending pain control system. However, it is unclear whether brain GPR40/FFAR1 contributes to emotional function. In this study, we investigated the involvement of GPR40/FFAR1 in emotional behavior using GPR40/FFAR1 deficient (knockout, KO) mice. The emotional behavior in wild and KO male mice was evaluated at 9-10 weeks of age by the elevated plus-maze test, open field test, social interaction test, and sucrose preference test. Brain monoamines levels were measured using LC-MS/MS. The elevated plus-maze test and open field tests revealed that the KO mice reduced anxiety-like behavior. There were no differences in locomotor activity or social behavior between the wild and KO mice. In the sucrose preference test, the KO mice showed reduction in sucrose preference and intake. The level of noradrenaline was higher in the hippocampus, medulla oblongata, hypothalamus and midbrain of KO mice. Therefore, these results suggest that brain GPR40/FFAR1 is associated with anxiety- and depression-related behavior regulated by the increment of noradrenaline in the brain. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

P2 receptor-stimulation influences axonal outgrowth in the developing hippocampus in vitro.

PubMed

Heine, C; Heimrich, B; Vogt, J; Wegner, A; Illes, P; Franke, Heike

2006-01-01

Extracellular ATP might act as a trophic factor on growing axons during development of the CNS via P2 receptors. In the present study the postnatal presence of selected P2 receptor subtypes was analyzed and their putative trophic capacity in entorhino-hippocampal slice co-cultures of mouse brain was tested. The effect of the P2 receptor ligands 2-methylthioadenosine-5'-triphosphate (P2X/Y receptor agonist) and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (P2X/Y receptor antagonist) on axonal growth and fiber density of biocytin-labeled hippocampal projections was compared both with untreated cultures and with cultures treated with artificial cerebrospinal fluid. After 10 days in vitro, double immunofluorescence labeling revealed the expression of P2X(1), P2X(2), P2X(4) as well as P2Y(1) and P2Y(2) receptors in the examined regions of entorhinal fiber termination. Further, quantitative analysis of identified biocytin-traced entorhinal fibers showed a significant increase in fiber density in the dentate gyrus after incubation of the slices with the P2 receptor agonist 2-methylthioadenosine-5'-triphosphate. This neurite outgrowth promoting effect was completely abolished by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid. Our in vitro data indicate that ATP via its P2X and P2Y receptors can shape hippocampal connectivity during development.

A Newly Discovered Antifibrotic Pathway Regulated by Two Fatty Acid Receptors: GPR40 and GPR84.

PubMed

Gagnon, Lyne; Leduc, Martin; Thibodeau, Jean-Francois; Zhang, Ming-Zhi; Grouix, Brigitte; Sarra-Bournet, Francois; Gagnon, William; Hince, Kathy; Tremblay, Mikaël; Geerts, Lilianne; Kennedy, Christopher R J; Hébert, Richard L; Gutsol, Alex; Holterman, Chet E; Kamto, Eldjonai; Gervais, Liette; Ouboudinar, Jugurtha; Richard, Jonathan; Felton, Alexandra; Laverdure, Alexandre; Simard, Jean-Christophe; Létourneau, Sylvie; Cloutier, Marie-Pier; Leblond, Francois A; Abbott, Shaun D; Penney, Christopher; Duceppe, Jean-Simon; Zacharie, Boulos; Dupuis, Jocelyn; Calderone, Angelino; Nguyen, Quang T; Harris, Raymond C; Laurin, Pierre

2018-05-01

Numerous clinical conditions can lead to organ fibrosis and functional failure. There is a great need for therapies that could effectively target pathophysiological pathways involved in fibrosis. GPR40 and GPR84 are G protein-coupled receptors with free fatty acid ligands and are associated with metabolic and inflammatory disorders. Although GPR40 and GPR84 are involved in diverse physiological processes, no evidence has demonstrated the relevance of GPR40 and GPR84 in fibrosis pathways. Using PBI-4050 (3-pentylbenzeneacetic acid sodium salt), a synthetic analog of a medium-chain fatty acid that displays agonist and antagonist ligand affinity toward GPR40 and GPR84, respectively, we uncovered an antifibrotic pathway involving these receptors. In experiments using Gpr40- and Gpr84-knockout mice in models of kidney fibrosis (unilateral ureteral obstruction, long-term post-acute ischemic injury, and adenine-induced chronic kidney disease), we found that GPR40 is protective and GPR84 is deleterious in these diseases. Moreover, through binding to GPR40 and GPR84, PBI-4050 significantly attenuated fibrosis in many injury contexts, as evidenced by the antifibrotic activity observed in kidney, liver, heart, lung, pancreas, and skin fibrosis models. Therefore, GPR40 and GPR84 may represent promising molecular targets in fibrosis pathways. We conclude that PBI-4050 is a first-in-class compound that may be effective for managing inflammatory and fibrosis-related diseases. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Arrestin Scaffolds NHERF1 to the P2Y12 Receptor to Regulate Receptor Internalization*

PubMed Central

Nisar, Shaista P.; Cunningham, Margaret; Saxena, Kunal; Pope, Robert J.; Kelly, Eamonn; Mundell, Stuart J.

2012-01-01

We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y12 receptor (P2Y12R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y12R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y12R internalization. In vitro and prior to agonist stimulation P2Y12R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization. PMID:22610101

Arrestin scaffolds NHERF1 to the P2Y12 receptor to regulate receptor internalization.

PubMed

Nisar, Shaista P; Cunningham, Margaret; Saxena, Kunal; Pope, Robert J; Kelly, Eamonn; Mundell, Stuart J

2012-07-13

We have recently shown in a patient with mild bleeding that the PDZ-binding motif of the platelet G protein-coupled P2Y(12) receptor (P2Y(12)R) is required for effective receptor traffic in human platelets. In this study we show for the first time that the PDZ motif-binding protein NHERF1 exerts a major role in potentiating G protein-coupled receptor (GPCR) internalization. NHERF1 interacts with the C-tail of the P2Y(12)R and unlike many other GPCRs, NHERF1 interaction is required for effective P2Y(12)R internalization. In vitro and prior to agonist stimulation P2Y(12)R/NHERF1 interaction requires the intact PDZ binding motif of this receptor. Interestingly on receptor stimulation NHERF1 no longer interacts directly with the receptor but instead binds to the receptor via the endocytic scaffolding protein arrestin. These findings suggest a novel model by which arrestin can serve as an adaptor to promote NHERF1 interaction with a GPCR to facilitate effective NHERF1-dependent receptor internalization.

Investigation of free fatty acid associated recombinant membrane receptor protein expression in HEK293 cells using Raman spectroscopy, calcium imaging, and atomic force microscopy.

PubMed

Lin, Juqiang; Xu, Han; Wu, Yangzhe; Tang, Mingjie; McEwen, Gerald D; Liu, Pin; Hansen, Dane R; Gilbertson, Timothy A; Zhou, Anhong

2013-02-05

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

Metalloprotease cleavage of the N terminus of the orphan G protein-coupled receptor GPR37L1 reduces its constitutive activity.

PubMed

Coleman, James L J; Ngo, Tony; Schmidt, Johannes; Mrad, Nadine; Liew, Chu Kong; Jones, Nicole M; Graham, Robert M; Smith, Nicola J

2016-04-12

Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gα(s) when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3',5'-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gα(s) or Gα(i) signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue. Copyright © 2016, American Association for the Advancement of Science.

GPR119 agonists: a promising approach for T2DM treatment? A SWOT analysis of GPR119.

PubMed

Kang, Sang-Uk

2013-12-01

Ever since its advent as a promising therapeutic target for type 2 diabetes mellitus (T2DM), G-protein-coupled receptor 119 (GPR119) has received much interest from the pharmaceutical industry. This interest peaked in June 2010, when Sanofi-Aventis agreed to pay Metabolex (Cymabay Therapeutics) US$375 million for MBX-2982, which was a representative orally active GPR119 agonist. However, Sanofi-Aventis opted to terminate the deal in May 2011 and another leading GPR119 agonist, GSK1292263, had a loss of efficacy during its clinical trial. In this review, I discuss the pros and cons of GPR119 through a strengths, weaknesses, opportunities, and threats (SWOT) analysis and propose development strategies for the eventual success of a GPR119 agonist development program. Copyright © 2013 Elsevier Ltd. All rights reserved.

MiR-338 controls BPA-triggered pancreatic islet insulin secretory dysfunction from compensation to decompensation by targeting Pdx-1.

PubMed

Wei, Jie; Ding, Dongxiao; Wang, Tao; Liu, Qiong; Lin, Yi

2017-12-01

Bisphenol A (BPA) can disrupt glucose homeostasis and impair pancreatic islet function; however, the mechanisms behind these effects are poorly understood. Male mice (4 wk old) were treated with BPA (50 or 500 μg/kg/d) for 8 wk. Whole-body glucose homeostasis, pancreatic islet morphology and function, and miR-338-mediated molecular signal transduction analyses were examined. We showed that BPA treatment led to a disruption of glucose tolerance and a compensatory increase of pancreatic islets insulin secretion and pancreatic and duodenal homeobox 1 ( Pdx1 ) expression in mice. Inhibition of Pdx1 reduced glucose-stimulated insulin secretion and ATP production in the islets of BPA-exposed mice. Based on primary pancreatic islets, we also confirmed that miR-338 regulated Pdx1 and thus contributed to BPA-induced insulin secretory dysfunction from compensation to decompensation. Short-term BPA exposure downregulated miR-338 through activation of G-protein-coupled estrogen receptor 1 (Gpr30), whereas long-term BPA exposure upregulated miR-338 through suppression of glucagon-like peptide 1 receptor (Glp1r). Taken together, our results reveal a molecular mechanism, whereby BPA regulates Gpr30/Glp1r to mediate the expression of miR-338, which acts to control Pdx1-dependent insulin secretion. The Gpr30/Glp1r-miR-338-Pdx1 axis should be represented as a novel mechanism by which BPA induces insulin secretory dysfunction in pancreatic islets.-Wei, J., Ding, D., Wang, T., Liu, Q., Lin, Y. MiR-338 controls BPA-triggered pancreatic islet insulin secretory dysfunction from compensation to decompensation by targeting Pdx-1. © FASEB.

Genetic background can result in a marked or minimal effect of gene knockout (GPR55 and CB2 receptor) in experimental autoimmune encephalomyelitis models of multiple sclerosis.

PubMed

Sisay, Sofia; Pryce, Gareth; Jackson, Samuel J; Tanner, Carolyn; Ross, Ruth A; Michael, Gregory J; Selwood, David L; Giovannoni, Gavin; Baker, David

2013-01-01

Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 (tm1Zim)) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 (Dgen)) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 (tm1Zim) mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

PubMed Central

Jackson, Samuel J.; Tanner, Carolyn; Ross, Ruth A.; Michael, Gregory J.; Selwood, David L.; Giovannoni, Gavin; Baker, David

2013-01-01

Endocannabinoids and some phytocannabinoids bind to CB1 and CB2 cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 tm1Zim) CB2 cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB2 receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB2 (Cnr2 Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB2 receptor deletion was lost. Likewise CB1 receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB1 receptor rather than a CB2 receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility. PMID:24130809

Nicotinic acid increases adiponectin secretion from differentiated bovine preadipocytes through G-protein coupled receptor signaling.

PubMed

Kopp, Christina; Hosseini, Afshin; Singh, Shiva P; Regenhard, Petra; Khalilvandi-Behroozyar, Hamed; Sauerwein, Helga; Mielenz, Manfred

2014-11-18

The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001) and the mRNA abundances of GPR109A (p ≤ 0.05) and chemerin (p ≤ 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.

The Orphan G Protein-coupled Receptor Gpr175 (Tpra40) Enhances Hedgehog Signaling by Modulating cAMP Levels.

PubMed

Singh, Jaskirat; Wen, Xiaohui; Scales, Suzie J

2015-12-04

The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain

PubMed Central

Andó, RD; Méhész, B; Gyires, K; Illes, P; Sperlágh, B

2010-01-01

Background and purpose: This study was undertaken to compare the analgesic activity of antagonists acting at P2X1, P2X7, and P2Y12 receptors and agonists acting at P2Y1, P2Y2, P2Y4, and P2Y6 receptors in neuropathic, acute, and inflammatory pain. Experimental approach: The effect of the wide spectrum P2 receptor antagonist PPADS, the selective P2X7 receptor antagonist Brilliant Blue G (BBG), the P2X1 receptor antagonist (4,4′,4″,4-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid, octasodium salt (NF449) and (8,8′-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-1,3,5-naphthalene-trisulphonic acid, hexasodium salt (NF023), the P2Y12 receptor antagonist (2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propylester (MRS2395), the selective P2Y1 receptor agonist ([[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS2365), the P2Y2/P2Y4 agonist uridine-5′-triphosphate (UTP), and the P2Y4/P2Y6 agonist uridine-5′-diphosphate (UDP) were examined on mechanical allodynia in the Seltzer model of neuropathic pain, on acute thermal nociception, and on the inflammatory pain and oedema induced by complete Freund's adjuvant (CFA). Key results: MRS2365, MRS2395 and UTP, but not the other compounds, significantly alleviated mechanical allodynia in the neuropathic pain model, with the following rank order of minimal effective dose (mED) values: MRS2365 > MRS2395 > UTP. All compounds had a dose-dependent analgesic action in acute pain except BBG, which elicited hyperalgesia at a single dose. The rank order of mED values in acute pain was the following: MRS2365 > MRS2395 > NF449 > NF023 > UDP = UTP > PPADS. MRS2365 and MRS2395 had a profound, while BBG had a mild effect on inflammatory pain, with a following rank order of mED values: MRS2395 > MRS2365 > BBG. None of the tested compounds had significant action on oedema evoked by intraplantar injection of CFA. Conclusions and implications: Our results show that antagonism at P2X1, P2Y12, and P2X7 receptors and agonism at P2Y1 receptors define promising therapeutic strategies in acute, neuropathic, and inflammatory pain respectively. PMID:20136836

A comparative analysis of the activity of ligands acting at P2X and P2Y receptor subtypes in models of neuropathic, acute and inflammatory pain.

PubMed

Andó, R D; Méhész, B; Gyires, K; Illes, P; Sperlágh, B

2010-03-01

This study was undertaken to compare the analgesic activity of antagonists acting at P2X1, P2X7, and P2Y12 receptors and agonists acting at P2Y1, P2Y2, P2Y4, and P2Y6 receptors in neuropathic, acute, and inflammatory pain. The effect of the wide spectrum P2 receptor antagonist PPADS, the selective P2X7 receptor antagonist Brilliant Blue G (BBG), the P2X1 receptor antagonist (4,4',4'',4-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid, octasodium salt (NF449) and (8,8'-[carbonylbis(imino-3,1-phenylenecarbonylimino)]bis-1,3,5-naphthalene-trisulphonic acid, hexasodium salt (NF023), the P2Y12 receptor antagonist (2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propylester (MRS2395), the selective P2Y1 receptor agonist ([[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS2365), the P2Y2/P2Y4 agonist uridine-5'-triphosphate (UTP), and the P2Y4/P2Y6 agonist uridine-5'-diphosphate (UDP) were examined on mechanical allodynia in the Seltzer model of neuropathic pain, on acute thermal nociception, and on the inflammatory pain and oedema induced by complete Freund's adjuvant (CFA). MRS2365, MRS2395 and UTP, but not the other compounds, significantly alleviated mechanical allodynia in the neuropathic pain model, with the following rank order of minimal effective dose (mED) values: MRS2365 > MRS2395 > UTP. All compounds had a dose-dependent analgesic action in acute pain except BBG, which elicited hyperalgesia at a single dose. The rank order of mED values in acute pain was the following: MRS2365 > MRS2395 > NF449 > NF023 > UDP = UTP > PPADS. MRS2365 and MRS2395 had a profound, while BBG had a mild effect on inflammatory pain, with a following rank order of mED values: MRS2395 > MRS2365 > BBG. None of the tested compounds had significant action on oedema evoked by intraplantar injection of CFA. Our results show that antagonism at P2X1, P2Y12, and P2X7 receptors and agonism at P2Y1 receptors define promising therapeutic strategies in acute, neuropathic, and inflammatory pain respectively.

Metabolic effects of orally administered small-molecule agonists of GPR55 and GPR119 in multiple low-dose streptozotocin-induced diabetic and incretin-receptor-knockout mice.

PubMed

McKillop, Aine M; Moran, Brian M; Abdel-Wahab, Yasser H A; Gormley, Noella M; Flatt, Peter R

2016-12-01

Abnormal cannabidiol (Abn-CBD) and AS-1269574 are potent selective agonists for GPR55 and GPR119, respectively. The present study evaluated the actions and ability of these small-molecule agonists to counteract experimental diabetes in mice. Diabetes was induced in NIH Swiss mice by five consecutive daily intraperitoneal injections of 40 mg/(kg body weight) streptozotocin. Diabetic mice received daily oral administration of Abn-CBD or AS-1269574 (0.1 μmol/kg) or saline vehicle (0.9% wt/vol. NaCl) over 28 days. Body weight, food intake, fluid intake, plasma glucose, insulin, glucose tolerance, insulin release, lipid profile and pancreatic morphology were examined. Mechanism of action of agonists was assessed in acute studies using incretin-receptor-knockout mice. Abn-CBD and AS-1269574 decreased plasma glucose (20-26%, p < 0.05) and increased circulating insulin (47-48%, p < 0.05) by 10-28 days, compared with saline-treated diabetic controls. Food intake and polydipsia were reduced by both agonists (21-23%, p < 0.05 and 33-35%, p < 0.01, respectively). After 28 days of treatment, plasma glucagon concentrations were reduced (p < 0.01) and glucose tolerance was enhanced by 19-44% by Abn-CBD (p < 0.05 or p < 0.001) and AS-1269574 (p < 0.05 to p < 0.001). Plasma insulin responses were improved (p < 0.01) and insulin resistance was decreased (p < 0.05 or p < 0.01) in both Abn-CBD- and AS-1269574-treated groups. Triacylglycerols were decreased by 19% with Abn-CBD (p < 0.05) and 32% with AS-1269574 (p < 0.01) while total cholesterol was reduced by 17% (p < 0.01) and 15% (p < 0.05), respectively. Both agonists enhanced beta cell proliferation (p < 0.001) although islet area was unchanged. Acute studies in Gipr- and Glp1r-knockout mice revealed an important role for the glucagon-like peptide 1 (GLP-1) receptor in the actions of both agonists, with the glucose-lowering effects of Abn-CBD also partly mediated through the glucose-dependent insulinotropic peptide (GIP) receptor. These data highlight the potential for fatty acid G-protein-coupled receptor-based therapies as novel insulinotropic and glucose-lowering agents acting partly through the activation of incretin receptors.

Agonist and antagonist effects of diadenosine tetraphosphate, a platelet dense granule constituent, on platelet P2Y1, P2Y12 and P2X1 receptors.

PubMed

Chang, Hung; Yanachkov, Ivan B; Michelson, Alan D; Li, YouFu; Barnard, M R; Wright, George E; Frelinger, Andrew L

2010-02-01

Diadenosine 5',5'''-P(1),P(4)- tetraphosphate (Ap(4)A) is stored in platelet dense granules, but its effects on platelet function are not well understood. We examined the effects of Ap(4)A on platelet purinergic receptors P2Y(1), P2Y(12) and P2X(1). Flow cytometry was used to measure the effects of Ap(4)A in the presence or absence of ADP on: a) P2Y(12)-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y(1)-mediated increase in platelet cytosolic Ca(2+), and c) P2X(1)-mediated intraplatelet entry of extracellular Ca(2+). ADP-stimulated platelet shape change (P2Y(1)-mediated) and aggregation (P2Y(1)- and P2Y(12)-mediated) were measured optically. Ap(4)A inhibited 3 microM ADP-induced: a) platelet aggregation (IC(50) 9.8+/-2.8 microM), b) P2Y(1)-mediated shape change, c) P2Y(1)-mediated increase in platelet cytosolic Ca(2+) (IC(50) 40.8+/-12.3 microM), and d) P2Y(12)-mediated decrease in VASP phosphorylation (IC(50)>250 microM). In the absence of added ADP, Ap(4)A had agonist effects on platelet P2X(1) and P2Y(12), but not P2Y(1), receptors. Ap(4)A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y(1) and P2Y(12) receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X(1) and P2Y(12) receptors. Copyright 2009 Elsevier Ltd. All rights reserved.

Agonist and Antagonist Effects of Diadenosine Tetraphosphate, a Platelet Dense Granule Constituent, on Platelet P2Y1, P2Y12 and P2X1 Receptors

PubMed Central

Chang, Hung; Yanachkov, Ivan B.; Michelson, Alan D.; Li, YouFu; Barnard, M.R.; Wright, George E.; Frelinger, Andrew L.

2010-01-01

Introduction Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) is stored in platelet dense granules, but its effects on platelet function are not well understood. Methods and Results We examined the effects of Ap4A on platelet purinergic receptors P2Y1, P2Y12 and P2X1. Flow cytometry was used to measure the effects of Ap4A in the presence or absence of ADP on: a) P2Y12-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y1-mediated increase in platelet cytosolic Ca2+, and c) P2X1-mediated intraplatelet entry of extracellular Ca2+. ADP-stimulated platelet shape change (P2Y1-mediated) and aggregation (P2Y1- and P2Y12-mediated) were measured optically. Ap4A inhibited 3 µM ADP-induced: a) platelet aggregation (IC50 9.8 ± 2.8 µM), b) P2Y1-mediated shape change, c) P2Y1-mediated increase in platelet cytosolic Ca2+ (IC50 40.8 ± 12.3 µM), and d) P2Y12-mediated decrease in VASP phosphorylation (IC50 >250 µM). In the absence of added ADP, Ap4A had agonist effects on platelet P2X1 and P2Y12, but not P2Y1, receptors. Conclusion Ap4A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y1 and P2Y12 receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X1 and P2Y12 receptors. PMID:19945153

An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets.

PubMed

Nisar, Shaista; Daly, Martina E; Federici, Augusto B; Artoni, Andrea; Mumford, Andrew D; Watson, Stephen P; Mundell, Stuart J

2011-11-17

The platelet P2Y(12) purinoceptor (P2Y(12)R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y(12)R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)-binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y(12)R (P341A) that is associated with reduced expression of the P2Y(12)R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y(12)R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic.

The metabotropic P2Y4 receptor participates in the commitment to differentiation and cell death of human neuroblastoma SH-SY5Y cells.

PubMed

Cavaliere, Fabio; Nestola, Valeria; Amadio, Susanna; D'Ambrosi, Nadia; Angelini, Daniela F; Sancesario, Giuseppe; Bernardi, Giorgio; Volonté, Cinzia

2005-02-01

Extracellular nucleotides exert a variety of biological actions through different subtypes of P2 receptors. Here we characterized in the human neuroblastoma SH-SY5Y cells the simultaneous presence of various P2 receptors, belonging to the P2X ionotropic and P2Y metabotropic families. Western blot analysis detected the P2X1,2,4,5,6,7 and P2Y1,2,4,6, but not the P2X3 and P2Y12 receptors. We then investigated which biological effects were mediated by the P2Y4 subtype and its physiological pyrimidine agonist UTP. We found that neuronal differentiation of the SH-SY5Y cells with dibutiryl-cAMP increased the expression of the P2Y4 protein and that UTP itself was able to positively interfere with neuritogenesis. Moreover, transient transfection and activation of P2Y4 also facilitated neuritogenesis in SH-SY5Y cells, as detected by morphological phase contrast analysis and confocal examination of neurofilament proteins NFL. This was concurrent with increased transcription of immediate-early genes linked to differentiation such as cdk-5 and NeuroD6, and activity of AP-1 transcription family members such as c-fos, fos-B, and jun-D. Nevertheless, a prolonged activation of the P2Y4 receptor by UTP also induced cell death, both in naive, differentiated, and P2Y4-transfected SH-SY5Y cells, as measured by direct count of intact nuclei and cytofluorimetric analysis of damaged DNA. Taken together, our data indicate that the high expression and activation of the P2Y4 receptor participates in the neuronal differentiation and commitment to death of SH-SY5Y cells.

Gene-by-environment effect of house dust mite on purinergic receptor P2Y12 (P2RY12) and lung function in children with asthma.

PubMed

Bunyavanich, S; Boyce, J A; Raby, B A; Weiss, S T

2012-02-01

Distinct receptors likely exist for leukotriene (LT)E(4), a potent mediator of airway inflammation. Purinergic receptor P2Y12 is needed for LTE(4)-induced airways inflammation, and P2Y12 antagonism attenuates house dust mite-induced pulmonary eosinophilia in mice. Although experimental data support a role for P2Y12 in airway inflammation, its role in human asthma has never been studied. To test for association between variants in the P2Y12 gene (P2RY12) and lung function in human subjects with asthma, and to examine for gene-by-environment interaction with house dust mite exposure. Nineteen single nucleotide polymorphisms (SNPs) in P2RY12 were genotyped in 422 children with asthma and their parents (n = 1266). Using family based methods, we tested for associations between these SNPs and five lung function measures. We performed haplotype association analyses and tested for gene-by-environment interactions using house dust mite exposure. We used the false discovery rate to account for multiple comparisons. Five SNPs in P2RY12 were associated with multiple lung function measures (P-values 0.006–0.025). Haplotypes in P2RY12 were also associated with lung function (P-values 0.0055–0.046). House dust mite exposure modulated associations between P2RY12 and lung function, with minor allele homozygotes exposed to house dust mite demonstrating worse lung function than those unexposed (significant interaction P-values 0.0028–0.040). The P2RY12 variants were associated with lung function in a large family-based asthma cohort. House dust mite exposure caused significant gene-by-environment effects. Our findings add the first human evidence to experimental data supporting a role for P2Y12 in lung function. P2Y12 could represent a novel target for asthma treatment.

Anti-Inflammatory and Insulin-Sensitizing Effects of Free Fatty Acid Receptors.

PubMed

Miyamoto, Junki; Kasubuchi, Mayu; Nakajima, Akira; Kimura, Ikuo

2017-01-01

Chronic low-grade inflammation in macrophages and adipose tissues can promote the development of obesity and type 2 diabetes. Free fatty acids (FFAs) have important roles in various tissues, acting as both essential energy sources and signaling molecules. FFA receptors (FFARs) can modulate inflammation in various types of cells and tissues; however the underlying mechanisms mediating these effects are unclear. FFARs are activated by specific FFAs; for example, GPR40 and GPR120 are activated by medium and long chain FFAs, GPR41 and GPR43 are activated by short chain FFAs, and GPR84 is activated by medium-chain FFAs. To date, a number of studies associated with the physiological functions of G protein-coupled receptors (GPCRs) have reported that these GPCRs are expressed in various tissues and involved in inflammatory and metabolic responses. Thus, the development of selective agonists or antagonists for various GPCRs may facilitate the establishment of novel therapies for the treatment of various diseases. In this review, we summarize current literature describing the potential of GPCRs as therapeutic targets for inflammatory and metabolic disorders.

FFA4 receptor (GPR120): A hot target for the development of anti-diabetic therapies.

PubMed

Liu, Hong-Da; Wang, Wen-bo; Xu, Zhi-gang; Liu, Chang-hong; He, Dong-fang; Du, Lv-Pei; Li, Min-Yong; Yu, Xiao; Sun, Jin-peng

2015-09-15

Free Fatty Acid 4 receptor (FFA4 receptor or GPR120), a rhodopsin-like G protein coupled receptor (GPCR) subfamily member, is a receptor that senses specific fatty acids such as ω-3 fatty acid in fish oil or the endogenous signaling lipid, PHASA. FFA4 receptor is enriched in lung, colon and adipose tissue but is also detected in many other tissues and cells. The activation of FFA4 receptor has multiple effects, including but not limited to inhibition of inflammation, improving insulin sensitivity and adipogenesis, and regulating hormone secretion from the gastro-intestinal system and pancreatic islets. The important role of FFA4 receptor in maintaining metabolic homeostasis strongly indicates the great potential of selective FFA4 receptor agonizts to treat diabetes and inflammation. In this review, we summarize recent research progress in the physiological and biochemical studies of FFA4 receptor and highlight its underlying signaling mechanisms and ligand identification to assist future research to exploit FFA4 receptor as a drug target. Copyright © 2015 Elsevier B.V. All rights reserved.

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

PubMed

Kanamarlapudi, Venkateswarlu; Owens, Sian E; Saha, Keya; Pope, Robert J; Mundell, Stuart J

2012-01-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

Twenty years of the G protein-coupled estrogen receptor GPER: Historical and personal perspectives.

PubMed

Barton, Matthias; Filardo, Edward J; Lolait, Stephen J; Thomas, Peter; Maggiolini, Marcello; Prossnitz, Eric R

2018-02-01

Estrogens play a critical role in many aspects of physiology, particularly female reproductive function, but also in pathophysiology, and are associated with protection from numerous diseases in premenopausal women. Steroids and the effects of estrogen have been known for ∼90 years, with the first evidence for a receptor for estrogen presented ∼50 years ago. The original ancestral steroid receptor, extending back into evolution more than 500 million years, was likely an estrogen receptor, whereas G protein-coupled receptors (GPCRs) trace their origins back into history more than one billion years. The classical estrogen receptors (ERα and ERβ) are ligand-activated transcription factors that confer estrogen sensitivity upon many genes. It was soon apparent that these, or novel receptors may also be responsible for the "rapid"/"non-genomic" membrane-associated effects of estrogen. The identification of an orphan GPCR (GPR30, published in 1996) opened a new field of research with the description in 2000 that GPR30 expression is required for rapid estrogen signaling. In 2005-2006, the field was greatly stimulated by two studies that described the binding of estrogen to GPR30-expressing cell membranes, followed by the identification of a GPR30-selective agonist (that lacked binding and activity towards ERα and ERβ). Renamed GPER (G protein-coupled estrogen receptor) by IUPHAR in 2007, the total number of articles in PubMed related to this receptor recently surpassed 1000. In this article, the authors present personal perspectives on how they became involved in the discovery and/or advancement of GPER research. These areas include non-genomic effects on vascular tone, receptor cloning, molecular and cellular biology, signal transduction mechanisms and pharmacology of GPER, highlighting the roles of GPER and GPER-selective compounds in diseases such as obesity, diabetes, and cancer and the obligatory role of GPER in propagating cardiovascular aging, arterial hypertension and heart failure through the stimulation of Nox expression. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions.

PubMed

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity.

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions

PubMed Central

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity. PMID:25191551

Investigation of the effects of P2 purinoceptor ligands on the micturition reflex in female urethane-anaesthetized rats.

PubMed

King, Brian F; Knowles, Ian D; Burnstock, Geoffrey; Ramage, Andrew G

2004-06-01

1 The effects of purinoceptor ligands for P2X1 and/or P2X3 receptors (alpha,beta-meATP, IP(5)I, TNP-ATP, MRS 2179, PPADS, Phenol red and RO116-6446/008; i.v., n=4-5) and for P2Y1 receptors (PPADS, MRS 2179 and MRS 2269; i.v., n=3-5) were investigated on the distension-evoked 'micturition reflex' in the urethane-anaesthetized female rat. 2 Alpha,beta-meATP (180 nmol kg(-1) min(-1)), IP5I (10, 30 and 100 nmol kg(-1)), TNP-ATP (1 micromol kg(-1)), MRS 2179 (1 micromol kg(-1)) and PPADS (17 micromol kg(-1)) each caused maintained bladder contractions to occur during the infusion of saline into the bladder. PPADS (17 micromol kg(-1) min(-1)) had a similar effect when infused intravesicularly. Regular bladder contractions were not observed until the infusion of saline was halted. For IP5I, TNP-ATP, MRS 2179 and PPADS, the magnitude of postinfusion isovolumetric contractions was significantly reduced and, for IP5I, this action was also associated with a significant reduction in urethral relaxation. Additionally, TNP-ATP caused a significant increase in the pressure and volume thresholds required to initiate a reflex. 3 Phenol red (a P2X1/P2X3 antagonist; 0.1 and 1 micromol kg(-1)) caused a significant increase in the pressure and volume thresholds required to initiate a reflex and, at the higher dose, also caused a reduction in postinfusion isovolumetric contractions. 4 RO116-6446/008 (a P2X1-selective antagonist; 1 and 10 micromol kg(-1)) only caused a reduction in postinfusion isovolumetric contractions. 5 It is concluded that P2X1 and P2X3 receptors play a fundamental role in the micturition reflex in urethane-anesthetized female rats. P2X3 receptor blockade raised the pressure and volume thresholds for the reflex, whereas P2X1 receptor blockade diminished motor activity associated with voiding. P2Y1 receptors may be involved in inhibition of rat detrusor tone.

Reconstructed Serine 288 in the Left Flipper Region of the Rat P2X7 Receptor Stabilizes Nonsensitized States.

PubMed

Ishchenko, Yevheniia; Novosolova, Nataliia; Khafizov, Kamil; Bart, Geneviève; Timonina, Arina; Fayuk, Dmitriy; Skorinkin, Andrei; Giniatullin, Rashid

2017-07-05

Serine 275, a conserved residue of the left flipper region of ATP-gated P2X3 receptors, plays a key role in both agonist binding and receptor desensitization. It is conserved in most of the P2X receptors except P2X7 and P2X6. By combining experimental patch-clamp and modeling approaches, we explored the role of the corresponding residue in the rat P2X7 receptor (rP2X7) by replacing the phenylalanine at position 288 with serine and characterizing the membrane currents generated by either the wild-type (WT) or the mutated rP2X7 receptor. F288S, an rP2X7 mutation, slowed the deactivation subsequent to 2 and 20 s applications of 1 mM ATP. F288S also prevented sensitization (a progressive current growth) observed with the WT in response to a 20 s application of 1 mM ATP. Increasing the ATP concentration to 5 mM promoted sensitization also in the mutated rP2X7 receptor, accelerating the deactivation rate to typical WT values. YO-PRO1 uptake in cells expressing either the WT or the F288S P2X7 receptor was consistent with recorded membrane current data. Interestingly, in the human P2X7 (hP2X7) receptor, substitution Y288S did not change the deactivation rate, while the Y288F mutant generated a "rat-like" phenotype with a fast deactivation rate. Our combined experimental, kinetic, and molecular modeling data suggest that the rat F288S novel phenotype is due to a slower rate of ATP binding and/or unbinding and stabilization of nonsensitized receptor states.

Activation of Adhesion G Protein-coupled Receptors: AGONIST SPECIFICITY OF STACHEL SEQUENCE-DERIVED PEPTIDES.

PubMed

Demberg, Lilian M; Winkler, Jana; Wilde, Caroline; Simon, Kay-Uwe; Schön, Julia; Rothemund, Sven; Schöneberg, Torsten; Prömel, Simone; Liebscher, Ines

2017-03-17

Members of the adhesion G protein-coupled receptor (aGPCR) family carry an agonistic sequence within their large ectodomains. Peptides derived from this region, called the Stachel sequence, can activate the respective receptor. As the conserved core region of the Stachel sequence is highly similar between aGPCRs, the agonist specificity of Stachel sequence-derived peptides was tested between family members using cell culture-based second messenger assays. Stachel peptides derived from aGPCRs of subfamily VI (GPR110/ADGRF1, GPR116/ADGRF5) and subfamily VIII (GPR64/ADGRG2, GPR126/ADGRG6) are able to activate more than one member of the respective subfamily supporting their evolutionary relationship and defining them as pharmacological receptor subtypes. Extended functional analyses of the Stachel sequences and derived peptides revealed agonist promiscuity, not only within, but also between aGPCR subfamilies. For example, the Stachel -derived peptide of GPR110 (subfamily VI) can activate GPR64 and GPR126 (both subfamily VIII). Our results indicate that key residues in the Stachel sequence are very similar between aGPCRs allowing for agonist promiscuity of several Stachel -derived peptides. Therefore, aGPCRs appear to be pharmacologically more closely related than previously thought. Our findings have direct implications for many aGPCR studies, as potential functional overlap has to be considered for in vitro and in vivo studies. However, it also offers the possibility of a broader use of more potent peptides when the original Stachel sequence is less effective. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The role of GPR1 signaling in mice corpus luteum

PubMed Central

Yang, Ya-Li; Ren, Li-Rong; Sun, Li-Feng; Huang, Chen; Xiao, Tian-Xia; Wang, Bao-Bei; Chen, Jie; Zabel, Brian A

2016-01-01

Chemerin, a chemokine, plays important roles in immune responses, inflammation, adipogenesis, and carbohydrate metabolism. Our recent research has shown that chemerin has an inhibitory effect on hormone secretion from the testis and ovary. However, whether G protein-coupled receptor 1 (GPR1), the active receptor for chemerin, regulates steroidogenesis and luteolysis in the corpus luteum is still unknown. In this study, we established a pregnant mare serum gonadotropin-human chorionic gonadotropin (PMSG-hCG) superovulation model, a prostaglandin F2α (PGF2α) luteolysis model, and follicle and corpus luteum culture models to analyze the role of chemerin signaling through GPR1 in the synthesis and secretion of gonadal hormones during follicular/luteal development and luteolysis. Our results, for the first time, show that chemerin and GPR1 are both differentially expressed in the ovary over the course of the estrous cycle, with highest levels in estrus and metestrus. GPR1 has been localized to granulosa cells, cumulus cells, and the corpus luteum by immunohistochemistry (IHC). In vitro, we found that chemerin suppresses hCG-induced progesterone production in cultured follicle and corpus luteum and that this effect is attenuated significantly by anti-GPR1 MAB treatment. Furthermore, when the phosphoinositide 3-kinase (PI3K) pathway was blocked, the attenuating effect of GPR1 MAB was abrogated. Interestingly, PGF2α induces luteolysis through activation of caspase-3, leading to a reduction in progesterone secretion. Treatment with GPR1 MAB blocked the PGF2α effect on caspase-3 expression and progesterone secretion. This study indicates that chemerin/GPR1 signaling directly or indirectly regulates progesterone synthesis and secretion during the processes of follicular development, corpus luteum formation, and PGF2α-induced luteolysis. PMID:27149986

β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1*

PubMed Central

Mancini, Arturo D.; Bertrand, Gyslaine; Vivot, Kevin; Carpentier, Éric; Tremblay, Caroline; Ghislain, Julien; Bouvier, Michel; Poitout, Vincent

2015-01-01

FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses (“biased agonism”). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes. PMID:26157145

β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1.

PubMed

Mancini, Arturo D; Bertrand, Gyslaine; Vivot, Kevin; Carpentier, Éric; Tremblay, Caroline; Ghislain, Julien; Bouvier, Michel; Poitout, Vincent

2015-08-21

FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins Gq/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses ("biased agonism"). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of Gq/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of Gq activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to Gq/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

75 FR 50768 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-17

.... N-Methanocarba Adenosine Derivatives and Their Dendrimer Conjugates as A 3 Receptor Agonists... receptors and agonists and antagonists of P2Y receptors, such as P2Y 1 and P2Y 14 . Dendrimer conjugates may...

Neuropeptide Y and peptide YY: important regulators of energy metabolism.

PubMed

Nguyen, Amy D; Herzog, Herbert; Sainsbury, Amanda

2011-02-01

An overview of recent developments documenting the neuropeptide Y (NPY) family's role in energy metabolism. Specifically focusing on site-specific functions of NPY and increasing evidence of peptide YY (PYY) as a weight loss therapeutic. Studying the NPY family in hypothalamic nuclei, other than the arcuate and paraventricular nuclei, is a recent shift in metabolic research. NPY overexpression in the dorsomedial hypothalamus increases food intake whereas its ablation in this area reduces hyperphagia and obesity. Similarly, NPY exerts orexigenic effects in the ventromedial nucleus. However, specific arcuate Y2 receptor ablation leads to positive energy balance, suggesting the NPY family demonstrates location-specific functions. Peripherally, dual blockade of cannabinoid and NPY pathways has synergistic effects on weight loss, as does combined administration of PYY3-36 and oxyntomodulin in reducing food intake, perhaps due to the recently discovered role of PYY in mediating intestinal Gpr119 activity and controlling glucose tolerance. Conditional Y receptor knockout models have provided deeper insights on NPY's functions according to location. Further study of PYY appears vital, due to recent evidence of its role in intestinal motility, with exercise positively influencing PYY levels.

The role of G-protein-coupled receptors in mediating the effect of fatty acids on inflammation and insulin sensitivity.

PubMed

Oh, Da Young; Lagakos, William S

2011-07-01

Chronic activation of inflammatory pathways mediates the pathogenesis of insulin resistance, and the macrophage/adipocyte nexus provides a key mechanism underlying decreased insulin sensitivity. Free fatty acids are important in the pathogenesis of insulin resistance, although their precise mechanisms of action have yet to be fully elucidated. Recently, a family of G-protein-coupled receptors has been identified that exhibits high affinity for fatty acids. This review summarizes recent findings on six of these receptors, their ligands, and their potential physiological functions in vivo. Upon activation, the free fatty acid receptors affect inflammation, glucose metabolism, and insulin sensitivity. Genetic deletion of GPR40 and GPR41, receptors for long-chain and short-chain fatty acids, respectively, results in resistance to diet-induced obesity. Deletion of GPR43 and GPR84 exacerbates inflammation, and deletion of the long-chain fatty acid receptors GPR119 and GPR120 reduces or is predicted to reduce glucose tolerance. These studies provide a new understanding of the general biology of gastric motility and also shed valuable insight into some potentially beneficial therapeutic targets. Furthermore, highly selective agonists or antagonists for the free fatty acid receptors have been developed and look promising for treating various metabolic diseases.

Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER).

PubMed

Thekkumkara, Thomas; Snyder, Russell; Karamyan, Vardan T

2016-01-01

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

GPR Technologies and Methodologies in Italy: A Review

NASA Astrophysics Data System (ADS)

Benedetto, Andrea; Frezza, Fabrizio; Manacorda, Guido; Massa, Andrea; Pajewski, Lara

2014-05-01

GPR techniques and technologies have been subject of intense research activities at the Italian level in the last 15 years because of their potential applications specifically to civil engineering. More in detail, several innovative approaches and models have been developed to inspect road pavements to measure the thickness of their layers as well as to diagnose or prevent damage. Moreover, new frontiers in bridge inspection as well as in geotechnical applications such as slides and flows have been investigated using GPR. From the methodological viewpoint, innovative techniques have been developed to solve GPR forward-scattering problems, as well to locate and classify subsurface targets in real-time and to retrieve their properties through multi-resolution strategies, and linear and non-linear methodologies. Furthermore, the application of GPR and other non-destructive testing methods in archaeological prospecting, cultural heritage diagnostics, and in the localization and detection of vital signs of trapped people has been widely investigated. More recently, new theoretical and empirical paradigms regarding water moisture evaluation in various porous media and soil characterization have been published as the results of long terms research activities. Pioneer studies are also currently under development with the scope to correlate GPR measurement with mechanical characteristics of bound and unbound construction materials. In such a framework, this abstract will be aimed at reviewing some of the most recent advances of GPR techniques and technologies within the Italian industrial and academic communities [also including their application within international projects such as FP7 ISTIMES (http://www.istimes.eu)], and at envisaging some of the most promising research trends currently under development. Acknowledgment - This work was supported by COST Action TU1208 'Civil Engineering Applications of Ground Penetrating Radar' References [1] M. Balsi, S. Esposito, F. Frezza, P. Nocito, L. Porrini, L. Pajewski, G. Schettini e C. Twizere, 'FDTD Simulation of GPR Measurements in a Laboratory Sandbox for Landmine Detection', Proc. IWAGPR 2009, Granada, Spagna, 27-29 maggio 2009, pp. 45-49. [2] M. Balsi, S. Esposito, F. Frezza, P. Nocito, P. M. Barone, S. E. Lauro, E. Mattei, E. Pettinelli, G. Schettini e C. Twizere, 'GPR Measurements and FDTD Simulations for Landmine Detection', Proc. XIII International Conference on Ground Penetrating Radar, 21-25 giugno 2010, Lecce, pp. 865-869. [3] M. Balsi, P. M. Barone, S. Esposito, F. Frezza, S. E. Lauro, P. Nocito, E. Pettinelli, G. Schettini e C. Twizere, 'FDTD Simulations and GPR Measurements for Land Mine Detection in a Controlled Environment', Atti XVIII Riunione Nazionale di Elettromagnetismo, Benevento, 6-10 settembre 2010, pp. 59-65. [4] M. Salucci, D. Sartori, N. Anselmi, A. Randazzo, G. Oliveri, and A. Massa, 'Imaging buried objects within the second-order born approximation through a multiresolution regularized Inexact-Newton method', 2013 International Symposium on Electromagnetic Theory (EMTS), (Hiroshima, Japan), May 20-24 2013. [5] L. Lizzi, F. Viani, P. Rocca, G. Oliveri, M. Benedetti and A. Massa, 'Three-dimensional real-time localization of subsurface objects - From theory to experimental validation,' 2009 IEEE International Geoscience and Remote Sensing Symposium, vol. 2, pp. II-121-II-124, 12-17 July 2009. [6] S. Meschino, L. Pajewski, M. Pastorino, A. Randazzo, and G. Schettini, 'Detection of Subsurface Metallic Utilities by Means of a SAP Technique: Comparing MUSIC- and SVM-Based Approaches', J. Appl. Geophy., vol. 97, pp. 60-68, Oct. 2013. [7] M. Pastorino, and A. Randazzo, 'Buried object detection by an Inexact-Newton applied to nonlinear inverse scattering', Int. J. of Microwave Sci. Technol., vol. 2012, Article ID 637301, 7 pages, 2012. [8] F. Soldovieri and L. Crocco, "Electromagnetic Tomography", in Subsurface Sensing, A. S. Turk, K. A. Hocaoglu, A. A. Vertiy ed., Wiley Series in Microwave and Optical Engineering (Volume 1), Sept. 2011, John Wiley & Sons, NY. [9] I. Catapano, L. Crocco, Y. Krellmann, G. Triltzsch, F. Soldovieri, "A Tomographic Approach for Helicopter-Borne Ground Penetrating Radar Imaging", IEEE Geosci. Rem. Sensing Lett., vol. 9, p. 378-382, 2012.

Diadenosine polyphosphates as antagonists of the endogenous P2Y1 receptor in rat brain capillary endothelial cells of the B7 and B10 clones

PubMed Central

Vigne, Paul; Breittmayer, Jean Philippe; Frelin, Christian

2000-01-01

Diadenosine polyphosphates (ApnAs, n=2–7) are considered as stress mediators in the cardiovascular system. They act both via identified P2 purinoceptors and via yet to be characterized receptors. This study analyses the actions of ApnAs in clones of rat brain capillary endothelial cells that express P2Y1 receptors (B10 cells) or both P2Y1 and P2Y2 receptors (B7 cells).B10 cells responded to Ap3A with rises in intracellular Ca2+ concentration ([Ca2+]i). This response was prevented by adenosine-3′-phosphate-5′-phosphate, an antagonist of P2Y1 receptors. It was largely suppressed by a treatment with apyrase VII or with creatine phosphokinase/creatine phosphate to degrade contaminating ADP.ApnAs inhibited ADP induced increases in [Ca2+]i mediated by P2Y1 receptors by shifting ADP concentration-response curves to larger concentrations. Apparent Ki values were estimated to be 6 μM for Ap4A, 10 μM for Ap5A and 47 μM for Ap6A. Ap2A and Ap3A were much less active.ApnAs were neither agonists nor antagonists of the endogenous P2Y2 receptor in B7 cells.ApnAs are neither agonists nor antagonists of the Gi-coupled, ADP receptor in B10 cells.The results suggest that most actions of ApnAs in B7 and B10 cells can be accounted for by endogenous P2Y1 receptors. Ap4A, Ap5A and Ap6A are specific antagonists of endogenous Ca2+-coupled P2Y1 receptors. PMID:10742308

Cloning, Phylogenetic Analysis, and Distribution of Free Fatty Acid Receptor GPR120 Expression along the Gastrointestinal Tract of Housing versus Grazing Kid Goats.

PubMed

Ran, Tao; Li, Hengzhi; Liu, Yong; Zhou, Chuanshe; Tang, Shaoxun; Han, Xuefeng; Wang, Min; He, Zhixiong; Kang, Jinghe; Yan, Qiongxian; Tan, Zhiliang; Beauchemin, Karen A

2016-03-23

G-protein-coupled receptor 120 (GPR120) is reported as a long-chain fatty acid (LCFA) receptor that elicits free fatty acid (FFA) regulation on metabolism homeostasis. The study aimed to clone the gpr120 gene of goats (g-GPR120) and subsequently investigate phylogenetic analysis and tissue distribution throughout the digestive tracts of kid goats, as well as the effect of housing versus grazing (H vs G) feeding systems on GPR120 expression. Partial coding sequence (CDS) of g-GPR120 was cloned and submitted to NCBI (accession no. KU161270 ). Phylogenetic analysis revealed that g-GPR120 shared higher homology in both mRNA and amino acid sequences for ruminants than nonruminants. Immunochemistry, real-time PCR, and Western blot analysis showed that g-GPR120 was expressed throughout the digestive tracts of goats. The expression of g-GPR120 was affected by feeding system and age, with greater expression of g-GPR120 in the G group. It was concluded that the g-GPR120-mediated LCFA chemosensing mechanism is widely present in the tongue and gastrointestinal tract of goats and that its expression can be affected by feeding system and age.

Comparative Long-Term Effect of Three Anti-P2Y12 Drugs after Percutaneous Angioplasty: An Observational Study Based on Electronic Drug Adherence Monitoring

PubMed Central

Forni Ogna, Valentina; Bassi, Isabelle; Menetrey, Isabelle; Muller, Olivier; Tousset, Eric; Fontana, Pierre; Eeckhout, Eric; Eap, Chin B.; Vrijens, Bernard; Burnier, Michel; Wuerzner, Grégoire

2017-01-01

Aims: Dual platelet inhibition using anti-P2Y12 drugs and aspirin is the standard of care in patients after percutaneous coronary interventions (PCI). Prasugrel and ticagrelor have been shown to be more potent than clopidogrel with less high on-treatment platelet reactivity. Whether differences in long-term adherence to these drugs can partly explain different antiplatelet efficacy has not been studied so far. The objective was to compare the long-term P2Y12 receptor inhibition and drug adherence to different anti-P2Y12 drugs, and to assess the impact of adherence on the pharmacodynamic effect. Methods: Monocentric, prospective, observational study. Stable outpatients treated with clopidogrel 75 mg once daily, prasugrel 10 mg once daily or ticagrelor 90 mg twice daily after PCI with stent implantation were included. Drug adherence was recorded during 6 months using electronic monitoring. Platelet responsiveness was assessed with the vasodilator-stimulated phosphoprotein platelet reactivity index (VASP-PRI) at inclusion, 3 and 6 months. Results: 120 patients had VASP-PRI and adherence data available. At 6-months, mean VASP-PRI (±SD) was 17.7 ± 11.0% with ticagrelor, 29.2 ± 15.5% with prasugrel and 47.2 ± 17.6% with clopidogrel (ANOVA, P < 0.0001). Median [IQR] taking adherence was 96 [82–100]% with ticagrelor, 100 [97–101]% with prasugrel and 100 [99–101]% with clopidogrel (p = 0.0001). Median [IQR] correct dosing was 88 [73–95]% with ticagrelor, 97 [92.5–98]% with prasugrel and 98 [96–99]% with clopidogrel (p = 0.0001). Anti-P2Y12 drug (p ≤ 0.001) and diabetes (p = 0.014) emerged as predictors of poor antiplatelet response after adjusting for age, BMI, sex, and CYP2C19∗2 carriers status. Conclusion: Drug adherence to anti-P2Y12 drugs assessed with electronic monitoring was very high. However, anti-P2Y12 drugs showed significant differences in antiplatelet activity, with newer anti-P2Y12 drugs ticagrelor and prasugrel exerting a stronger P2Y12 receptor inhibition. These data suggest that pharmacokinetic-pharmacodynamic differences between oral anti-P2Y12 drugs are more important than adherence in determining antiplatelet efficacy when adherence to prescription is high. The study was registered (Current Controlled Trials ISRCTN85949729). PMID:29118712

GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells.

PubMed

Wiley, Shu Z; Sriram, Krishna; Liang, Wenjing; Chang, Sarah E; French, Randall; McCann, Thalia; Sicklick, Jason; Nishihara, Hiroshi; Lowy, Andrew M; Insel, Paul A

2018-03-01

The microenvironment of pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma (desmoplasia) generated by pancreatic cancer-associated fibroblasts (CAFs) derived from pancreatic stellate cells (PSCs) and pancreatic fibroblasts (PFs). Using an unbiased GPCRomic array approach, we identified 82 G-protein-coupled receptors (GPCRs) commonly expressed by CAFs derived from 5 primary PDAC tumors. Compared with PSCs and PFs, CAFs have increased expression of GPR68 (a proton-sensing GPCR), with the results confirmed by immunoblotting, The Cancer Genome Atlas data, and immunohistochemistry of PDAC tumors. Co-culture of PSCs with PDAC cells, or incubation with TNF-α, induced GPR68 expression. GPR68 activation (by decreasing the extracellular pH) enhanced IL-6 expression via a cAMP/PKA/cAMP response element binding protein signaling pathway. Knockdown of GPR68 by short interfering RNA diminished low pH-induced production of IL-6 and enhancement of PDAC cell proliferation by CAF conditioned media. CAFs from other gastrointestinal cancers also express GPR68. PDAC cells thus induce expression by CAFs of GPR68, which senses the acidic microenvironment, thereby increasing production of fibrotic markers and IL-6 and promoting PDAC cell proliferation. CAF-expressed GPR68 is a mediator of low-pH-promoted regulation of the tumor microenvironments, in particular to PDAC cell-CAF interaction and may be a novel therapeutic target for pancreatic and perhaps other types of cancers.-Wiley, S. Z., Sriram, K., Liang, W., Chang, S. E., French, R., McCann, T., Sicklick, J., Nishihara, H., Lowy, A. M., Insel, P. A. GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells.

Stimulating the GPR30 estrogen receptor with a novel tamoxifen analogue activates SF-1 and promotes endometrial cell proliferation.

PubMed

Lin, Benjamin C; Suzawa, Miyuki; Blind, Raymond D; Tobias, Sandra C; Bulun, Serdar E; Scanlan, Thomas S; Ingraham, Holly A

2009-07-01

Estrogens and selective estrogen receptor (ER) modulators such as tamoxifen are known to increase uterine cell proliferation. Mounting evidence suggests that estrogen signaling is mediated not only by ERalpha and ERbeta nuclear receptors, but also by GPR30 (GPER), a seven transmembrane (7TM) receptor. Here, we report that primary human endometriotic H-38 cells express high levels of GPR30 with no detectable ERalpha or ERbeta. Using a novel tamoxifen analogue, STX, which activates GPR30 but not ERs, significant stimulation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways was observed in H-38 cells and in Ishikawa endometrial cancer cells expressing GPR30; a similar effect was observed in JEG3 choriocarcinoma cells. STX treatment also increased cellular pools of phosphatidylinositol (3,4,5) triphosphate, a proposed ligand for the nuclear hormone receptor SF-1 (NR5A1). Consistent with these findings, STX, tamoxifen, and the phytoestrogen genistein were able to increase SF-1 transcription, promote Ishikawa cell proliferation, and induce the SF-1 target gene aromatase in a GPR30-dependent manner. Our findings suggest a novel signaling paradigm that is initiated by estrogen activation of the 7TM receptor GPR30, with signal transduction cascades (PI3K and MAPK) converging on nuclear hormone receptors (SF-1/LRH-1) to modulate their transcriptional output. We propose that this novel GPR30/SF-1 pathway increases local concentrations of estrogen, and together with classic ER signaling, mediate the proliferative effects of synthetic estrogens such as tamoxifen, in promoting endometriosis and endometrial cancers.

Role of the metabotropic P2Y(4) receptor during hypoglycemia: cross talk with the ionotropic NMDAR1 receptor.

PubMed

Cavaliere, Fabio; Amadio, Susanna; Angelini, Daniela F; Sancesario, Giuseppe; Bernardi, Giorgio; Volonté, Cinzia

2004-10-15

It is well established that both extracellular ATP and glutamate exert a critical role during metabolic impairment, that several P2 receptor subunits are directly involved in this action and that a strong relationship exists between glutamatergic and purinergic signals. Therefore, here we studied the molecular behavior of the purinergic metabotropic P2Y(4) and the glutamatergic ionotropic NMDAR1 receptors during hypoglycemic cell death. We find that these proteins are oppositely modulated during glucose starvation (P2Y(4) is induced, whereas NMDAR1 is inhibited) and that both P2 and NMDA antagonists can restore basal protein expression levels. Moreover, double immunofluorescence experiments with confocal laser microscopy reveal co-localization at the membrane level between the P2Y(4) and NMDAR1 receptors, in both homologous (cerebellar granule neurons) and heterologous (Hek-293) cellular systems. This is furthermore confirmed by co-immunoprecipitation experiments. Finally, when we express the P2Y(4) receptor in the heterologous SH-SY5Y neuronal cell line, hypoglycemia then causes severe cell death and simultaneous downregulation of the NMDAR1 protein. In summary, our work establishes a potential molecular interplay between P2Y(4) and NMDAR1 receptors during glucose deprivation and the causative role of the P2Y(4) during cell death.

Role of estrogen receptors alpha, beta and GPER1/GPR30 in pancreatic beta-cells.

PubMed

Nadal, Angel; Alonso-Magdalena, Paloma; Soriano, Sergi; Ripoll, Cristina; Fuentes, Esther; Quesada, Ivan; Ropero, Ana Belen

2011-01-01

Estrogen receptors (ER) are emerging as important molecules involved in the adaptation of beta-cells to insulin resistance. The onset of type 2 diabetes is marked by insulin secretory dysfunction and decreased beta-cell mass. During pregnancy, puberty and obesity there is increased metabolic demand and insulin resistance is developed. This metabolic state increases the demand on beta-cells to augment insulin biosynthesis and release. In this respect, ERalpha is directly implicated in the E2-regulation of insulin content and secretion, while ERbeta is in the E2-potentiation of glucose-induced insulin release. Both receptors develop their actions within the physiological range of E2. In addition, the G protein-coupled estrogen receptor (GPER1/GPR30) seems to be implicated in the E2-regulation of stimulus-secretion coupling in the three cell types of the islet. The increased demand of insulin production for long time may lead to beta-cell stress and apoptosis. ERalpha, ERbeta and GPER1/GPR30 are involved in preventing beta-cell apoptosis, impeding the loss of critical beta-cell mass. Therefore, estrogen receptors may play an essential role in the adaptation of the pancreas to insulin resistant periods.

GPER/GPR30 knockout mice: effects of GPER on metabolism

PubMed Central

Sharma, Geetanjali; Prossnitz, Eric R.

2015-01-01

i. Summary Endogenous estrogens, predominantly 17β-estradiol (E2), mediate various very diverse effects throughout the body in both normal physiology and disease. Actions include development (including puberty) and reproduction as well as additional effects throughout life in the metabolic, endocrine, musculoskeletal, nervous, cardiovascular and immune systems. The actions of E2 have traditionally been attributed to the classical nuclear estrogen receptors (ERα and ERβ) that largely mediate transcriptional/genomic activities. However, more recently the G protein-coupled estrogen receptor GPER/GPR30 has become recognized as an essential mediator of certain, and particularly rapid, signaling events in response to E2. Murine genetic knockout (KO) models represent an important approach to understand the mechanisms of E2 action in physiology and disease. Studies of GPER KO mice over the last years have revealed functions for GPER in the regulation of obesity, insulin resistance and glucose intolerance, among other areas of (patho)physiology. This chapter focuses on methods for the evaluation of metabolic parameters in vivo and ex vivo with an emphasis on glucose homeostasis and metabolism through the use of glucose and insulin tolerance tests, pancreatic islet and adipocyte isolation and characterization. PMID:26585159

GPER/GPR30 Knockout Mice: Effects of GPER on Metabolism.

PubMed

Sharma, Geetanjali; Prossnitz, Eric R

2016-01-01

Endogenous estrogens, predominantly 17β-estradiol (E2), mediate various diverse effects throughout the body in both normal physiology and disease. Actions include development (including puberty) and reproduction as well as additional effects throughout life in the metabolic, endocrine, musculoskeletal, nervous, cardiovascular, and immune systems. The actions of E2 have traditionally been attributed to the classical nuclear estrogen receptors (ERα and ERβ) that largely mediate transcriptional/genomic activities. However, more recently the G protein-coupled estrogen receptor GPER/GPR30 has become recognized as an essential mediator of certain, and particularly rapid, signaling events in response to E2. Murine genetic knockout (KO) models represent an important approach to understand the mechanisms of E2 action in physiology and disease. Studies of GPER KO mice over the last years have revealed functions for GPER in the regulation of obesity, insulin resistance and glucose intolerance, among other areas of (patho)physiology. This chapter focuses on methods for the evaluation of metabolic parameters in vivo and ex vivo with an emphasis on glucose homeostasis and metabolism through the use of glucose and insulin tolerance tests, pancreatic islet and adipocyte isolation and characterization.

Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi

2013-01-04

Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delaymore » of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.« less

Dietary Fiber Intake is Associated with Increased Colonic Mucosal GPR43+ Polymorphonuclear Infiltration in Active Crohn’s Disease

PubMed Central

Zhao, Mingli; Zhu, Weiming; Gong, Jianfeng; Zuo, Lugen; Zhao, Jie; Sun, Jing; Li, Ning; Li, Jieshou

2015-01-01

G protein-coupled receptor 43/free fatty acid receptor 2 (GPR43/FFAR2) is essential for polymorphonuclear (PMN) recruitment. We investigated the expression of GPR43/FFAR2 in the colon from Crohn’s disease patients and whether dietary fiber in enteral nutrition increases GPR43+ polymorphonuclear infiltration in mucosa. Segments of ascending colon and white blood cells from peripheral blood were obtained from 46 Crohn’s disease patients and 10 colon cancer patients. The Crohn’s disease patients were grouped by the activity of disease (active or remission) and enteral nutrition with or without dietary fiber. Histological feature, expression and location of GPR43/FFAR2 and level of tumor necrosis factor-α (TNF-α), interleukine-6 (IL-6) and myeloperoxidase were assessed. The results of hematoxylin-eosin and immunohistochemistry staining revealed that the infiltration of immune cells, including GPR43+ PMN, was more severe in active Crohn’s disease patients who consumed normal food or enteral nutrition with dietary fiber than in remission patients and colon cancer patients. This finding was supported by the results of GPR43 and myeloperoxidase expression. Active Crohn’s disease (CD) patients who consumed enteral nutrition without dietary fiber exhibited severe immune cell infiltration similar to the other active CD patients, but GPR43+ PMNs were rarely observed. The level of TNF-α mRNA in active Crohn’s disease patients was higher than those of the other patients. In conclusion, the use of dietary fiber in enteral nutrition by active Crohn’s disease patients might increase GPR43+ PMNs infiltration in colon mucosa. This effect was not observed in Crohn’s disease patients in remission. PMID:26140540

Dietary Fiber Intake is Associated with Increased Colonic Mucosal GPR43+ Polymorphonuclear Infiltration in Active Crohn's Disease.

PubMed

Zhao, Mingli; Zhu, Weiming; Gong, Jianfeng; Zuo, Lugen; Zhao, Jie; Sun, Jing; Li, Ning; Li, Jieshou

2015-07-01

G protein-coupled receptor 43/free fatty acid receptor 2 (GPR43/FFAR2) is essential for polymorphonuclear (PMN) recruitment. We investigated the expression of GPR43/FFAR2 in the colon from Crohn's disease patients and whether dietary fiber in enteral nutrition increases GPR43+ polymorphonuclear infiltration in mucosa. Segments of ascending colon and white blood cells from peripheral blood were obtained from 46 Crohn's disease patients and 10 colon cancer patients. The Crohn's disease patients were grouped by the activity of disease (active or remission) and enteral nutrition with or without dietary fiber. Histological feature, expression and location of GPR43/FFAR2 and level of tumor necrosis factor-α (TNF-α), interleukine-6 (IL-6) and myeloperoxidase were assessed. The results of hematoxylin-eosin and immunohistochemistry staining revealed that the infiltration of immune cells, including GPR43+ PMN, was more severe in active Crohn's disease patients who consumed normal food or enteral nutrition with dietary fiber than in remission patients and colon cancer patients. This finding was supported by the results of GPR43 and myeloperoxidase expression. Active Crohn's disease (CD) patients who consumed enteral nutrition without dietary fiber exhibited severe immune cell infiltration similar to the other active CD patients, but GPR43+ PMNs were rarely observed. The level of TNF-α mRNA in active Crohn's disease patients was higher than those of the other patients. In conclusion, the use of dietary fiber in enteral nutrition by active Crohn's disease patients might increase GPR43+ PMNs infiltration in colon mucosa. This effect was not observed in Crohn's disease patients in remission.

Tyrosine phosphorylation of the orphan receptor ESDN/DCBLD2 serves as a scaffold for the signaling adaptor CrkL.

PubMed

Aten, Tyler M; Redmond, Miranda M; Weaver, Sheila O; Love, Collin C; Joy, Ryan M; Lapp, Aliya S; Rivera, Osvaldo D; Hinkle, Karen L; Ballif, Bryan A

2013-08-02

A quantitative proteomics screen to identify substrates of the Src family of tyrosine kinases (SFKs) whose phosphorylation promotes CrkL-SH2 binding identified the known Crk-associated substrate (Cas) of Src as well as the orphan receptor endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). Mutagenesis analysis of ESDN's seven intracellular tyrosines in YxxP motifs found several contribute to the binding of ESDN to the SH2 domains of both CrkCT10 regulator of kinase Crk-Like (CrkL) and a representative SFK Fyn. Quantitative mass spectrometry showed that at least three of these (Y565, Y621 and Y750), as well as non-YxxP Y715, are reversibly phosphorylated. SFK activity was shown to be sufficient, but not required for the interaction between ESDN and the CrkL-SH2 domain. Finally, antibody-mediated ESDN clustering induces ESDN tyrosine phosphorylation and CrkL-SH2 binding. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Painful purinergic receptors.

PubMed

Donnelly-Roberts, Diana; McGaraughty, Steve; Shieh, Char-Chang; Honore, Prisca; Jarvis, Michael F

2008-02-01

Multiple P2 receptor-mediated mechanisms exist by which ATP can alter nociceptive sensitivity following tissue injury. Evidence from a variety of experimental strategies, including genetic disruption studies and the development of selective antagonists, has indicated that the activation of P2X receptor subtypes, including P2X(3), P2X(2/3), P2X(4) and P2X(7), and P2Y (e.g., P2Y(2)) receptors, can modulate pain. For example, administration of a selective P2X(3) antagonist, A-317491, has been shown to effectively block both hyperalgesia and allodynia in different animal models of pathological pain. Intrathecally delivered antisense oligonucleotides targeting P2X(4) receptors decrease tactile allodynia following nerve injury. Selective antagonists for the P2X(7) receptor also reduce sensitization in animal models of inflammatory and neuropathic pain, providing evidence that purinergic glial-neural interactions are important modulators of noxious sensory neurotransmission. Furthermore, activation of P2Y(2) receptors leads to sensitization of polymodal transient receptor potential-1 receptors. Thus, ATP acting at multiple purinergic receptors, either directly on neurons (e.g., P2X(3), P2X(2/3), and P2Y receptors) or indirectly through neural-glial cell interactions (P2X(4) and P2X(7) receptors), alters nociceptive sensitivity. The development of selective antagonists for some of these P2 receptors has greatly aided investigations into the nociceptive role of ATP. This perspective highlights some of the recent advances to identify selective P2 receptor ligands, which has enhanced the investigation of ATP-related modulation of pain sensitivity.

An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets

PubMed Central

Nisar, Shaista; Daly, Martina E.; Federici, Augusto B.; Artoni, Andrea; Mumford, Andrew D.; Watson, Stephen P.

2011-01-01

The platelet P2Y12 purinoceptor (P2Y12R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y12R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)–binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y12R (P341A) that is associated with reduced expression of the P2Y12R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y12R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic. PMID:21937696

Remodeling of Sensorimotor Brain Connectivity in Gpr88-Deficient Mice.

PubMed

Arefin, Tanzil Mahmud; Mechling, Anna E; Meirsman, Aura Carole; Bienert, Thomas; Hübner, Neele Saskia; Lee, Hsu-Lei; Ben Hamida, Sami; Ehrlich, Aliza; Roquet, Dan; Hennig, Jürgen; von Elverfeldt, Dominik; Kieffer, Brigitte Lina; Harsan, Laura-Adela

2017-10-01

Recent studies have demonstrated that orchestrated gene activity and expression support synchronous activity of brain networks. However, there is a paucity of information on the consequences of single gene function on overall brain functional organization and connectivity and how this translates at the behavioral level. In this study, we combined mouse mutagenesis with functional and structural magnetic resonance imaging (MRI) to determine whether targeted inactivation of a single gene would modify whole-brain connectivity in live animals. The targeted gene encodes GPR88 (G protein-coupled receptor 88), an orphan G protein-coupled receptor enriched in the striatum and previously linked to behavioral traits relevant to neuropsychiatric disorders. Connectivity analysis of Gpr88-deficient mice revealed extensive remodeling of intracortical and cortico-subcortical networks. Most prominent modifications were observed at the level of retrosplenial cortex connectivity, central to the default mode network (DMN) whose alteration is considered a hallmark of many psychiatric conditions. Next, somatosensory and motor cortical networks were most affected. These modifications directly relate to sensorimotor gating deficiency reported in mutant animals and also likely underlie their hyperactivity phenotype. Finally, we identified alterations within hippocampal and dorsal striatum functional connectivity, most relevant to a specific learning deficit that we previously reported in Gpr88 -/- animals. In addition, amygdala connectivity with cortex and striatum was weakened, perhaps underlying the risk-taking behavior of these animals. This is the first evidence demonstrating that GPR88 activity shapes the mouse brain functional and structural connectome. The concordance between connectivity alterations and behavior deficits observed in Gpr88-deficient mice suggests a role for GPR88 in brain communication.

IL-33 stimulates expression of the GPR84 (EX33) fatty acid receptor gene and of cytokine and chemokine genes in human adipocytes.

PubMed

Zaibi, Mohamed S; Kępczyńska, Małgorzata A; Harikumar, Parvathy; Alomar, Suliman Y; Trayhurn, Paul

2018-05-15

Expression of GPCR fatty acid sensor/receptor genes in adipocytes is modulated by inflammatory mediators, particularly IL-1β. In this study we examined whether the IL-1 gene superfamily member, IL-33, also regulates expression of the fatty acid receptor genes in adipocytes. Human fat cells, differentiated from preadipocytes, were incubated with IL-33 at three different dose levels for 3 or 24 h and mRNA measured by qPCR. Treatment with IL-33 induced a dose-dependent increase in GPR84 mRNA at 3 h, the level with the highest dose being 13.7-fold greater than in controls. Stimulation of GPR84 expression was transitory; the mRNA level was not elevated at 24 h. In contrast to GPR84, IL-33 had no effect on GPR120 expression. IL-33 markedly stimulated expression of the IL1B, CCL2, IL6, CXCL2 and CSF3 genes, but there was no effect on ADIPOQ expression. The largest effect was on CSF3, the mRNA level of which increased 183-fold over controls at 3 h with the highest dose of IL-33; there was a parallel increase in the secretion of G-CSF protein into the medium. It is concluded that in human adipocytes IL-33, which is synthesised in adipose tissue, has a strong stimulatory effect on the expression of cytokine and chemokine genes, particularly CSF3, and on the expression of GPR84, a pro-inflammatory fatty acid receptor. Copyright © 2018 Elsevier Ltd. All rights reserved.

Regulation of ERRα Gene Expression by Estrogen Receptor Agonists and Antagonists in SKBR3 Breast Cancer Cells: Differential Molecular Mechanisms Mediated by G Protein-Coupled Receptor GPR30/GPER-1

PubMed Central

Li, Yin; Birnbaumer, Lutz; Teng, Christina T.

2010-01-01

In selected tissues and cell lines, 17β-estradiol (E2) regulates the expression of estrogen-related receptor α (ERRα), a member of the orphan nuclear receptor family. This effect is thought to be mediated by the estrogen receptor α (ERα). However in the ERα- and ERβ-negative SKBR3 breast cancer cell line, physiological levels of E2 also stimulate ERRα expression. Here, we explored the molecular mechanism that mediates estrogen action in ER-negative breast cancer cells. We observed that E2, the ERα agonist, as well as the ERα antagonists ICI 182,780 and tamoxifen (TAM), a selective ER modulator, stimulate the transcriptional activity of the ERRα gene and increase the production of ERRα protein in SKBR3 cells. Moreover, the ERRα downstream target genes expression and cellular proliferation are also increased. We show further that the G protein-coupled receptor GPR30/GPER-1 (GPER-1) mediates these effects. The GPER-1 specific ligand G-1 mimics the actions of E2, ICI 182,780, and TAM on ERRα expression, and changing the levels of GPER-1 mRNA by overexpression or small interfering RNA knockdown affected the expression of ERRα accordingly. Utilizing inhibitors, we delineate a different downstream pathway for ER agonist and ER antagonist-triggered signaling through GPER-1. We also find differential histone acetylation and transcription factor recruitment at distinct nucleosomes of the ERRα promoter, depending on whether the cells are activated with E2 or with ER antagonists. These findings provide insight into the molecular mechanisms of GPER-1/ERRα-mediated signaling and may be relevant to what happens in breast cancer cells escaping inhibitory control by TAM. PMID:20211987

Novel Therapeutic GPCRs for Psychiatric Disorders

PubMed Central

Komatsu, Hidetoshi

2015-01-01

G protein-coupled receptors (GPCRs) are the most common targets of the neuropharmacological drugs in the central nervous system (CNS). GPCRs are activated by manifold neurotransmitters, and their activation in turn evokes slow synaptic transmission. They are deeply involved in multiple neurological and psychiatric disorders such as Parkinson’s disease and schizophrenia. In the brain, the striatum is strongly innervated by the ventral tegmental area (VTA) and plays a central role in manifestation of psychiatric disorders. Recently, anatomical and comprehensive transcriptome analysis of the non-odorant GPCR superfamily revealed that the orphan GPCRs GPR88, GPR6, and GPR52, as well as dopamine D1 and D2 receptors and the adenosine A2a receptor, are the most highly enriched in the rodent striatum. Genetically engineered animal models and molecular biological studies have suggested that these striatally enriched GPCRs have a potential to be therapeutic psychiatric receptors. This review summarizes the current understanding of the therapeutic GPCR candidates for psychiatric disorders. PMID:26101869

Novel Therapeutic GPCRs for Psychiatric Disorders.

PubMed

Komatsu, Hidetoshi

2015-06-19

G protein-coupled receptors (GPCRs) are the most common targets of the neuropharmacological drugs in the central nervous system (CNS). GPCRs are activated by manifold neurotransmitters, and their activation in turn evokes slow synaptic transmission. They are deeply involved in multiple neurological and psychiatric disorders such as Parkinson's disease and schizophrenia. In the brain, the striatum is strongly innervated by the ventral tegmental area (VTA) and plays a central role in manifestation of psychiatric disorders. Recently, anatomical and comprehensive transcriptome analysis of the non-odorant GPCR superfamily revealed that the orphan GPCRs GPR88, GPR6, and GPR52, as well as dopamine D1 and D2 receptors and the adenosine A2a receptor, are the most highly enriched in the rodent striatum. Genetically engineered animal models and molecular biological studies have suggested that these striatally enriched GPCRs have a potential to be therapeutic psychiatric receptors. This review summarizes the current understanding of the therapeutic GPCR candidates for psychiatric disorders.

Activation of P2Y6 Receptors Facilitates Nonneuronal Adenosine Triphosphate and Acetylcholine Release from Urothelium with the Lamina Propria of Men with Bladder Outlet Obstruction.

PubMed

Silva, Isabel; Ferreirinha, Fátima; Magalhães-Cardoso, Maria Teresa; Silva-Ramos, Miguel; Correia-de-Sá, Paulo

2015-10-01

Deregulation of purinergic bladder signaling may contribute to persistent detrusor overactivity in patients with bladder outlet obstruction. Activation of uridine diphosphate sensitive P2Y6 receptors increases voiding frequency in rats indirectly by releasing adenosine triphosphate from the urothelium. To our knowledge this mechanism has never been tested in the human bladder. We examined the role of the uridine diphosphate sensitive P2Y6 receptor on tetrodotoxin insensitive nonneuronal adenosine triphosphate and [(3)H]acetylcholine release from the human urothelium with the lamina propria of control organ donors and patients with benign prostatic hyperplasia. The adenosine triphosphate-to-[(3)H]acetylcholine ratio was fivefold higher in mucosal urothelium/lamina propria strips from benign prostatic hyperplasia patients than control men. The selective P2Y6 receptor agonist PSB0474 (100 nM) augmented by a similar amount adenosine triphosphate and [(3)H]acetylcholine release from mucosal urothelium/lamina propria strips from both groups of individuals. The facilitatory effect of PSB0474 was prevented by MRS2578 (50 nM) and by carbenoxolone (10 μM), which block P2Y6 receptor and pannexin-1 hemichannels, respectively. Blockade of P2X3 (and/or P2X2/3) receptors with A317491 (100 nM) also attenuated release facilitation by PSB0474 in control men but not in patients with benign prostatic hyperplasia. Immunolocalization studies showed that P2Y6, P2X2 and P2X3 receptors were present in choline acetyltransferase positive urothelial cells. In contrast to P2Y6 staining, choline acetyltransferase, P2X2 and P2X3 immunoreactivity decreased in the urothelium of benign prostatic hyperplasia patients. Activation of P2Y6 receptor amplifies mucosal adenosine triphosphate release underlying bladder overactivity in patients with benign prostatic hyperplasia. Therefore, we propose selective P2Y6 receptor blockade as a novel therapeutic strategy to control persistent storage symptoms in obstructed patients. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Structure Based Virtual Screening Studies to Identify Novel Potential Compounds for GPR142 and Their Relative Dynamic Analysis for Study of Type 2 Diabetes

NASA Astrophysics Data System (ADS)

Kaushik, Aman C.; Kumar, Sanjay; Wei, Dong Q.; Sahi, Shakti

2018-02-01

GPR142 (G protein receptor 142) is a novel orphan GPCR (G protein coupled receptor) belonging to ‘Class A’ of GPCR family and expressed in beta cells of pancreas. In this study, we reported the structure based virtual screening to identify the hit compounds which can be developed as leads for potential agonists. The results were validated through induced fit docking, pharmacophore modeling and system biology approaches. Since, there is no solved crystal structure of GPR142, we attempted to predict the 3D structure followed by validation and then identification of active site using threading and ab initio methods. Also, structure based virtual screening was performed against a total of 1171519 compounds from different libraries and only top 20 best hit compounds were screened and analyzed. Moreover, the biochemical pathway of GPR142 complex with screened compound2 was also designed and compared with experimental data. Interestingly, compound2 showed an increase in insulin production via Gq mediated signaling pathway suggesting the possible role of novel GPR142 agonists in therapy against type 2 diabetes.

SRC-like adaptor protein 2 (SLAP2) is a negative regulator of KIT-D816V-mediated oncogenic transformation.

PubMed

Rupar, Kaja; Moharram, Sausan A; Kazi, Julhash U; Rönnstrand, Lars

2018-04-23

KIT is a receptor tyrosine kinase (RTK) involved in several cellular processes such as regulation of proliferation, survival and differentiation of early hematopoietic cells, germ cells and melanocytes. Activation of KIT results in phosphorylation of tyrosine residues in the receptor, and recruitment of proteins that mediate downstream signaling and also modulate receptor signaling. Here we show that the SRC-like adaptor protein 2 (SLAP2) binds to wild-type KIT in a ligand-dependent manner and is furthermore found constitutively associated with the oncogenic mutant KIT-D816V. Peptide fishing analysis mapped pY568 and pY570 as potential SLAP2 association sites in KIT, which overlaps with the SRC binding sites in KIT. Expression of SLAP2 in cells expressing the transforming mutant KIT-D816V led to reduced cell viability and reduced colony formation. SLAP2 also partially blocked phosphorylation of several signal transduction molecules downstream of KIT such as AKT, ERK, p38 and STAT3. Finally, SLAP2 expression enhanced ubiquitination of KIT and its subsequent degradation. Taken together, our data demonstrate that SLAP2 negatively modulates KIT-D816V-mediated transformation by enhancing degradation of the receptor.

Development of [3H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([3H]PSB-12150): A Useful Tool for Studying GPR17

PubMed Central

2014-01-01

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [3H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities. PMID:24900835

Development of [(3)H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([(3)H]PSB-12150): A Useful Tool for Studying GPR17.

PubMed

Köse, Meryem; Ritter, Kirsten; Thiemke, Katharina; Gillard, Michel; Kostenis, Evi; Müller, Christa E

2014-04-10

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [(3)H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities.

Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

PubMed Central

Kopp, Christina; Hosseini, Afshin; Singh, Shiva P.; Regenhard, Petra; Khalilvandi-Behroozyar, Hamed; Sauerwein, Helga; Mielenz, Manfred

2014-01-01

The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001) and the mRNA abundances of GPR109A (p ≤ 0.05) and chemerin (p ≤ 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows. PMID:25411802

P2Y12 receptor upregulation in satellite glial cells is involved in neuropathic pain induced by HIV glycoprotein 120 and 2',3'-dideoxycytidine.

PubMed

Yi, Zhihua; Xie, Lihui; Zhou, Congfa; Yuan, Huilong; Ouyang, Shuai; Fang, Zhi; Zhao, Shanhong; Jia, Tianyu; Zou, Lifang; Wang, Shouyu; Xue, Yun; Wu, Bing; Gao, Yun; Li, Guilin; Liu, Shuangmei; Xu, Hong; Xu, Changshui; Zhang, Chunping; Liang, Shangdong

2018-03-01

The direct neurotoxicity of HIV and neurotoxicity of combination antiretroviral therapy medications both contribute to the development of neuropathic pain. Activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) plays a crucial role in mechanical and thermal hyperalgesia. The P2Y 12 receptor expressed in SGCs of the DRG is involved in pain transmission. In this study, we explored the role of the P2Y 12 receptor in neuropathic pain induced by HIV envelope glycoprotein 120 (gp120) combined with ddC (2',3'-dideoxycytidine). A rat model of gp120+ddC-induced neuropathic pain was used. Peripheral nerve exposure to HIV-gp120+ddC increased mechanical and thermal hyperalgesia in gp120+ddC-treated model rats. The gp120+ddC treatment increased expression of P2Y 12 receptor mRNA and protein in DRG SGCs. In primary cultured DRG SGCs treated with gp120+ddC, the levels of [Ca 2+ ] i activated by the P2Y 12 receptor agonist 2-(Methylthio) adenosine 5'-diphosphate trisodium salt (2-MeSADP) were significantly increased. P2Y 12 receptor shRNA treatment inhibited 2-MeSADP-induced [Ca 2+ ] i in primary cultured DRG SGCs treated with gp120+ddC. Intrathecal treatment with a shRNA against P2Y 12 receptor in DRG SGCs reduced the release of pro-inflammatory cytokines, decreased phosphorylation of p38 MAPK in the DRG of gp120+ddC-treated rats. Thus, downregulating the P2Y 12 receptor relieved mechanical and thermal hyperalgesia in gp120+ddC-treated rats.

Structure of FcRY, an avian immunoglobulin receptor related to mammalian mannose receptors, and its complex with IgY

PubMed Central

He, Yongning; Bjorkman, Pamela J.

2011-01-01

Fc receptors transport maternal antibodies across epithelial cell barriers to passively immunize newborns. FcRY, the functional counterpart of mammalian FcRn (a major histocompatibility complex homolog), transfers IgY across the avian yolk sac, and represents a new class of Fc receptor related to the mammalian mannose receptor family. FcRY and FcRn bind immunoglobulins at pH ≤6.5, but not pH ≥7, allowing receptor–ligand association inside intracellular vesicles and release at the pH of blood. We obtained structures of monomeric and dimeric FcRY and an FcRY–IgY complex and explored FcRY's pH-dependent binding mechanism using electron cryomicroscopy (cryoEM) and small-angle X-ray scattering. The cryoEM structure of FcRY at pH 6 revealed a compact double-ring “head,” in which the N-terminal cysteine-rich and fibronectin II domains were folded back to contact C-type lectin-like domains 1–6, and a “tail” comprising C-type lectin-like domains 7–8. Conformational changes at pH 8 created a more elongated structure that cannot bind IgY. CryoEM reconstruction of FcRY dimers at pH 6 and small-angle X-ray scattering analysis at both pH values confirmed both structures. The cryoEM structure of the FcRY–IgY revealed symmetric binding of two FcRY heads to the dimeric FcY, each head contacting the CH4 domain of one FcY chain. FcRY shares structural properties with mannose receptor family members, including a head and tail domain organization, multimerization that may regulate ligand binding, and pH-dependent conformational changes. Our results facilitate understanding of immune recognition by the structurally related mannose receptor family and comparison of diverse methods of Ig transport across evolution. PMID:21746914

G-protein coupled receptor 30 (GPR30): a novel regulator of endothelial inflammation.

PubMed

Chakrabarti, Subhadeep; Davidge, Sandra T

2012-01-01

Estrogen, the female sex hormone, is known to exert anti-inflammatory and anti-atherogenic effects. Traditionally, estrogen effects were believed to be largely mediated through the classical estrogen receptors (ERs). However, there is increasing evidence that G-protein coupled receptor 30 (GPR30), a novel estrogen receptor, can mediate many estrogenic effects on the vasculature. Despite this, the localization and functional significance of GPR30 in the human vascular endothelium remains poorly understood. Given this background, we examined the subcellular location and potential anti-inflammatory roles of GPR30 using human umbilical vein endothelial cells as a model system. Inflammatory changes were induced by treatment with tumor necrosis factor (TNF), a pro-inflammatory cytokine involved in atherogenesis and many other inflammatory conditions. We found that GPR30 was located predominantly in the endothelial cell nuclei. Treatment with the selective GPR30 agonist G-1 partially attenuated the TNF induced upregulation of pro-inflammatory proteins such as intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was completely abolished by the selective GPR30 antagonist G-15, suggesting that it was indeed mediated in a GPR30 dependent manner. Interestingly, estrogen alone had no effects on TNF-treated endothelium. Concomitant activation of the classical ERs blocked the anti-inflammatory effects of G-1, indicating opposing effects of GPR30 and the classical ERs. Our findings demonstrate that endothelial GPR30 is a novel regulator of the inflammatory response which could be a potential therapeutic target against atherosclerosis and other inflammatory diseases.

Butyl benzyl phthalate suppresses the ATP-induced cell proliferation in human osteosarcoma HOS cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, P.-S., E-mail: pslediting@mail.scu.edu.t; Chen, C.-Y.

2010-05-01

Butyl benzyl phthalate (BBP), an endocrine disruptor present in the environment, exerts its genomic effects via intracellular steroid receptors and elicits non-genomic effects by interfering with membrane ion-channel receptors. We previously found that BBP blocks the calcium signaling coupled with P2X receptors in PC12 cells (Liu and Chen, 2006). Osteoblast P2X receptors were recently reported to play a role in cell proliferation and bone remodeling. In this present study, the effects of BBP on ATP-induced responses were investigated in human osteosarcoma HOS cells. These receptors mRNA had been detected, named P2X4, P2X7, P2Y2, P2Y4, P2Y5, P2Y9, and P2Y11, in humanmore » osteosarcoma HOS cells by RT-PCR. The enhancement of cell proliferation and the decrease of cytoviability had both been shown to be coupled to stimulation via different concentrations of ATP. BBP suppressed the ATP-induced calcium influx (mainly coupled with P2X) and cell proliferation but not the ATP-induced intracellular calcium release (mainly coupled with P2Y) and cytotoxicity in human osteosarcoma HOS cells. Suramin, a common P2 receptor's antagonist, blocked the ATP-induced calcium signaling, cell proliferation, and cytotoxicity. We suggest that P2X is mainly responsible for cell proliferation, and P2Y might be partially responsible for the observed cytotoxicity. BBP suppressed the calcium signaling coupled with P2X, suppressing cell proliferation. Since the importance of P2X receptors during bone metastasis has recently become apparent, the possible toxic risk of environmental BBP during bone remodeling is a public problem of concern.« less

Characterization of P2Y receptors mediating ATP induced relaxation in guinea pig airway smooth muscle: involvement of prostaglandins and K+ channels.

PubMed

Montaño, Luis M; Cruz-Valderrama, José E; Figueroa, Alejandra; Flores-Soto, Edgar; García-Hernández, Luz M; Carbajal, Verónica; Segura, Patricia; Méndez, Carmen; Díaz, Verónica; Barajas-López, Carlos

2011-10-01

In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,β-methylene-ATP was inactive from 1 to 100 μM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.

Identification of Novel G Protein–Coupled Receptor 143 Ligands as Pharmacologic Tools for Investigating X-Linked Ocular Albinism

PubMed Central

De Filippo, Elisabetta; Manga, Prashiela; Schiedel, Anke C.

2017-01-01

Purpose GPR143 regulates melanosome biogenesis and organelle size in pigment cells. The mechanisms underlying receptor function remain unclear. G protein–coupled receptors (GPCRs) are excellent pharmacologic targets; thus, we developed and applied a screening approach to identify potential GPR143 ligands and chemical modulators. Methods GPR143 interacts with β-arrestin; we therefore established a β-arrestin recruitment assay to screen for compounds that modulate activity. Because GPR143 is localized intracellularly, screening with the wild-type receptor would be restricted to agents absorbed by the cell. For the screen we used a mutant receptor, which shows similar basal activity as the wild type but traffics to the plasma membrane. We tested two compound libraries and investigated validated hits for their effects on melanocyte pigmentation. Results GPR143, which showed high constitutive activity in the β-arrestin assay, was inhibited by several compounds. The three validated inhibitors (pimozide, niclosamide, and ethacridine lactate) were assessed for impact on melanocytes. Pigmentation and expression of tyrosinase, a key melanogenic enzyme, were reduced by all compounds. Because GPR143 appears to be constitutively active, these compounds may turn off its activity. Conclusions X-linked ocular albinism type I, characterized by developmental eye defects, results from GPR143 mutations. Identifying pharmacologic agents that modulate GPR143 activity will contribute significantly to our understanding of its function and provide novel tools with which to study GPCRs in melanocytes and retinal pigment epithelium. Pimozide, one of three GPR143 inhibitors identified in this study, maybe be a good lead structure for development of more potent compounds and provide a platform for design of novel therapeutic agents. PMID:28632878

Pilocarpine-Induced Status Epilepticus Increases the Sensitivity of P2X7 and P2Y1 Receptors to Nucleotides at Neural Progenitor Cells of the Juvenile Rodent Hippocampus.

PubMed

Rozmer, Katalin; Gao, Po; Araújo, Michelle G L; Khan, Muhammad Tahir; Liu, Juan; Rong, Weifang; Tang, Yong; Franke, Heike; Krügel, Ute; Fernandes, Maria José S; Illes, Peter

2017-07-01

Patch-clamp recordings indicated the presence of P2X7 receptors at neural progenitor cells (NPCs) in the subgranular zone of the dentate gyrus in hippocampal brain slices prepared from transgenic nestin reporter mice. The activation of these receptors caused inward current near the resting membrane potential of the NPCs, while P2Y1 receptor activation initiated outward current near the reversal potential of the P2X7 receptor current. Both receptors were identified by biophysical/pharmacological methods. When the brain slices were prepared from mice which underwent a pilocarpine-induced status epilepticus or when brain slices were incubated in pilocarpine-containing external medium, the sensitivity of P2X7 and P2Y1 receptors was invariably increased. Confocal microscopy confirmed the localization of P2X7 and P2Y1 receptor-immunopositivity at nestin-positive NPCs. A one-time status epilepticus in rats caused after a latency of about 5 days recurrent epileptic fits. The blockade of central P2X7 receptors increased the number of seizures and their severity. It is hypothesized that P2Y1 receptors after a status epilepticus may increase the ATP-induced proliferation/ectopic migration of NPCs; the P2X7 receptor-mediated necrosis/apoptosis might counteract these effects, which would otherwise lead to a chronic manifestation of recurrent epileptic fits. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Acidosis Activates Endoplasmic Reticulum Stress Pathways through GPR4 in Human Vascular Endothelial Cells

PubMed Central

Dong, Lixue; Krewson, Elizabeth A.; Yang, Li V.

2017-01-01

Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the “Warburg effect”), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4-induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment. PMID:28134810

Acidosis Activates Endoplasmic Reticulum Stress Pathways through GPR4 in Human Vascular Endothelial Cells.

PubMed

Dong, Lixue; Krewson, Elizabeth A; Yang, Li V

2017-01-27

Acidosis commonly exists in the tissue microenvironment of various pathophysiological conditions such as tumors, inflammation, ischemia, metabolic disease, and respiratory disease. For instance, the tumor microenvironment is characterized by acidosis and hypoxia due to tumor heterogeneity, aerobic glycolysis (the "Warburg effect"), and the defective vasculature that cannot efficiently deliver oxygen and nutrients or remove metabolic acid byproduct. How the acidic microenvironment affects the function of blood vessels, however, is not well defined. GPR4 (G protein-coupled receptor 4) is a member of the proton-sensing G protein-coupled receptors and it has high expression in endothelial cells (ECs). We have previously reported that acidosis induces a broad inflammatory response in ECs. Acidosis also increases the expression of several endoplasmic reticulum (ER) stress response genes such as CHOP (C/EBP homologous protein) and ATF3 (activating transcription factor 3). In the current study, we have examined acidosis/GPR4- induced ER stress pathways in human umbilical vein endothelial cells (HUVEC) and other types of ECs. All three arms of the ER stress/unfolded protein response (UPR) pathways were activated by acidosis in ECs as an increased expression of phosphorylated eIF2α (eukaryotic initiation factor 2α), phosphorylated IRE1α (inositol-requiring enzyme 1α), and cleaved ATF6 upon acidic pH treatment was observed. The expression of other downstream mediators of the UPR, such as ATF4, ATF3, and spliced XBP-1 (X box-binding protein 1), was also induced by acidosis. Through genetic and pharmacological approaches to modulate the expression level or activity of GPR4 in HUVEC, we found that GPR4 plays an important role in mediating the ER stress response induced by acidosis. As ER stress/UPR can cause inflammation and cell apoptosis, acidosis/GPR4-induced ER stress pathways in ECs may regulate vascular growth and inflammatory response in the acidic microenvironment.

Genistein regulates the IL-1 beta induced activation of MAPKs in human periodontal ligament cells through G protein-coupled receptor 30.

PubMed

Luo, Li-Jun; Liu, Feng; Lin, Zhi-Kai; Xie, Yu-Feng; Xu, Jia-Li; Tong, Qing-Chun; Shu, Rong

2012-06-01

Periodontal ligament (PDL) cells are fibroblasts that play key roles in tissue integrity, periodontal inflammation and tissue regeneration in the periodontium. The periodontal tissue destruction in periodontitis is mediated by host tissue-produced inflammatory cytokines, including interleukin-1β (IL-1β). Here, we report the expression of G protein-coupled receptor 30 (GPR30, also known as G protein-coupled estrogen receptor 1 GPER) in human PDL cells and its regulation by IL-1β. IL-1β-induced GPR30 expression in human PDL cells leads to the activation of multiple signaling pathways, including MAPK, NF-κB and PI3K. In contrast, genistein, an estrogen receptor ligand, postpones the activation of MAPKs induced by IL-1β. Moreover, the inhibition of GPR30 by G15, a GPR30-specific antagonist, eliminates this delay. Thus, genistein plays a role in the regulation of MAPK activation via GPR30, and GPR30 represents a novel target regulated by steroid hormones in PDL cells. Copyright © 2012 Elsevier Inc. All rights reserved.

The P2X7 Receptor in Inflammatory Diseases: Angel or Demon?

PubMed Central

Savio, Luiz E. B.; de Andrade Mello, Paola; da Silva, Cleide Gonçalves; Coutinho-Silva, Robson

2018-01-01

Under physiological conditions, adenosine triphosphate (ATP) is present at low levels in the extracellular milieu, being massively released by stressed or dying cells. Once outside the cells, ATP and related nucleotides/nucleoside generated by ectonucleotidases mediate a high evolutionary conserved signaling system: the purinergic signaling, which is involved in a variety of pathological conditions, including inflammatory diseases. Extracellular ATP has been considered an endogenous adjuvant that can initiate inflammation by acting as a danger signal through the activation of purinergic type 2 receptors—P2 receptors (P2Y G-protein coupled receptors and P2X ligand-gated ion channels). Among the P2 receptors, the P2X7 receptor is the most extensively studied from an immunological perspective, being involved in both innate and adaptive immune responses. P2X7 receptor activation induces large-scale ATP release via its intrinsic ability to form a membrane pore or in association with pannexin hemichannels, boosting purinergic signaling. ATP acting via P2X7 receptor is the second signal to the inflammasome activation, inducing both maturation and release of pro-inflammatory cytokines, such as IL-1β and IL-18, and the production of reactive nitrogen and oxygen species. Furthermore, the P2X7 receptor is involved in caspases activation, as well as in apoptosis induction. During adaptive immune response, P2X7 receptor modulates the balance between the generation of T helper type 17 (Th17) and T regulatory (Treg) lymphocytes. Therefore, this receptor is involved in several inflammatory pathological conditions. In infectious diseases and cancer, P2X7 receptor can have different and contrasting effects, being an angel or a demon depending on its level of activation, cell studied, type of pathogen, and severity of infection. In neuroinflammatory and neurodegenerative diseases, P2X7 upregulation and function appears to contribute to disease progression. In this review, we deeply discuss P2X7 receptor dual function and its pharmacological modulation in the context of different pathologies, and we also highlight the P2X7 receptor as a potential target to treat inflammatory related diseases. PMID:29467654

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

PubMed Central

Shabir, Saqib; Cross, William; Kirkwood, Lisa A.; Pearson, Joanna F.; Appleby, Peter A.; Walker, Dawn; Eardley, Ian

2013-01-01

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. PMID:23720349

Nucleotide-mediated relaxation in guinea-pig aorta: selective inhibition by MRS2179

PubMed Central

Kaiser, Robert A; Buxton, Iain L O

2002-01-01

The vasodilatory effects of nucleotides in the guinea-pig thoracic aorta were examined to determine the relationship between molecular expression and function of P2Y receptors. In aortic rings precontracted with norepinephrine, vasodilatory responses to purine nucleotides exhibited a rank-order of potency of 2-methylthio-ATP>ADP>ATP. Responses to UTP, but not UDP suggested a functional role for P2Y4 but not P2Y6 receptors. Aortic endothelial cells express at least four P2Y receptors; P2Y1, P2Y2, P2Y4 and P2Y6. In primary culture, these cells exhibit desensitizing transient calcium responses characteristic of P2Y1, P2Y2 and P2Y4, but not P2Y6 receptors. UDP had no effect on endothelial cell calcium. The pyrimidinergic receptor agonist UTP is capable of eliciting robust vasodilation in aortic rings and causing calcium responses in cultured guineapig aortic endothelial cells. These responses are equivalent to the maximum responses observed to ATP and ADP. Measurement of intracellular calcium release in response to ATP and 2-methylthio-ATP were similar, however only the 2-methylthio-ATP response was sensitive to the P2Y1 antagonist N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179). In aortic rings, vasodilatory responses to 2-methylthio-ATP, ATP and ADP were all blocked by pre-incubation of tissues with MRS2179. MRS2179 pretreatment had no effect of the ability of UTP to cause relaxation of norepinephrine responses in aortic rings or the ability of UTP to cause calcium release in aortic endothelial cells. We demonstrate robust effects of purine and pyrimidine nucleotides in guineapig aorta and provide functional and biochemical evidence that MRS2179 is a selective P2Y1 antagonist. PMID:11815389

Anatomical and histological profiling of orphan G-protein-coupled receptor expression in gastrointestinal tract of C57BL/6J mice.

PubMed

Ito, Junko; Ito, Masahiko; Nambu, Hirohide; Fujikawa, Toru; Tanaka, Kenichi; Iwaasa, Hisashi; Tokita, Shigeru

2009-11-01

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors and regulate a variety of physiological and disease processes. Although the roles of many non-odorant GPCRs have been identified in vivo, several GPCRs remain orphans (oGPCRs). The gastrointestinal (GI) tract is the largest endocrine organ and is a promising target for drug discovery. Given their close link to physiological function, the anatomical and histological expression profiles of benchmark GI-related GPCRs, such as the cholecystokinin-1 receptor and GPR120, and 106 oGPCRs were investigated in the mucosal and muscle-myenteric nerve layers in the GI tract of C57BL/6J mice by quantitative real-time polymerase chain reaction. The mRNA expression patterns of these benchmark molecules were consistent with previous in situ hybridization and immunohistochemical studies, validating the experimental protocols in this study. Of 96 oGPCRs with significant mRNA expression in the GI tract, several oGPCRs showed unique expression patterns. GPR85, GPR37, GPR37L1, brain-specific angiogenesis inhibitor (BAI) 1, BAI2, BAI3, and GPRC5B mRNAs were preferentially expressed in the muscle-myenteric nerve layer, similar to GPCRs that are expressed in both the central and enteric nerve systems and that play multiple regulatory roles throughout the gut-brain axis. In contrast, GPR112, trace amine-associated receptor (TAAR) 1, TAAR2, and GPRC5A mRNAs were preferentially expressed in the mucosal layer, suggesting their potential roles in the regulation of secretion, immunity, and epithelial homeostasis. These anatomical and histological mRNA expression profiles of oGPCRs provide useful clues about the physiological roles of oGPCRs in the GI tract.

Expression of Estrogen Receptors Alpha (ER-α), Beta (ER-β), and G Protein-Coupled Receptor 30 (GPR30) in Testicular Tissue of Men with Klinefelter Syndrome.

PubMed

Bernardino, R L; Alves, M G; Silva, J; Barros, A; Ferraz, L; Sousa, M; Sá, R; Oliveira, P F

2016-06-01

Men with Klinefelter syndrome (KS) present severe hormonal dysregulation, particularly elevated serum estradiol concentration. Estrogens act through specific receptors and regulate testes development and spermatogenesis. Herein, we evaluated GPR30, ERα, and ERβ mRNA expression in testis of KS men and men with 46XY karyotype by reverse transcriptase and quantitative PCR. ERβ transcripts are the most abundant in testicular tissue of 46XY men. Notably, testicular GPR30 transcription in KS men was approximately 12 times higher. Since GPR30 is essential to mediate estrogen effects over steroidogenesis, our data illustrate that GPR30 may underpin the testicular alterations observed in KS men. © Georg Thieme Verlag KG Stuttgart · New York.

Lactate produced during labor modulates uterine inflammation via GPR81 (HCA1).

PubMed

Madaan, Ankush; Nadeau-Vallée, Mathieu; Rivera, Jose Carlos; Obari, Dima; Hou, Xin; Sierra, Estefania Marin; Girard, Sylvie; Olson, David M; Chemtob, Sylvain

2017-01-01

Uterine inflammatory processes trigger prolabor pathways and orchestrate on-time labor onset. Although essential for successful labor, inflammation needs to be regulated to avoid uncontrolled amplification and resolve postpartum. During labor, myometrial smooth muscle cells generate ATP mainly via anaerobic glycolysis, resulting in accumulation of lactate. Aside from its metabolic function, lactate has been shown to activate a G protein-coupled receptor, GPR81, reported to regulate inflammation. We therefore hypothesize that lactate produced during labor may act via GPR81 in the uterus to exert in a feedback manner antiinflammatory effects, to resolve or mitigate inflammation. We sought to investigate the role of lactate produced during labor and its receptor, GPR81, in regulating inflammation in the uterus. We investigated the expression of GPR81 in the uterus and the pharmacological role of lactate acting via GPR81 during labor, using shRNA-GPR81 and GPR81 -/- mice. (1) Uterine lactate levels increased substantially from 2 to 9 mmol/L during labor. (2) Immunohistological analysis revealed expression of GPR81 in the uterus with high expression in myometrium. (3) GPR81 expression increased during gestation, and peaked near labor. (4) In primary myometrial smooth muscle cell and ex vivo uteri from wild-type mice, lactate decreased interleukin-1β-induced transcription of key proinflammatory Il1b, Il6, Ccl2, and Pghs2; suppressive effects of lactate were not observed in cells and tissues from GPR81 -/- mice. (5) Conversely, proinflammatory gene expression was augmented in the uterus at term in GPR81 -/- mice and wild-type mice treated intrauterine with lentiviral-encoded shRNA-GPR81; GPR81 silencing also induced proinflammatory gene transcription in the uterus when labor was induced by endotoxin (lipopolysaccharide). (6) Importantly, administration to pregnant mice of a metabolically stable specific GPR81 agonist, 3,5-dihydroxybenzoic acid, decreased endotoxin-induced uterine inflammation, preterm birth, and associated neonatal mortality. Collectively, our data uncover a novel link between the anaerobic glycolysis and the control of uterine inflammation wherein the high levels of lactate produced during labor act on uterine GPR81 to down-regulate key proinflammatory genes. This discovery may represent a novel feedback mechanism to regulate inflammation during labor, and conveys a potential rationale for the use of GPR81 agonists to attenuate inflammation and resulting preterm birth. Copyright © 2016 Elsevier Inc. All rights reserved.

A Functional Landscape of Resistance to ALK Inhibition in Lung Cancer

PubMed Central

Wilson, Frederick H.; Johannessen, Cory M.; Piccioni, Federica; Tamayo, Pablo; Kim, Jong Wook; Van Allen, Eliezer M.; Corsello, Steven M.; Capelletti, Marzia; Calles, Antonio; Butaney, Mohit; Sharifnia, Tanaz; Gabriel, Stacey B.; Mesirov, Jill P.; Hahn, William C.; Engelman, Jeffrey A.; Meyerson, Matthew; Root, David E.; Jänne, Pasi A.; Garraway, Levi A.

2015-01-01

Summary We conducted a large-scale functional genetic study to characterize mechanisms of resistance to ALK inhibition in ALK-dependent lung cancer cells. We identify members of known resistance pathways and additional putative resistance drivers. Among the latter were members of the P2Y purinergic receptor family of G-protein coupled receptors (P2Y1, P2Y2, and P2Y6). P2Y receptors mediated resistance in part through a protein kinase C (PKC)-dependent mechanism. Moreover, PKC activation alone was sufficient to confer resistance to ALK inhibitors whereas combined ALK and PKC inhibition restored sensitivity. We observed enrichment of gene signatures associated with several resistance drivers (including P2Y receptors) in crizotinib-resistant ALK-rearranged lung tumors compared to treatment-naïve controls, supporting a role for identified resistance mechanisms in clinical resistance. PMID:25759024

The P2Y12 Antagonists, 2-Methylthioadenosine 5′-Monophosphate Triethylammonium Salt and Cangrelor (ARC69931MX), Can Inhibit Human Platelet Aggregation through a Gi-independent Increase in cAMP Levels*

PubMed Central

Srinivasan, Subhashini; Mir, Fozia; Huang, Jin-Sheng; Khasawneh, Fadi T.; Lam, Stephen C.-T.; Le Breton, Guy C.

2009-01-01

ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y1 and P2Y12. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y1 receptors are involved in the initiation of platelet aggregation, P2Y12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y12 antagonists (2-methylthioadenosine 5′-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require Gi signaling or functional P2Y12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving Gs. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP2 receptors, which are known to couple to Gs. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y12 receptors. PMID:19346255

Stimulation of GPR30 increases release of EMMPRIN-containing microvesicles in human uterine epithelial cells.

PubMed

Burnett, Lindsey A; Light, Mallory M; Mehrotra, Pavni; Nowak, Romana A

2012-12-01

Uterine remodeling is highly dependent on the glycosylated transmembrane protein extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN). Previous studies indicate estradiol can increase EMMPRIN expression in uterine cells and promote subsequent induction of MMP production. The aim of this study was to investigate the role of G protein-coupled receptor 30 (GPR30) stimulation on EMMPRIN microvesicle release in the human uterine epithelial cell line hTERT-EEC (EECs). We examined EMMPRIN release by human EECs in response to GPR30 stimulation by microvesicle isolation, Western blot, and immunocytochemistry. We employed a pharmacological approach using the GPR30-selective agonist G1 and the antagonist G15 to determine the receptor specificity of this response. We demonstrated GPR30 expression in EECs and release of EMMPRIN in microvesicles in response to stimulation of GPR30. G1, estradiol, and cholera toxin stimulated EMMPRIN release in microvesicles as detected by Western blot and immunocytochemistry, indicating that stimulation of GPR30 can induce EMMPRIN microvesicle release. These data indicate that EMMPRIN release in microvesicles can be mediated by stimulation of GPR30 in human EECs, suggesting that inappropriate stimulation or expression of this receptor may be significant in uterine pathology.

Metabolism meets immunity: The role of free fatty acid receptors in the immune system.

PubMed

Alvarez-Curto, Elisa; Milligan, Graeme

2016-08-15

There are significant numbers of nutrient sensing G protein-coupled receptors (GPCRs) that can be found in cells of the immune system and in tissues that are involved in metabolic function, such as the pancreas or the intestinal epithelium. The family of free fatty acid receptors (FFAR1-4, GPR84), plus a few other metabolite sensing receptors (GPR109A, GPR91, GPR35) have been for this reason the focus of studies linking the effects of nutrients with immunological responses. A number of the beneficial anti-inflammatory effects credited to dietary fats such as omega-3 fatty acids are attributed to their actions on FFAR4.This might play an important protective role in the development of obesity, insulin resistance or asthma. The role of the short-chain fatty acids resulting from fermentation of fibre by the intestinal microbiota in regulating acute inflammatory responses is also discussed. Finally we assess the therapeutic potential of this family of receptors to treat pathologies where inflammation is a major factor such as type 2 diabetes, whether by the use of novel synthetic molecules or by the modulation of the individual's diet. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Concurrent activation of β2-adrenergic receptor and blockage of GPR55 disrupts pro-oncogenic signaling in glioma cells.

PubMed

Wnorowski, Artur; Such, Justyna; Paul, Rajib K; Wersto, Robert P; Indig, Fred E; Jozwiak, Krzysztof; Bernier, Michel; Wainer, Irving W

2017-08-01

Activation of β 2 -adrenergic receptor (β 2 AR) and deorphanized GPR55 has been shown to modulate cancer growth in diverse tumor types in vitro and in xenograft models in vivo. (R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a bivalent compound that agonizes β 2 AR but inhibits GPR55-mediated pro-oncogenic responses. Here, we investigated the molecular mechanisms underlying the anti-tumorigenic effects of concurrent β 2 AR activation and GPR55 blockade in C6 glioma cells using (R,R')-MNF as a marker ligand. Our data show that (R,R')-MNF elicited G1-phase cell cycle arrest and apoptosis, reduced serum-inducible cell motility, promoted the phosphorylation of PKA target proteins, and inhibited constitutive activation of ERK and AKT in the low nanomolar range, whereas high nanomolar levels of (R,R')-MNF were required to block GPR55-mediated cell motility. siRNA knockdown and pharmacological inhibition of β 2 AR activity were accompanied by significant upregulation of AKT and ERK phosphorylation, and selective alteration in (R,R')-MNF responsiveness. The effects of agonist stimulation of GPR55 on various readouts, including cell motility assays, were suppressed by (R,R')-MNF. Lastly, a significant increase in phosphorylation-mediated inactivation of β-catenin occurred with (R,R')-MNF, and we provided new evidence of (R,R')-MNF-mediated inhibition of oncogenic β-catenin signaling in a C6 xenograft tumor model. Thus, simultaneous activation of β 2 AR and blockade of GPR55 may represent a novel therapeutic approach to combat the progression of glioblastoma cancer. Published by Elsevier Inc.

GPR-4 Is a Predicted G-Protein-Coupled Receptor Required for Carbon Source-Dependent Asexual Growth and Development in Neurospora crassa

PubMed Central

Li, Liande; Borkovich, Katherine A.

2006-01-01

The filamentous fungus Neurospora crassa is able to utilize a wide variety of carbon sources. Here, we examine the involvement of a predicted G-protein-coupled receptor (GPCR), GPR-4, during growth and development in the presence of different carbon sources in N. crassa. Δgpr-4 mutants have reduced mass accumulation compared to the wild type when cultured on high levels of glycerol, mannitol, or arabinose. The defect is most severe on glycerol and is cell density dependent. The genetic and physical relationship between GPR-4 and the three N. crassa Gα subunits (GNA-1, GNA-2, and GNA-3) was explored. All three Gα mutants are defective in mass accumulation when cultured on glycerol. However, the phenotypes of Δgna-1 and Δgpr-4 Δgna-1 mutants are identical, introduction of a constitutively activated gna-1 allele suppresses the defects of the Δgpr-4 mutation, and the carboxy terminus of GPR-4 interacts most strongly with GNA-1 in the yeast two-hybrid assay. Although steady-state cyclic AMP (cAMP) levels are normal in Δgpr-4 strains, exogenous cAMP partially remediates the dry mass defects of Δgpr-4 mutants on glycerol medium and Δgpr-4 strains lack the transient increase in cAMP levels observed in the wild type after addition of glucose to glycerol-grown liquid cultures. Our results support the hypothesis that GPR-4 is coupled to GNA-1 in a cAMP signaling pathway that regulates the response to carbon source in N. crassa. GPR-4-related GPCRs are present in the genomes of several filamentous ascomycete fungal pathogens, raising the possibility that a similar pathway regulates carbon sensing in these organisms. PMID:16896213

An Rgd Sequence in the P2y2 Receptor Interacts with αVβ3 Integrins and Is Required for Go-Mediated Signal Transduction

PubMed Central

Erb, Laurie; Liu, Jun; Ockerhausen, Jonathan; Kong, Qiongman; Garrad, Richard C.; Griffin, Korey; Neal, Chris; Krugh, Brent; Santiago-Pérez, Laura I.; González, Fernando A.; Gresham, Hattie D.; Turner, John T.; Weisman, Gary A.

2001-01-01

The P2Y2 nucleotide receptor (P2Y2R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein–coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y2R to K562 erythroleukemia cells was inhibited by antibodies against αVβ3/β5 integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y2Rs indicated that αV integrins colocalized 10-fold better with the wild-type P2Y2R than with a mutant P2Y2R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y2R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal–regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca2+. Furthermore, an anti-αV integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y2R. Pertussis toxin, an inhibitor of Gi/o proteins, partially inhibited Ca2+ mobilization mediated by the wild-type P2Y2R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y2R-mediated activation of Go, but not Gq. Since CD47 has been shown to associate directly with Gi/o family proteins, these results suggest that interactions between P2Y2Rs, integrins, and CD47 may be important for coupling the P2Y2R to Go. PMID:11331301

Targeted Inactivation of GPR26 Leads to Hyperphagia and Adiposity by Activating AMPK in the Hypothalamus

PubMed Central

Zhang, Weiping; Shi, Yuguang

2012-01-01

G-protein coupled receptor 26 (GPR26) is a brain-specific orphan GPCR with high expression in the brain region that controls satiety. Depletion of GPR26 has been shown to increase fat storage in C. elegans, whereas GPR26 deficiency in the hypothalamus is associated with high genetic susceptibility to the onset of obesity in mice. However, the metabolic function of GPR26 in mammals remains elusive. Herein, we investigated a role of GPR26 in regulating energy homeostasis by generating mice with targeted deletion of the GPR26 gene. We show that GPR26 deficiency causes hyperphagia and hypometabolism, leading to early onset of diet-induced obesity. Accordingly, GPR26 deficiency also caused metabolic complications commonly associated with obesity, including glucose intolerance, hyperinsulinemia, and dyslipidemia. Moreover, consistent with hyperphagia in GPR26 null mice, GPR26 deficiency significantly increased hypothalamic activity of AMPK, a key signaling event that stimulates appetite. In further support of a regulatory role of GPR26 in satiety, GPR26 knockout mice also demonstrate hypersensitivity to treatment of rimonabant, an endocannabinoid receptor-1 antagonist commonly used to treat obesity by suppressing appetite in humans. Together, these findings identified a key role of GPR26 as a central regulator of energy homeostasis though modulation of hypothalamic AMPK activation. PMID:22815809

Targeted inactivation of GPR26 leads to hyperphagia and adiposity by activating AMPK in the hypothalamus.

PubMed

Chen, Daohong; Liu, Xiaolei; Zhang, Weiping; Shi, Yuguang

2012-01-01

G-protein coupled receptor 26 (GPR26) is a brain-specific orphan GPCR with high expression in the brain region that controls satiety. Depletion of GPR26 has been shown to increase fat storage in C. elegans, whereas GPR26 deficiency in the hypothalamus is associated with high genetic susceptibility to the onset of obesity in mice. However, the metabolic function of GPR26 in mammals remains elusive. Herein, we investigated a role of GPR26 in regulating energy homeostasis by generating mice with targeted deletion of the GPR26 gene. We show that GPR26 deficiency causes hyperphagia and hypometabolism, leading to early onset of diet-induced obesity. Accordingly, GPR26 deficiency also caused metabolic complications commonly associated with obesity, including glucose intolerance, hyperinsulinemia, and dyslipidemia. Moreover, consistent with hyperphagia in GPR26 null mice, GPR26 deficiency significantly increased hypothalamic activity of AMPK, a key signaling event that stimulates appetite. In further support of a regulatory role of GPR26 in satiety, GPR26 knockout mice also demonstrate hypersensitivity to treatment of rimonabant, an endocannabinoid receptor-1 antagonist commonly used to treat obesity by suppressing appetite in humans. Together, these findings identified a key role of GPR26 as a central regulator of energy homeostasis though modulation of hypothalamic AMPK activation.

Inflammatory changes in adipose tissue enhance expression of GPR84, a medium-chain fatty acid receptor: TNFα enhances GPR84 expression in adipocytes.

PubMed

Nagasaki, Hiroshi; Kondo, Takaaki; Fuchigami, Masahiro; Hashimoto, Hiroyuki; Sugimura, Yoshihisa; Ozaki, Nobuaki; Arima, Hiroshi; Ota, Akira; Oiso, Yutaka; Hamada, Yoji

2012-02-17

In this study we aimed to identify the physiological roles of G protein-coupled receptor 84 (GPR84) in adipose tissue, together with medium-chain fatty acids (MCFAs), the specific ligands for GPR84. In mice, high-fat diet up-regulated GPR84 expression in fat pads. In 3T3-L1 adipocytes, co-culture with a macrophage cell line, RAW264, or TNFα remarkably enhanced GPR84 expression. In the presence of TNFα, MCFAs down-regulated adiponectin mRNA expression in 3T3-L1 adipocytes. Taken together, our results suggest that GPR84 emerges in adipocytes in response to TNFα from infiltrating macrophages and exacerbates the vicious cycle between adiposity and diabesity. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

The l-α-Lysophosphatidylinositol/GPR55 System and Its Potential Role in Human Obesity

PubMed Central

Moreno-Navarrete, José María; Catalán, Victoria; Whyte, Lauren; Díaz-Arteaga, Adenis; Vázquez-Martínez, Rafael; Rotellar, Fernando; Guzmán, Rocío; Gómez-Ambrosi, Javier; Pulido, Marina R.; Russell, Wendy R.; Imbernón, Mónica; Ross, Ruth A.; Malagón, María M.; Dieguez, Carlos; Fernández-Real, José Manuel; Frühbeck, Gema; Nogueiras, Ruben

2012-01-01

GPR55 is a putative cannabinoid receptor, and l-α-lysophosphatidylinositol (LPI) is its only known endogenous ligand. We investigated 1) whether GPR55 is expressed in fat and liver; 2) the correlation of both GPR55 and LPI with several metabolic parameters; and 3) the actions of LPI on human adipocytes. We analyzed CB1, CB2, and GPR55 gene expression and circulating LPI levels in two independent cohorts of obese and lean subjects, with both normal or impaired glucose tolerance and type 2 diabetes. Ex vivo experiments were used to measure intracellular calcium and lipid accumulation. GPR55 levels were augmented in the adipose tissue of obese subjects and further so in obese patients with type 2 diabetes when compared with nonobese subjects. Visceral adipose tissue GPR55 correlated positively with weight, BMI, and percent fat mass, particularly in women. Hepatic GPR55 gene expression was similar in obese and type 2 diabetic subjects. Circulating LPI levels were increased in obese patients and correlated with fat percentage and BMI in women. LPI increased the expression of lipogenic genes in visceral adipose tissue explants and intracellular calcium in differentiated visceral adipocytes. These findings indicate that the LPI/GPR55 system is positively associated with obesity in humans. PMID:22179809

Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB

PubMed Central

Chiadak, Jeanne Durendale; Arsenijevic, Tatjana; Verstrepen, Kevin; Gregoire, Françoise; Bolaky, Nargis; Delforge, Valérie; Flamand, Véronique

2016-01-01

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes. PMID:27881903

Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB.

PubMed

Chiadak, Jeanne Durendale; Arsenijevic, Tatjana; Verstrepen, Kevin; Gregoire, Françoise; Bolaky, Nargis; Delforge, Valérie; Flamand, Véronique; Perret, Jason; Delporte, Christine

2016-01-01

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (I κ B α ). In DC, LPS increased MCP-1, TLR-4, and nuclear factor- κ B1 (NF κ B1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NF κ B1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NF κ B may be involved in the LPS-induced regulation of these genes.

GPR55: Metabolic Help or Hindrance?

PubMed

Henstridge, Christopher M; Brown, Andrew J; Waldhoer, Maria

2016-09-01

Since the discovery of the lysophospholipid-sensitive receptor GPR55, hopes have been raised that targeting this G protein-coupled receptor (GPCR) may represent a novel approach for the treatment of metabolic disorders. We discuss conflicting evidence surrounding GPR55 physiology and highlight its potential as a novel target for the treatment of obesity and diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gpr109a Limits Microbiota-Induced IL-23 Production To Constrain ILC3-Mediated Colonic Inflammation.

PubMed

Bhatt, Brinda; Zeng, Peng; Zhu, Huabin; Sivaprakasam, Sathish; Li, Siyi; Xiao, Haiyan; Dong, Lixin; Shiao, Pamela; Kolhe, Ravindra; Patel, Nikhil; Li, Honglin; Levy-Bercowski, Daniel; Ganapathy, Vadivel; Singh, Nagendra

2018-04-15

A set of coordinated interactions between gut microbiota and the immune cells surveilling the intestine play a key role in shaping local immune responses and intestinal health. Gpr109a is a G protein-coupled receptor expressed at a very high level on innate immune cells and previously shown to play a key role in the induction of colonic regulatory T cells. In this study, we show that Gpr109a -/- Rag1 -/- mice exhibit spontaneous rectal prolapse and colonic inflammation, characterized by the presence of an elevated number of IL-17-producing Rorγt + innate lymphoid cells (ILCs; ILC3). Genetic deletion of Rorγt alleviated the spontaneous colonic inflammation in Gpr109a -/- Rag1 -/- mice. Gpr109a-deficient colonic dendritic cells produce higher amounts of IL-23 and thereby promote ILC3. Moreover, the depletion of gut microbiota by antibiotics treatment decreased IL-23 production, ILC3, and colonic inflammation in Gpr109a -/- Rag1 -/- mice. The ceca of Gpr109a -/- Rag1 -/- mice showed significantly increased colonization by members of Bacteroidaceae , Porphyromonadaceae , Prevotellaceae, Streptococcaceae , Christensenellaceae , and Mogibacteriaceae , as well as IBD-associated microbiota such as Enterobacteriaceae and Mycoplasmataceae , compared with Rag1 -/- mice, housed in a facility positive for Helicobacter and murine norovirus. Niacin, a Gpr109a agonist, suppressed both IL-23 production by colonic DCs and ILC3 number in a Gpr109a-dependent manner. Collectively, our data present a model suggesting that targeting Gpr109a will be potentially beneficial in the suppression of IL-23-mediated immunopathologies. Copyright © 2018 by The American Association of Immunologists, Inc.

RNAi targeting GPR4 influences HMEC-1 gene expression by microarray analysis

PubMed Central

Ren, Juan; Zhang, Yuelang; Cai, Hui; Ma, Hongbing; Zhao, Dongli; Zhang, Xiaozhi; Li, Zongfang; Wang, Shufeng; Wang, Jiangsheng; Liu, Rui; Li, Yi; Qian, Jiansheng; Wei, Hongxia; Niu, Liying; Liu, Yan; Xiao, Lisha; Ding, Muyang; Jiang, Shiwen

2014-01-01

G-protein coupled receptor 4 (GPR4) belongs to a protein family comprised of 3 closely related G protein-coupled receptors. Recent studies have shown that GPR4 plays important roles in angiogenesis, proton sensing, and regulating tumor cells as an oncogenic gene. How GPR4 conducts its functions? Rare has been known. In order to detect the genes related to GPR4, microarray technology was employed. GPR4 is highly expressed in human vascular endothelial cell HMEC-1. Small interfering RNA against GPR4 was used to knockdown GPR4 expression in HMEC-1. Then RNA from the GPR4 knockdown cells and control cells were analyzed through genome microarray. Microarray results shown that among the whole genes and expressed sequence tags, 447 differentially expressed genes were identified, containing 318 up-regulated genes and 129 down-regulated genes. These genes whose expression dramatically changed may be involved in the GPR4 functions. These genes were related to cell apoptosis, cytoskeleton and signal transduction, cell proliferation, differentiation and cell-cycle regulation, gene transcription and translation and cell material and energy metabolism. PMID:24753754

Protease-activated receptor-4 and purinergic receptor P2Y12 dimerize, co-internalize, and activate Akt signaling via endosomal recruitment of β-arrestin.

PubMed

Smith, Thomas H; Li, Julia G; Dores, Michael R; Trejo, JoAnn

2017-08-18

Vascular inflammation and thrombosis require the concerted actions of several different agonists, many of which act on G protein-coupled receptors (GPCRs). GPCR dimerization is a well-established phenomenon that can alter protomer function. In platelets and other cell types, protease-activated receptor-4 (PAR4) has been shown to dimerize with the purinergic receptor P2Y12 to coordinate β-arrestin-mediated Akt signaling, an important mediator of integrin activation. However, the mechanism by which the PAR4-P2Y12 dimer controls β-arrestin-dependent Akt signaling is not known. We now report that PAR4 and P2Y12 heterodimer internalization is required for β-arrestin recruitment to endosomes and Akt signaling. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, and co-immunoprecipitation in cells expressing receptors exogenously and endogenously, we demonstrate that PAR4 and P2Y12 specifically interact and form dimers expressed at the cell surface. We also found that activation of PAR4 but not of P2Y12 drives internalization of the PAR4-P2Y12 heterodimer. Remarkably, activated PAR4 internalization was required for recruitment of β-arrestin to endocytic vesicles, which was dependent on co-expression of P2Y12. Interestingly, stimulation of the PAR4-P2Y12 heterodimer promotes β-arrestin and Akt co-localization to intracellular vesicles. Moreover, activated PAR4-P2Y12 internalization is required for sustained Akt activation. Thus, internalization of the PAR4-P2Y12 heterodimer is necessary for β-arrestin recruitment to endosomes and Akt signaling and lays the foundation for examining whether blockade of PAR4 internalization reduces integrin and platelet activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Augmentation of cutaneous immune responses by ATP gamma S: purinergic agonists define a novel class of immunologic adjuvants.

PubMed

Granstein, Richard D; Ding, Wanhong; Huang, Jing; Holzer, Aton; Gallo, Richard L; Di Nardo, Anna; Wagner, John A

2005-06-15

Extracellular nucleotides activate ligand-gated P2XR ion channels and G protein-coupled P2YRs. In this study we report that intradermal administration of ATPgammaS, a hydrolysis-resistant P2 agonist, results in an enhanced contact hypersensitivity response in mice. Furthermore, ATPgammaS enhanced the induction of delayed-type hypersensitivity to a model tumor vaccine in mice and enhanced the Ag-presenting function of Langerhans cells (LCs) in vitro. Exposure of a LC-like cell line to ATPgammaS in the presence of LPS and GM-CSF augmented the induction of I-A, CD80, CD86, IL-1beta, and IL-12 p40 while inhibiting the expression of IL-10, suggesting that the immunostimulatory activities of purinergic agonists in the skin are mediated at least in part by P2Rs on APCs. In this regard, an LC-like cell line was found to express mRNA for P2X(1), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(9), and P2Y(11) receptors. We suggest that ATP, when released after trauma or infection, may act as an endogenous adjuvant to enhance the immune response, and that P2 agonists may augment the efficacy of vaccines.

Receptor homodimerization plays a critical role in a novel dominant negative P2RY12 variant identified in a family with severe bleeding.

PubMed

Mundell, S J; Rabbolini, D; Gabrielli, S; Chen, Q; Aungraheeta, R; Hutchinson, J L; Kilo, T; Mackay, J; Ward, C M; Stevenson, W; Morel-Kopp, M-C

2018-01-01

Essentials Three dominant variants for the autosomal recessive bleeding disorder type-8 have been described. To date, there has been no phenotype/genotype correlation explaining their dominant transmission. Proline plays an important role in P2Y12R ligand binding and signaling defects. P2Y12R homodimer formation is critical for the receptor function and signaling. Background Although inherited platelet disorders are still underdiagnosed worldwide, advances in molecular techniques are improving disease diagnosis and patient management. Objective To identify and characterize the mechanism underlying the bleeding phenotype in a Caucasian family with an autosomal dominant P2RY12 variant. Methods Full blood counts, platelet aggregometry, flow cytometry and western blotting were performed before next-generation sequencing (NGS). Detailed molecular analysis of the identified variant of the P2Y12 receptor (P2Y12R) was subsequently performed in mammalian cells overexpressing receptor constructs. Results All three referred individuals had markedly impaired ADP-induced platelet aggregation with primary wave only, despite normal total and surface P2Y12R expression. By NGS, a single P2RY12:c.G794C substitution (p.R265P) was identified in all affected individuals, and this was confirmed by Sanger sequencing. Mammalian cell experiments with the R265P-P2Y12R variant showed normal receptor surface expression versus wild-type (WT) P2Y12R. Agonist-stimulated R265P-P2Y12R function (both signaling and surface receptor loss) was reduced versus WT P2Y12R. Critically, R265P-P2Y12R acted in a dominant negative manner, with agonist-stimulated WT P2Y12R activity being reduced by variant coexpression, suggesting dramatic loss of WT homodimers. Importantly, platelet P2RY12 cDNA cloning and sequencing in two affected individuals also revealed three-fold mutant mRNA overexpression, decreasing even further the likelihood of WT homodimer formation. R265 located within extracellular loop 3 (EL3) is one of four residues that are important for receptor functional integrity, maintaining the binding pocket conformation and allowing rotation following ligand binding. Conclusion This novel dominant negative variant confirms the important role of R265 in EL3 in the functional integrity of P2Y12R, and suggests that pathologic heterodimer formation may underlie this family bleeding phenotype. © 2017 International Society on Thrombosis and Haemostasis.

The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease

PubMed Central

Karmouty-Quintana, Harry; Cicko, Sanja; Ayata, Korcan; Zissel, Gernot; Goldmann, Torsten; Lungarella, Giuseppe; Ferrari, Davide; Di Virgilio, Francesco; Robaye, Bernard; Boeynaems, Jean-Marie; Blackburn, Michael R.; Idzko, Marco

2017-01-01

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with few available treatment options. Recently, the involvement of purinergic receptor subtypes in the pathogenesis of different lung diseases has been demonstrated. Here we investigated the role of the purinergic receptor subtype P2Y2 in the context of fibrotic lung diseases. The concentration of different nucleotides was measured in the broncho-alveolar lavage (BAL) fluid derived from IPF patients and animals with bleomycin-induced pulmonary fibrosis. In addition expression of P2Y2 receptors by different cell types was determined. To investigate the functional relevance of P2Y2 receptors for the pathogenesis of the disease the bleomycin model of pulmonary fibrosis was used. Finally, experiments were performed in pursuit of the involved mechanisms. Compared to healthy individuals or vehicle treated animals, extracellular nucleotide levels in the BAL fluid were increased in patients with IPF and in mice after bleomycin administration, paralleled by a functional up-regulation of P2Y2R expression. Both bleomycin-induced inflammation and fibrosis were reduced in P2Y2R-deficient compared to wild type animals. Mechanistic studies demonstrated that recruitment of neutrophils into the lungs, proliferation and migration of lung fibroblasts as well as IL6 production are key P2Y2R mediated processes. Our results clearly demonstrate the involvement of P2Y2R subtypes in the pathogenesis of fibrotic lung diseases in humans and mice and hence support the development of selective P2Y2R antagonists for the treatment of IPF. PMID:28415591

Actions of subtype-specific purinergic ligands on rat spiral ganglion neurons.

PubMed

Ito, Ken; Iwasaki, Shinichi; Kondo, Kenji; Dulon, Didier; Kaga, Kimitaka

2004-08-01

In a previous study we showed that, in rat spiral ganglion neurons (SGNs), the adenosine 5'-triphosphate (ATP)-evoked currents were a combination of the activation of ionotropic receptors (the first fast current) and the activation of metabotropic receptors which secondarily opened non-selective cation channels. These two conductances imply the involvement of different receptor subtypes. In the present study, we tested three subtype-specific purinergic ligands: alpha,beta-methylene ATP (a;pha,beta-meATP) for P2X receptors, uridine 5'-triphosphate (UTP) for P2Y receptors and 2'-3'-O-(4-benzoylbenzoyl) ATP (Bz-ATP) for P2Z (P2X(7)) receptors. Application of 100 microM alpha,beta-meATP did not trigger any significant change in membrane conductance, while the SGNs were responsive to ATP. Pressure application of UTP (100 microM, 1 s) evoked an inward current averaging 344+/-169 pA at a holding potential of -50 mV. The conductance developed after a latency averaging 1.5+/-0.6 s, took 4-6 s to peak and reversed slowly within 15-30 s. The current-voltage curve reversed near 0 mV, suggesting a non-selective cation conductance, like the second component of the ATP conductance. Bz-ATP evoked an inward current which developed without latency, was sustained during ligand application and was rapidly inactivated at the end of application: the same characteristics as the first component of the ATP-evoked current. The Bz-ATP conductance reversed around -10 mV, indicating also a non-selective cation conductance. These results suggest that, in SGNs, ATP acts via two different receptor subtypes, ionotropic P2Z receptors and metabotropic P2Y receptors, and that these two receptor subtypes can assume different physiological roles.

The P2Y(1) and P2Y(12) receptors mediate autoinhibition of transmitter release in sympathetic innervated tissues.

PubMed

Quintas, Clara; Fraga, Sónia; Gonçalves, Jorge; Queiroz, Glória

2009-12-01

In the sympathetic nervous system, ATP is a co-transmitter and modulator of transmitter release, inhibiting noradrenaline release by acting on P2Y autoreceptors, but in peripheral tissues the subtypes involved have only scarcely been identified. We investigated the identity of the noradrenaline release-inhibiting P2Y subtypes in the epididymal portion of vas deferens and tail artery of the rat. The subtypes operating as autoreceptors, the signalling mechanism and cross-talk with alpha(2)-autoreceptors, was also investigated in the epididymal portion. In both tissues, the nucleotides 2-methylthioATP, 2-methylthioADP, ADP and ATP inhibited noradrenaline release up to 68%, with the following order of potency: 2-methylthioADP=2-methylthioATP>ADP=ATP in the epididymal portion and 2-methylthioADP=2-methylthioATP=ADP>ATP in the tail artery. The selective P2Y(1) antagonist 2'-deoxy-N(6)-methyladenosine 3',5'-bisphosphate (30microM) and the P2Y(12) antagonist 2,2-dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl ester (30microM) increased noradrenaline release per se by 25+/-8% and 18+/-3%, respectively, in the epididymal portion but not in tail artery. Both antagonists attenuated the effect of nucleotides in the epididymal portion whereas in tail artery only the P2Y(1) antagonist was effective. The agonist of P2Y(1) and P2Y(12) receptors, 2-methylthioADP, caused an inhibition of noradrenaline release that was not prevented by inhibition of phospholipase C or protein kinase C but was abolished by pertussis toxin. 2-methylthioADP and the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine were less potent at inhibiting noradrenaline release under marked influence of alpha(2)-autoinhibition. In both tissues, nucleotides modulate noradrenaline release by activation of inhibitory P2Y(1) receptors but in the epididymal portion P2Y(12) receptors also participate. P2Y(1) and P2Y(12) receptors are coupled to G(i/o)-proteins and operate as autoreceptors in the vas deferens where they interact with alpha(2)-adrenoceptors on the modulation of noradrenaline release.

Attenuation of inflammatory and neuropathic pain behaviors in mice through activation of free fatty acid receptor GPR40.

PubMed

Karki, Prasanna; Kurihara, Takashi; Nakamachi, Tomoya; Watanabe, Jun; Asada, Toshihide; Oyoshi, Tatsuki; Shioda, Seiji; Yoshimura, Megumu; Arita, Kazunori; Miyata, Atsuro

2015-02-12

The G-protein-coupled receptor 40 (GPR40) is suggested to function as a transmembrane receptor for medium- to long-chain free fatty acids and is implicated to play a role in free fatty acids-mediated enhancement of glucose-stimulated insulin secretion from pancreas. However, the functional role of GPR40 in nervous system including somatosensory pain signaling has not been fully examined yet. Intrathecal injection of GPR40 agonist (MEDICA16 or GW9508) dose-dependently reduced ipsilateral mechanical allodynia in CFA and SNL models and thermal hyperalgesia in carrageenan model. These anti-allodynic and anti-hyperalgesic effects were almost completely reversed by a GPR40 antagonist, GW1100. Immunohistochemical analysis revealed that GPR40 is expressed in spinal dorsal horn and dorsal root ganglion neurons, and immunoblot analysis showed that carrageenan or CFA inflammation or spinal nerve injury resulted in increased expression of GPR40 in these areas. Patch-clamp recordings from spinal cord slices exhibited that bath-application of either MEDICA16 or GW9508 significantly decreased the frequency of spontaneous excitatory postsynaptic currents in the substantia gelatinosa neurons of the three pain models. Our results indicate that GPR40 signaling pathway plays an important suppressive role in spinal nociceptive processing after inflammation or nerve injury, and that GPR40 agonists might serve as a new class of analgesics for treating inflammatory and neuropathic pain.

17beta-estradiol stimulates the growth of human keratinocytes by inducing cyclin D2 expression.

PubMed

Kanda, Naoko; Watanabe, Shinichi

2004-08-01

Estrogen is reported to prevent age-associated epidermal thinning in the skin. We examined if 17beta-estradiol (E2) may enhance the growth of human keratinocytes, focusing on its effects on the expression of cell cycle-regulatory proteins. E2 enhanced proliferation, bromodeoxyuridine incorporation of keratinocytes, and increased the proportion of cells in the S phase. The E2-induced stimulation of proliferation and bromodeoxyuridine incorporation was suppressed by antisense oligonucleotide against cyclin D2, which induces G1 to S phase progression. E2 increased protein and mRNA levels of cyclin D2, and resultantly enhanced assembly and kinase activities of cyclin D2-cyclin-dependent kinases 4 or 6 complexes. E2 enhanced cyclin D2 promoter activity, and the element homologous to cAMP response element (CRE) on the promoter was responsible for the effect. Cyclin D2 expression was enhanced by antiestrogens, ICI 182,780 and 4-hydroxytamoxifen, and membrane-impermeable bovine serum albumin-conjugated E2, indicating the effects via membrane E2-binding sites. E2 increased the enhancer activity of CRE-like element and the amount of phosphorylated cAMP response element binding protein (CREB) binding this element, and the increases were suppressed by H-89, an inhibitor of cAMP-dependent protein kinase A. H-89 also suppressed E2-induced cyclin D2 expression, proliferation, and bromodeoxyuridine incorporation in keratinocytes. Antisense oligonucleotide against G-protein-coupled receptor GPR30 suppressed the E2-induced increases of phosphorylated CREB, cyclin D2 level, proliferation, and bromodeoxyuridine incorporation in keratinocytes. These results suggest that E2 may stimulate the growth of keratinocytes by inducing cyclin D2 expression via CREB phosphorylation by protein kinase A, dependent on cAMP. These effects of E2 may be mediated via cell surface GPR30.

Differential phosphorylation signals control endocytosis of GPR15

PubMed Central

Okamoto, Yukari; Shikano, Sojin

2017-01-01

GPR15 is an orphan G protein–coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T-cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin dependent and partially dependent on β-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane. An Ala mutation of the distal C-terminal Arg-354 or Ser-357, which forms a consensus phosphorylation site for basophilic kinases, markedly reduced the endocytosis, whereas phosphomimetic mutation of Ser-357 to Asp did not. Ser-357 was phosphorylated in vitro by multiple kinases, including PKA and PKC, and pharmacological activation of these kinases enhanced both phosphorylation of Ser-357 and endocytosis of GPR15. These results suggested that Ser-357 phosphorylation critically controls the ligand-independent endocytosis of GPR15. The functional role of Ser-357 in endocytosis was distinct from that of a conserved Ser/Thr cluster in the more proximal C-terminus, which was responsible for the β-arrestin– and GPCR kinase–dependent endocytosis of GPR15. Thus phosphorylation signals may differentially control cell surface density of GPR15 through endocytosis. PMID:28615320

Stimulation of GPR30 Increases Release of EMMPRIN-Containing Microvesicles in Human Uterine Epithelial Cells

PubMed Central

Light, Mallory M.; Mehrotra, Pavni; Nowak, Romana A.

2012-01-01

Context: Uterine remodeling is highly dependent on the glycosylated transmembrane protein extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN). Previous studies indicate estradiol can increase EMMPRIN expression in uterine cells and promote subsequent induction of MMP production. Objective: The aim of this study was to investigate the role of G protein-coupled receptor 30 (GPR30) stimulation on EMMPRIN microvesicle release in the human uterine epithelial cell line hTERT-EEC (EECs). Design: We examined EMMPRIN release by human EECs in response to GPR30 stimulation by microvesicle isolation, Western blot, and immunocytochemistry. We employed a pharmacological approach using the GPR30-selective agonist G1 and the antagonist G15 to determine the receptor specificity of this response. Results: We demonstrated GPR30 expression in EECs and release of EMMPRIN in microvesicles in response to stimulation of GPR30. G1, estradiol, and cholera toxin stimulated EMMPRIN release in microvesicles as detected by Western blot and immunocytochemistry, indicating that stimulation of GPR30 can induce EMMPRIN microvesicle release. Conclusions: These data indicate that EMMPRIN release in microvesicles can be mediated by stimulation of GPR30 in human EECs, suggesting that inappropriate stimulation or expression of this receptor may be significant in uterine pathology. PMID:23012390

Endogenous 17β-estradiol is required for activity-dependent long-term potentiation in the striatum: interaction with the dopaminergic system

PubMed Central

Tozzi, Alessandro; de Iure, Antonio; Tantucci, Michela; Durante, Valentina; Quiroga-Varela, Ana; Giampà, Carmela; Di Mauro, Michela; Mazzocchetti, Petra; Costa, Cinzia; Di Filippo, Massimiliano; Grassi, Silvarosa; Pettorossi, Vito Enrico; Calabresi, Paolo

2015-01-01

17β-estradiol (E2), a neurosteroid synthesized by P450-aromatase (ARO), modulates various brain functions. We characterized the role of the locally synthesized E2 on striatal long-term synaptic plasticity and explored possible interactions between E2 receptors (ERs) and dopamine (DA) receptors in the dorsal striatum of adult male rats. Inhibition of E2 synthesis or antagonism of ERs prevented the induction of long-term potentiation (LTP) in both medium spiny neurons (MSNs) and cholinergic interneurons (ChIs). Activation of a D1-like DA receptor/cAMP/PKA-dependent pathway restored LTP. In MSNs exogenous E2 reversed the effect of ARO inhibition. Also antagonism of M1 muscarinic receptors prevented the D1-like receptor-mediated restoration of LTP confirming a role for ChIs in controlling the E2-mediated LTP of MSNs. A novel striatal interaction, occurring between ERs and D1-like receptors in both MSNs and ChIs, might be critical to regulate basal ganglia physiology and to compensate synaptic alterations in Parkinson’s disease. PMID:26074768

Endogenous 17β-estradiol is required for activity-dependent long-term potentiation in the striatum: interaction with the dopaminergic system.

PubMed

Tozzi, Alessandro; de Iure, Antonio; Tantucci, Michela; Durante, Valentina; Quiroga-Varela, Ana; Giampà, Carmela; Di Mauro, Michela; Mazzocchetti, Petra; Costa, Cinzia; Di Filippo, Massimiliano; Grassi, Silvarosa; Pettorossi, Vito Enrico; Calabresi, Paolo

2015-01-01

17β-estradiol (E2), a neurosteroid synthesized by P450-aromatase (ARO), modulates various brain functions. We characterized the role of the locally synthesized E2 on striatal long-term synaptic plasticity and explored possible interactions between E2 receptors (ERs) and dopamine (DA) receptors in the dorsal striatum of adult male rats. Inhibition of E2 synthesis or antagonism of ERs prevented the induction of long-term potentiation (LTP) in both medium spiny neurons (MSNs) and cholinergic interneurons (ChIs). Activation of a D1-like DA receptor/cAMP/PKA-dependent pathway restored LTP. In MSNs exogenous E2 reversed the effect of ARO inhibition. Also antagonism of M1 muscarinic receptors prevented the D1-like receptor-mediated restoration of LTP confirming a role for ChIs in controlling the E2-mediated LTP of MSNs. A novel striatal interaction, occurring between ERs and D1-like receptors in both MSNs and ChIs, might be critical to regulate basal ganglia physiology and to compensate synaptic alterations in Parkinson's disease.

Three Mutations in the Bilateral Frontoparietal Polymicrogyria Gene GPR56 in Pakistani Intellectual Disability Families.

PubMed

Sawal, Humaira Aziz; Harripaul, Ricardo; Mikhailov, Anna; Vleuten, Kayla; Naeem, Farooq; Nasr, Tanveer; Hassan, Muhammad Jawad; Vincent, John B; Ayub, Muhammad; Rafiq, Muhammad Arshad

2018-06-01

Bilateral frontoparietal polymicrogyria (BFPP, MIM 606854) is a heterogeneous autosomal recessive disorder of abnormal cortical lamination, leading to moderate-to-severe intellectual disability (ID), seizure disorder, and motor difficulties, and caused by mutations in the G protein-coupled receptor 56 ( GPR56 ) gene. Twenty-eight mutations in 40 different families have been reported in the literature. The clinical and neuroimaging phenotype is consistent in these cases. The BFPP cortex consists of numerous small gyral cells, with scalloping of the cortical-white matter junction. There are also associated white matter, brain stem, and cerebellar changes. GPR56 is a member of an adhesion G protein-coupled receptor family with a very long N-terminal stalk and seven transmembrane domains. In this study, we identified three families from Pakistan, ascertained primarily for ID, with overlapping approximately 1 Mb region (chr16:56,973,335-57,942,866) of homozygosity by descent, including 24 RefSeq genes. We found three GPR56 homozygous mutations, using next-generation sequencing. These mutations include a substitutional variant, c.1460T > C; p.L487P, (chr16:57693480 T > C), a 13-bp insertion causing the frameshift and truncating mutation, p.Leu269Hisfs*21 (NM_005682.6:c.803_804insCCATGGAGGTGCT; Chr16: 57689345_57689346insCCATGGAGGTGCT), and a truncating mutation c.1426C > T; p.Arg476* (Chr16:57693446C > T). These mutations fully segregated with ID in these families and were absent in the Exome Aggregation Consortium database that has approximately 8,000 control samples of South Asian origin. Two of these mutations have been reported in ClinVar database, and the third one has not been reported before. Three families from Pakistan with GPR56 mutations have been reported before. With the addition of our findings, the total number of mutations reported in Pakistani patients now is six. These results increase our knowledge regarding the mutational spectrum of the GPR56 gene causing BFPP/ID.

Characterization of the Mel1c melatoninergic receptor in platypus (Ornithorhynchus anatinus)

PubMed Central

Gautier, Célia; Guenin, Sophie-Penelope; Riest-Fery, Isabelle; Perry, Tahlia Jade; Legros, Céline; Nosjean, Olivier; Simonneaux, Valerie; Grützner, Frank

2018-01-01

Melatonin is a neurohormone produced in both animals and plants. It binds at least three G-protein-coupled receptors: MT1 and MT2, and Mel1cGPR. Mammalian GPR50 evolved from the reptilian/avian Mel1c and lost its capacity to bind melatonin in all the therian mammal species that have been tested. In order to determine if binding is lost in the oldest surviving mammalian lineage of monotremes we investigated whether the melatonin receptor has the ability to bind melatonin in the platypus (Ornithorhynchus anatinus), and evaluated its pharmacological profile. Sequence and phylogenetic analysis showed that platypus has in fact retained the ancestral Mel1c and has the capacity to bind melatonin similar to other mammalian melatonin receptors (MT1 and MT2), with an affinity in the 1 nM range. We also investigated the binding of a set of melatoninergic ligands used previously to characterize the molecular pharmacology of the melatonin receptors from sheep, rats, mice, and humans and found that the general profiles of these compounds make Mel1c resemble human MT1 more than MT2. This work shows that the loss of GPR50 binding evolved after the divergence of monotremes less than 190MYA in therian mammals. PMID:29529033

Characterization of the Mel1c melatoninergic receptor in platypus (Ornithorhynchus anatinus).

PubMed

Gautier, Célia; Guenin, Sophie-Penelope; Riest-Fery, Isabelle; Perry, Tahlia Jade; Legros, Céline; Nosjean, Olivier; Simonneaux, Valerie; Grützner, Frank; Boutin, Jean A

2018-01-01

Melatonin is a neurohormone produced in both animals and plants. It binds at least three G-protein-coupled receptors: MT1 and MT2, and Mel1cGPR. Mammalian GPR50 evolved from the reptilian/avian Mel1c and lost its capacity to bind melatonin in all the therian mammal species that have been tested. In order to determine if binding is lost in the oldest surviving mammalian lineage of monotremes we investigated whether the melatonin receptor has the ability to bind melatonin in the platypus (Ornithorhynchus anatinus), and evaluated its pharmacological profile. Sequence and phylogenetic analysis showed that platypus has in fact retained the ancestral Mel1c and has the capacity to bind melatonin similar to other mammalian melatonin receptors (MT1 and MT2), with an affinity in the 1 nM range. We also investigated the binding of a set of melatoninergic ligands used previously to characterize the molecular pharmacology of the melatonin receptors from sheep, rats, mice, and humans and found that the general profiles of these compounds make Mel1c resemble human MT1 more than MT2. This work shows that the loss of GPR50 binding evolved after the divergence of monotremes less than 190MYA in therian mammals.

Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation.

PubMed

Emgård, Johanna; Kammoun, Hana; García-Cassani, Bethania; Chesné, Julie; Parigi, Sara M; Jacob, Jean-Marie; Cheng, Hung-Wei; Evren, Elza; Das, Srustidhar; Czarnewski, Paulo; Sleiers, Natalie; Melo-Gonzalez, Felipe; Kvedaraite, Egle; Svensson, Mattias; Scandella, Elke; Hepworth, Matthew R; Huber, Samuel; Ludewig, Burkhard; Peduto, Lucie; Villablanca, Eduardo J; Veiga-Fernandes, Henrique; Pereira, João P; Flavell, Richard A; Willinger, Tim

2018-01-16

Group 3 innate lymphoid cells (ILC3s) sense environmental signals and are critical for tissue integrity in the intestine. Yet, which signals are sensed and what receptors control ILC3 function remain poorly understood. Here, we show that ILC3s with a lymphoid-tissue-inducer (LTi) phenotype expressed G-protein-coupled receptor 183 (GPR183) and migrated to its oxysterol ligand 7α,25-hydroxycholesterol (7α,25-OHC). In mice lacking Gpr183 or 7α,25-OHC, ILC3s failed to localize to cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Gpr183 deficiency in ILC3s caused a defect in CP and ILF formation in the colon, but not in the small intestine. Localized oxysterol production by fibroblastic stromal cells provided an essential signal for colonic lymphoid tissue development, and inflammation-induced increased oxysterol production caused colitis through GPR183-mediated cell recruitment. Our findings show that GPR183 promotes lymphoid organ development and indicate that oxysterol-GPR183-dependent positioning within tissues controls ILC3 activity and intestinal homeostasis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

17β-estradiol-induced regulation of the novel 5-HT1A-related transcription factors NUDR and Freud-1 in SH SY5Y cells.

PubMed

Adeosun, Samuel O; Albert, Paul R; Austin, Mark C; Iyo, Abiye H

2012-05-01

Nuclear deformed epidermal autoregulatory factor-1 (NUDR/Deaf-1) and five prime repressor element under dual repression (Freud-1) are novel transcriptional regulators of the 5-HT(1A) receptor, a receptor that has been implicated in the pathophysiology of various psychiatric illnesses. The antidepressant effect of 17β-Estradiol (17βE(2)) is purported to involve the downregulation of this receptor. We investigated the possible role of NUDR and Freud-1 in 17βE(2)-induced downregulation of the 5-HT(1A) receptor in the neuroblastoma cell line SH SY5Y. Cells were treated with 10 nM of 17βE(2) for 3 or 48 h, followed by a 24-h withdrawal period. Proteins were isolated and analyzed by western blotting. 17βE(2) treatment increased NUDR immunoreactivity while Freud-1 and the 5-HT(1A) receptor showed significant decreases. Upon withdrawal of 17βE(2), protein expression returned to control levels, except for NUDR, which remained significantly elevated in the 3-h treatment. Taken together, these data support a non-genomic downregulation of 5-HT(1A) receptor protein by 17βE(2), which does not involve NUDR and Freud-1. Rather, changes in both transcription factors seem to be compensatory/homeostatic responses to changes in 5-HT(1A) receptor induced by 17βE(2). These observations further highlight the importance of NUDR and Freud-1 in regulating 5-HT(1A) receptor expression.

P2Y6 Receptor Activation Promotes Inflammation and Tissue Remodeling in Pulmonary Fibrosis

PubMed Central

Müller, Tobias; Fay, Susanne; Vieira, Rodolfo Paula; Karmouty-Quintana, Harry; Cicko, Sanja; Ayata, Cemil Korcan; Zissel, Gernot; Goldmann, Torsten; Lungarella, Giuseppe; Ferrari, Davide; Di Virgilio, Francesco; Robaye, Bernard; Boeynaems, Jean-Marie; Lazarowski, Eduardo R.; Blackburn, Michael R.; Idzko, Marco

2017-01-01

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and very few available treatment options. The involvement of the purinergic receptor subtypes P2Y2 and P2X7 in fibrotic lung disease has been demonstrated recently. In this study, we investigated the role of P2Y6 receptors in the pathogenesis of IPF in humans and in the animal model of bleomycin-induced lung injury. P2Y6R expression was upregulated in lung structural cells but not in bronchoalveolar lavage (BAL) cells derived from IPF patients as well as in animals following bleomycin administration. Furthermore, BAL fluid levels of the P2Y6R agonist uridine-5′-diphosphate were elevated in animals with bleomycin-induced pulmonary fibrosis. Inflammation and fibrosis following bleomycin administration were reduced in P2Y6R-deficient compared to wild-type animals confirming the pathophysiological relevance of P2Y6R subtypes for fibrotic lung diseases. Experiments with bone marrow chimeras revealed the importance of P2Y6R expression on lung structural cells for pulmonary inflammation and fibrosis. Similar effects were obtained when animals were treated with the P2Y6R antagonist MRS2578. In vitro studies demonstrated that proliferation and secretion of the pro-inflammatory/pro-fibrotic cytokine IL-6 by lung fibroblasts are P2Y6R-mediated processes. In summary, our results clearly demonstrate the involvement of P2Y6R subtypes in the pathogenesis of pulmonary fibrosis. Thus, blocking pulmonary P2Y6 receptors might be a new target for the treatment of IPF. PMID:28878780

Silencing of P2Y2 receptor delays Ap4A-corneal re-epithelialization process

PubMed Central

Crooke, Almudena; Mediero, Aránzazu; Guzmán-Aránguez, Ana

2009-01-01

Purpose There are no selective antagonists for the metabotropic nucleotide P2Y2 receptor subtype. This implies that it is not possible to demonstrate the importance of such a receptor in the relevant process of corneal wound healing. Therefore, we have cloned and designed a small interference RNA (siRNA) against the rabbit P2Y2 receptor (P2Y2-R) mRNA, which clearly demonstrates the importance of this receptor in the process of wound healing triggered by nucleotides and dinucleotides both in vitro and in vivo. Methods Rabbit P2Y2-R cDNA was cloned using a combination of degenerate reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). To test the efficacy of synthesized siRNAs targeting P2Y2-R, immunocytochemistry, immunohistochemistry, and quantitative RT-PCR (qRT–PCR) assays were performed. Migration assays were performed both in vitro and in vivo by wounding the epithelium with a pipette tip and n-heptanol, respectively. These wounds were performed 72 h after siRNA transfection either in the presence or the absence of the P2Y2 agonist, 100 μM Ap4A (diadenosine tetraphosphate). Results The cloned receptor presents 93% homology compared to the human gene. Two siRNAs were designed and synthesized against a rabbit P2Y2-R sequence. After transfection (in vitro assays) or topical instillation (in vivo assays), we demonstrated P2Y2-R siRNA efficient transfection/delivery and its efficient gene silencing. Clear reduction of P2Y2-R expression was observed at both the mRNA and protein levels in corneas treated with siRNA. In vitro and in vivo migration analysis showed that the silencing process has concomitantly reduced the ability of corneal cells to close the wounds in the presence of the Ap4A. In addition, both synthesized siRNAs exert a delay effect on the Ap4A-induced migration rate in vitro. These results suggest the absence of non-specific (off-target) effects by our siRNA. Conclusions The application of P2Y2-R siRNA has demonstrated the role of this receptor in the accelerating effect of diadenosine tetraphosphate (Ap4A) on the corneal wound healing process. PMID:19521552

Silencing of P2Y2 receptor delays Ap4A-corneal re-epithelialization process.

PubMed

Crooke, Almudena; Mediero, Aránzazu; Guzmán-Aránguez, Ana; Pintor, Jesús

2009-06-11

There are no selective antagonists for the metabotropic nucleotide P2Y(2) receptor subtype. This implies that it is not possible to demonstrate the importance of such a receptor in the relevant process of corneal wound healing. Therefore, we have cloned and designed a small interference RNA (siRNA) against the rabbit P2Y(2) receptor (P2Y(2)-R) mRNA, which clearly demonstrates the importance of this receptor in the process of wound healing triggered by nucleotides and dinucleotides both in vitro and in vivo. Rabbit P2Y(2)-R cDNA was cloned using a combination of degenerate reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). To test the efficacy of synthesized siRNAs targeting P2Y(2)-R, immunocytochemistry, immunohistochemistry, and quantitative RT-PCR (qRT-PCR) assays were performed. Migration assays were performed both in vitro and in vivo by wounding the epithelium with a pipette tip and n-heptanol, respectively. These wounds were performed 72 h after siRNA transfection either in the presence or the absence of the P2Y(2) agonist, 100 muM Ap(4)A (diadenosine tetraphosphate). The cloned receptor presents 93% homology compared to the human gene. Two siRNAs were designed and synthesized against a rabbit P2Y(2)-R sequence. After transfection (in vitro assays) or topical instillation (in vivo assays), we demonstrated P2Y(2)-R siRNA efficient transfection/delivery and its efficient gene silencing. Clear reduction of P2Y(2)-R expression was observed at both the mRNA and protein levels in corneas treated with siRNA. In vitro and in vivo migration analysis showed that the silencing process has concomitantly reduced the ability of corneal cells to close the wounds in the presence of the Ap(4)A. In addition, both synthesized siRNAs exert a delay effect on the Ap(4)A-induced migration rate in vitro. These results suggest the absence of non-specific (off-target) effects by our siRNA. The application of P2Y(2)-R siRNA has demonstrated the role of this receptor in the accelerating effect of diadenosine tetraphosphate (Ap(4)A) on the corneal wound healing process.

A New Pain Regulatory System via the Brain Long Chain Fatty Acid Receptor GPR40/FFA1 Signal.

PubMed

Nakamoto, Kazuo

2017-01-01

An increasingly large number of pharmacological and physiological works on fatty acids have shown that the functional properties of fatty acids are regulated by the amount of individual fatty acid intake and the distribution of fatty acids among organs. Recently, it has been determined that G-protein-coupled receptor 40/free fatty acid receptor 1 (GPR40/FFA1) is activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). GPR40/FFA1 is mainly expressed in the β cell of the pancreas, spinal cord and brain. It is reported that this receptor has a functional role in controlling blood glucose levels via the modulation of insulin secretion. However, its physiological function in the brain remains unknown. Our previous studies have shown that GPR40/FFA1 is expressed in pro-opiomelanocortin (POMC)-positive neurons of the arcuate nucleus, serotonergic neurons in the nucleus raphe magnus, and in noradrenergic neurons in the locus coeruleus. Furthermore, the intracerebroventricular injection of DHA or GW9508, which is a selective GPR40/FFA1 agonist, attenuates formalin-induced inflammatory pain behavior through increasing β-endorphin release in the hypothalamus. It also suppresses complete Freund's adjuvant-induced mechanical allodynia and thermal hyperalgesia. Our findings suggest that brain free long-chain fatty acids-GPR40/FFA1 signaling might have an important role in the modulation of endogenous pain control systems. In this review, I discuss the current status and our recent study regarding a new pain regulatory system via the brain long chain fatty acid receptor GPR40/FFA1 signal.

Functionalized Congeners of P2Y1 Receptor Antagonists:

DOE Office of Scientific and Technical Information (OSTI.GOV)

de Castro, Sonia; Maruoka, Hiroshi; Hong, Kunlun

2010-01-01

The P2Y{sub 1} receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y{sub 1} receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N{sup 6}-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y{sub 1} receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of anmore » intermediate amide group revealed high affinity of carboxylic congener 8 (K{sub i} 23 nM) and extended amine congener 15 (K{sub i} 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended {epsilon}-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y{sub 1} receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y{sub 1} receptor modeling and ligand docking. Attempted P2Y{sub 1} antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM dendrimer to produce a multivalent conjugate exhibiting a desired biological effect, i.e., antithrombotic action.« less

[Impact of novel P2Y12 receptor inhibitors on platelet reactivity in acute coronary syndrome patients undergoing percutaneous coronary intervention].

PubMed

Chong Tou, T J; Liu, P M; Wang, J F; Sio Cham, Z C; O U, Y F; Lei Sio, Z W; Lei Put, P Z; Lei Sok, S M; Zhou, S X; Wu, W

2016-02-01

To investigate the impact of novel P2Y(12) receptor inhibitors including prasugrel or ticagrelor on platelet reactivity in patients with acute coronary syndrome (ACS) receiving percutaneous coronary intervention (PCI), and provide clinical data for novel oral P2Y(12) receptor inhibitors use among Chinese patients. Between October 2011 to February 2014, 174 consecutive patients (135 males; (67.8±11.8) years old) with ACS undergoing PCI in Kiang Wu Hospital, Macau were prospectively enrolled in this study. Oral aspirin and one P2Y(12) receptor inhibitor were administered for 5 days or above after PCI, patients were divided into clopidogrel, prasugrel and ticagrelor groups in accordance with the agent administered. Platelet reactivity of the patients was detected by VerifyNow P2Y(12) reaction unit (PRU); and the high on-treatment platelet reactivity (HPR) and non-HPR were defined as PRU≥208 and PRU<208 respectively. Patients with HPR during clopidogrel therapy were switched either to prasugrel or ticagrelor, or continued the same treatment; and then the platelet reactivity was monitored again. There were 113 clopidogrel cases (64.9%), 20 prasugrel cases (11.5%) and 41 ticagrelor cases (23.6%). Fifty-seven cases (32.8%) were defined as HPR post P2Y(12) receptor inhibitor use, in which 55 cases (55/113, 48.7%) were treated with clopidogrel. The degree of inhibition of platelet reactivity was significantly different in patients on clopidogrel, prasugrel and ticagrelor therapy, percent inhibition assayed by the VerifyNow P2Y(12) system was 28.2%±23.5%, 61.4%±26.7% and 81.3%±19.8% respectively (P<0.05). Different degree of platelet reactivity was achieved by the 3 P2Y(12) receptor inhibitors at multiple time points. The among-group differences in platelet reactivity became apparent at the early treatment stage (P<0.05). Platelet aggregation decreased significantly in patients switched from clopidogrel to prasugrel or ticagrelor (P<0.05). Novel oral P2Y(12) receptor inhibitors are more effective in inhibiting platelet reactivity in ACS patients, and our results show that novel oral P2Y(12) receptor inhibitors provide a new option for ACS patients with HPR post clopidogrel or high-risk features of ischemic complications, including stent thrombosis and post-PCI ischemic events.

Deletion of Gpr55 Results in Subtle Effects on Energy Metabolism, Motor Activity and Thermal Pain Sensation.

PubMed

Bjursell, Mikael; Ryberg, Erik; Wu, Tingting; Greasley, Peter J; Bohlooly-Y, Mohammad; Hjorth, Stephan

2016-01-01

The G-protein coupled receptor 55 (GPR55) is activated by cannabinoids and non-cannabinoid molecules and has been speculated to play a modulatory role in a large variety of physiological and pathological processes, including in metabolically perturbed states. We therefore generated male mice deficient in the gene coding for the cannabinoid/lysophosphatidylinositol (LPI) receptor Gpr55 and characterized them under normal dietary conditions as well as during high energy dense diet feeding followed by challenge with the CB1 receptor antagonist/GPR55 agonist rimonabant. Gpr55 deficient male mice (Gpr55 KO) were phenotypically indistinguishable from their wild type (WT) siblings for the most part. However, Gpr55 KO animals displayed an intriguing nocturnal pattern of motor activity and energy expenditure (EE). During the initial 6 hours of the night, motor activity was significantly elevated without any significant effect observed in EE. Interestingly, during the last 6 hours of the night motor activity was similar but EE was significantly decreased in the Gpr55 KO mice. No significant difference in motor activity was detected during daytime, but EE was lower in the Gpr55 KO compared to WT mice. The aforementioned patterns were not associated with alterations in energy intake, daytime core body temperature, body weight (BW) or composition, although a non-significant tendency to increased adiposity was seen in Gpr55 KO compared to WT mice. Detailed analyses of daytime activity in the Open Field paradigm unveiled lower horizontal activity and rearing time for the Gpr55 KO mice. Moreover, the Gpr55 KO mice displayed significantly faster reaction time in the tail flick test, indicative of thermal hyperalgesia. The BW-decreasing effect of rimonabant in mice on long-term cafeteria diet did not differ between Gpr55 KO and WT mice. In conclusion, Gpr55 deficiency is associated with subtle effects on diurnal/nocturnal EE and motor activity behaviours but does not appear per se critically required for overall metabolism or behaviours.

Deciphering the GPER/GPR30-agonist and antagonists interactions using molecular modeling studies, molecular dynamics, and docking simulations.

PubMed

Méndez-Luna, D; Martínez-Archundia, M; Maroun, Rachid C; Ceballos-Reyes, G; Fragoso-Vázquez, M J; González-Juárez, D E; Correa-Basurto, J

2015-01-01

The G-protein coupled estrogen receptor 1 GPER/GPR30 is a transmembrane seven-helix (7TM) receptor involved in the growth and proliferation of breast cancer. Due to the absence of a crystal structure of GPER/GPR30, in this work, molecular modeling studies have been carried out to build a three-dimensional structure, which was subsequently refined by molecular dynamics (MD) simulations (up to 120 ns). Furthermore, we explored GPER/GPR30's molecular recognition properties by using reported agonist ligands (G1, estradiol (E2), tamoxifen, and fulvestrant) and the antagonist ligands (G15 and G36) in subsequent docking studies. Our results identified the E2 binding site on GPER/GPR30, as well as other receptor cavities for accepting large volume ligands, through GPER/GPR30 π-π, hydrophobic, and hydrogen bond interactions. Snapshots of the MD trajectory at 14 and 70 ns showed almost identical binding motifs for G1 and G15. It was also observed that C107 interacts with the acetyl oxygen of G1 (at 14 ns) and that at 70 ns the residue E275 interacts with the acetyl group and with the oxygen from the other agonist whereas the isopropyl group of G36 is oriented toward Met141, suggesting that both C107 and E275 could be involved in the protein activation. This contribution suggest that GPER1 has great structural changes which explain its great capacity to accept diverse ligands, and also, the same ligand could be recognized in different binding pose according to GPER structural conformations.

Identification of 6H1 as a P2Y purinoceptor: P2Y5.

PubMed

Webb, T E; Kaplan, M G; Barnard, E A

1996-02-06

We have determined the identity of the orphan G-protein coupled receptor cDNA, 6H1, present in activated chicken T cells, as a subtype of P2Y purinoceptor. This identification is based on first on the degree of sequence identity shared with recently cloned members of the P2Y receptor family and second on the pharmacological profile. Upon transient expression in COS-7 cells the 6H1 receptor bound the radiolabel [35S]dATP alpha S specifically and with high affinity (Kd, 10 nM). This specific binding could be competitively displaced by a range of ligands active at P2 purinoceptors, with ATP being the most active (K (i)), 116 nM). Such competition studies have established the following rank order of activity: ATP ADP 2-methylthioATP alpha, beta-methylene ATP, UTP, thus confirming 6H1 as a member of the growing family of P2Y purinoceptors. As the fifth receptor of this type to be identified we suggest that it be named P2Y5.

Gpr124 is essential for blood-brain barrier integrity in central nervous system disease.

PubMed

Chang, Junlei; Mancuso, Michael R; Maier, Carolina; Liang, Xibin; Yuki, Kanako; Yang, Lu; Kwong, Jeffrey W; Wang, Jing; Rao, Varsha; Vallon, Mario; Kosinski, Cynthia; Zhang, J J Haijing; Mah, Amanda T; Xu, Lijun; Li, Le; Gholamin, Sharareh; Reyes, Teresa F; Li, Rui; Kuhnert, Frank; Han, Xiaoyuan; Yuan, Jenny; Chiou, Shin-Heng; Brettman, Ari D; Daly, Lauren; Corney, David C; Cheshier, Samuel H; Shortliffe, Linda D; Wu, Xiwei; Snyder, Michael; Chan, Pak; Giffard, Rona G; Chang, Howard Y; Andreasson, Katrin; Kuo, Calvin J

2017-04-01

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation

PubMed Central

Bermudez, Yira; Benavente, Claudia A.; Meyer, Ralph G.; Coyle, W. Russell; Jacobson, Myron K.; Jacobson, Elaine L.

2011-01-01

Background Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through Gi-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. Results Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional Gi-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional. Conclusions The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s) of nicotinic acid receptors in human skin homeostasis. PMID:21655214

Expression pattern of G protein-coupled receptor 30 in human seminiferous tubular cells.

PubMed

Oliveira, Pedro F; Alves, Marco G; Martins, Ana D; Correia, Sara; Bernardino, Raquel L; Silva, Joaquina; Barros, Alberto; Sousa, Mário; Cavaco, José E; Socorro, Sílvia

2014-05-15

The role of estrogens in male reproductive physiology has been intensively studied over the last few years. Yet, the involvement of their specific receptors has long been a matter of debate. The selective testicular expression of the classic nuclear estrogen receptors (ERα and ERβ) argues in favor of ER-specific functions in the spermatogenic event. Recently, the existence of a G protein-coupled estrogen receptor (GPR30) mediating non-genomic effects of estrogens has also been described. However, little is known about the specific testicular expression pattern of GPR30, as well as on its participation in the control of male reproductive function. Herein, by means of immunohistochemical and molecular biology techniques (RT-PCR and Western blot), we aimed to present the first exhaustive evaluation of GPR30 expression in non-neoplastic human testicular cells. Indeed, we were able to demonstrate that GPR30 was expressed in human testicular tissue and that the staining pattern was consistent with its cytoplasmic localization. Additionally, by using cultured human Sertoli cells (SCs) and isolated haploid and diploid germ cells fractions, we confirmed that GPR30 is expressed in SCs and diploid germ cells but not in haploid germ cells. This specific expression pattern suggests a role for GPR30 in spermatogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells

PubMed Central

Song, Shanshan; Jacobson, Krista N.; McDermott, Kimberly M.; Reddy, Sekhar P.; Cress, Anne E.; Tang, Haiyang; Dudek, Steven M.; Black, Stephen M.; Garcia, Joe G. N.; Makino, Ayako

2015-01-01

Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca2+ signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca2+] ([Ca2+]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca2+ eliminated the plateau phase increase of [Ca2+]cyt in lung cancer cells, indicating that the plateau phase of [Ca2+]cyt increase is due to Ca2+ influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca2+ or chelating intracellular Ca2+ with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca2+]cyt through Ca2+ influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. PMID:26491047

Activation of a P2Y4-like purinoceptor triggers an increase in cytosolic [Ca2+] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae).

PubMed

Sartorello, R; Garcia, C R S

2005-01-01

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ss-ATP, and ADP failed to do so in a 1- to 200-microm con- centration. The EC50 obtained for the compounds tested was 41.77 microM for UTP, 48.11 microM for GTP, 53.11 microM for UDP, and 30.78 microM for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.

Neuropharmacology of purinergic receptors in human submucous plexus: Involvement of P2X₁, P2X₂, P2X₃ channels, P2Y and A₃ metabotropic receptors in neurotransmission.

PubMed

Liñán-Rico, A; Wunderlich, J E; Enneking, J T; Tso, D R; Grants, I; Williams, K C; Otey, A; Michel, K; Schemann, M; Needleman, B; Harzman, A; Christofi, F L

2015-08-01

The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 > P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology. Published by Elsevier Ltd.

P2Y12 shRNA treatment decreases SGC activation to relieve diabetic neuropathic pain in type 2 diabetes mellitus rats.

PubMed

Wang, Shouyu; Wang, Zilin; Li, Lin; Zou, Lifang; Gong, Yingxin; Jia, Tianyu; Zhao, Shanhong; Yuan, Huilong; Shi, Liran; Liu, Shuangmei; Wu, Bing; Yi, Zhihua; Liu, Hui; Gao, Yun; Li, Guilin; Deussing, Jan M; Li, Man; Zhang, Chunping; Liang, Shangdong

2018-06-26

Diabetic neuropathic pain is a common complication of type 2 diabetes mellitus (DM). Activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) plays a crucial role in neuropathic pain through the release of proinflammatory cytokines. The P2Y12 receptor is expressed in SGCs of the DRG. In this study, our aim was to investigate the role of the P2Y12 receptor on the pathological changes in diabetic neuropathic pain. The present study showed that diabetic neuropathic pain increased mechanical and thermal hyperalgesia in type 2 DM model rats. The results showed that the expression levels of P2Y12 messenger RNA (mRNA) and protein in DRG SGCs were increased in DM model rats compared with control rats. Glial fibrillary acidic protein (GFAP) and interleukin-1β (IL-1β) expression levels in the DRG were increased in DM rats. Upregulation of GFAP is a marker of SGC activation. Targeting the P2Y12 receptor by short hairpin RNA (shRNA) decreased the upregulated expression of P2Y12 mRNA and protein, coexpression of P2Y12 and GFAP, the expression of GFAP, IL-1β, and tumor necrosis factor-receptor 1 in the DRG of DM rats, and relieved mechanical and thermal hyperalgesia in DM rats. After treatment with the P2Y12 receptor shRNA, the enhancing integrated OPTICAL density (IOD) ratios of p-P38 MAPK to P38 mitogen activated protein kinase (MAPK) in the DM rats treated with P2Y12 shRNA were significantly lower than that in the untreated DM rats. Therefore, P2Y12 shRNA treatment decreased SGC activation to relieve mechanical and thermal hyperalgesia in DM rats. © 2018 Wiley Periodicals, Inc.

Three classes of ligands each bind to distinct sites on the orphan G protein-coupled receptor GPR84.

PubMed

Mahmud, Zobaer Al; Jenkins, Laura; Ulven, Trond; Labéguère, Frédéric; Gosmini, Romain; De Vos, Steve; Hudson, Brian D; Tikhonova, Irina G; Milligan, Graeme

2017-12-20

Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so also can molecules related to 3,3'-diindolylmethane. 3,3'-Diindolylmethane and decanoic acid acted as strong positive allosteric modulators of the function of each other and analysis showed the affinity of 3,3'-diindolylmethane to be at least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies a key role in binding for the carboxylic acid of the fatty acid. Via homology modelling we predicted and confirmed an integral role of arginine 172 , located in the 2nd extracellular loop, in the action of decanoic acid but not of 3,3'-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were able to block agonist actions of both decanoic acid and 3,3'-diindolylmethane at GPR84. However, although a radiolabelled form of a related antagonist, [ 3 H]G9543, was able to bind with high affinity to GPR84, this was not competed for by increasing concentrations of either decanoic acid or 3,3'-diindolylmethane and was not affected adversely by mutation of arginine 172 . These studies identify three separable ligand binding sites within GPR84 and suggest that if medium chain fatty acids are true endogenous regulators then co-binding with a positive allosteric modulator would greatly enhance their function in physiological settings.

Purinergic Signalling: Therapeutic Developments

PubMed Central

Burnstock, Geoffrey

2017-01-01

Purinergic signalling, i.e., the role of nucleotides as extracellular signalling molecules, was proposed in 1972. However, this concept was not well accepted until the early 1990’s when receptor subtypes for purines and pyrimidines were cloned and characterised, which includes four subtypes of the P1 (adenosine) receptor, seven subtypes of P2X ion channel receptors and 8 subtypes of the P2Y G protein-coupled receptor. Early studies were largely concerned with the physiology, pharmacology and biochemistry of purinergic signalling. More recently, the focus has been on the pathophysiology and therapeutic potential. There was early recognition of the use of P1 receptor agonists for the treatment of supraventricular tachycardia and A2A receptor antagonists are promising for the treatment of Parkinson’s disease. Clopidogrel, a P2Y12 antagonist, is widely used for the treatment of thrombosis and stroke, blocking P2Y12 receptor-mediated platelet aggregation. Diquafosol, a long acting P2Y2 receptor agonist, is being used for the treatment of dry eye. P2X3 receptor antagonists have been developed that are orally bioavailable and stable in vivo and are currently in clinical trials for the treatment of chronic cough, bladder incontinence, visceral pain and hypertension. Antagonists to P2X7 receptors are being investigated for the treatment of inflammatory disorders, including neurodegenerative diseases. Other investigations are in progress for the use of purinergic agents for the treatment of osteoporosis, myocardial infarction, irritable bowel syndrome, epilepsy, atherosclerosis, depression, autism, diabetes, and cancer. PMID:28993732

GPR30: a novel therapeutic target in estrogen-related disease.

PubMed

Prossnitz, Eric R; Sklar, Larry A; Oprea, Tudor I; Arterburn, Jeffrey B

2008-03-01

Estrogen is a crucial hormone in human physiology that regulates a multitude of biological processes. It is also an important target in many diseases such as cancer and skeletal, neurological and immunological conditions. The actions of estrogen have traditionally been ascribed to one of two closely related classical nuclear hormone receptors, ERalpha and ERbeta, which are best characterized for regulating gene expression. Recent studies have revealed the contribution of a novel estrogen receptor GPR30, which belongs to the family of seven-transmembrane G-protein-coupled receptors, to many of the rapid biological responses to estrogen. Many drugs, such as tamoxifen and fulvestrant, which seem to selectively inhibit the activities of the classical estrogen receptors, are in widespread clinical use. However, recent results indicate that these same drugs activate multiple cellular-signaling pathways via GPR30. Unraveling the pharmacological profiles and specificities of ERalpha, ERbeta and GPR30 will be vital for understanding not only the physiological roles of each receptor but also for the development of the next generation of receptor-specific drugs.

Ground penetrating radar examination of thin tsunami beds - A case study from Phra Thong Island, Thailand

NASA Astrophysics Data System (ADS)

Gouramanis, Chris; Switzer, Adam D.; Polivka, Peter M.; Bristow, Charles S.; Jankaew, Kruawun; Dat, Pham T.; Pile, Jeremy; Rubin, Charles M.; Yingsin, Lee; Ildefonso, Sorvigenaleon R.; Jol, Harry M.

2015-11-01

Coastal overwash deposits from tsunamis and storms have been identified and characterised from many coastal environments. To date, these investigations have utilised ad-hoc time, energy and cost intensive invasive techniques, such as, pits and trenches or taking core samples. Here, we present the application of high-frequency ground penetrating radar (GPR) to identify and characterise the 2004 Indian Ocean Tsunami (IOT) and palaeotsunami deposits from Phra Thong Island, Thailand. This site is one of the most intensively studied palaeotsunami sites globally and preserves a series of late-Holocene stacked sandy tsunami deposits within an organic, muddy low-energy backbeach environment. Using 100, 500 and 1000 MHz GPR antennas, 29 reflection profiles were collected from two swales (X and Y) inland of the modern beach, and two common mid-point (CMP) profiles using the 200 MHz antennas were collected from Swale Y. Detailed examination of the CMPs allowed accurate velocity estimates to be applied to each profile. The reflection profiles included across-swale profiles and a high-resolution grid in Swale X, and were collected to investigate the feasibility of GPR to image the palaeotsunami deposits, and two profiles from Swale Y where the tsunami deposits are poorly known. The 500 MHz antennas provided the best stratigraphic resolution which was independently validated from the stratigraphy and sedimentology recovered from 17 auger cores collected along the profiles. It is clear from the augers and GPR data, that the different dielectric properties of the individual layers allow the identification of the IOT and earlier tsunami deposits on Phra Thong Island. Although applied in a coastal setting here, this technique can be applied to other environments where thin sand beds are preserved, in order to prioritise sites for detailed examination.

Sex- and age-dependent effects of Gpr30 genetic deletion on the metabolic and cardiovascular profiles of diet-induced obese mice.

PubMed

Meoli, Luca; Isensee, Jörg; Zazzu, Valeria; Nabzdyk, Christoph S; Soewarto, Dian; Witt, Henning; Foryst-Ludwig, Anna; Kintscher, Ulrich; Noppinger, Patricia Ruiz

2014-05-01

The G protein-coupled receptor 30 (GPR30) has been claimed as an estrogen receptor. However, the literature reports controversial findings and the physiological function of GPR30 is not fully understood yet. Consistent with studies assigning a role of GPR30 in the cardiovascular and metabolic systems, GPR30 expression has been reported in small arterial vessels, pancreas and chief gastric cells of the stomach. Therefore, we hypothesized a role of GPR30 in the onset and progression of cardiovascular and metabolic diseases. In order to test our hypothesis, we investigated the effects of a high-fat diet on the metabolic and cardiovascular profiles of Gpr30-deficient mice (GPR30-lacZ mice). We found that GPR30-lacZ female, rather than male, mice had significant lower levels of HDL along with an increase in fat liver accumulation as compared to control mice. However, two indicators of cardiac performance assessed by echocardiography, ejection fraction and fractional shortening were both decreased in an age-dependent manner only in Gpr30-lacZ male mice. Collectively our results point to a potential role of Gpr30 in preserving lipid metabolism and cardiac function in a sex- and age-dependent fashion. Copyright © 2014 Elsevier B.V. All rights reserved.

Sleep deprivation and divergent toll-like receptor-4 activation of cellular inflammation in aging.

PubMed

Carroll, Judith E; Carrillo, Carmen; Olmstead, Richard; Witarama, Tuff; Breen, Elizabeth C; Yokomizo, Megumi; Seeman, Teresa; Irwin, Michael R

2015-02-01

Sleep disturbance and aging are associated with increases in inflammation, as well as increased risk of infectious disease. However, there is limited understanding of the role of sleep loss on age-related differences in immune responses. This study examines the effects of sleep deprivation on toll-like receptor activation of monocytic inflammation in younger compared to older adults. Community-dwelling adults (n = 70) who were categorized as younger (25-39 y old, n = 21) and older (60-84 y old, n = 49) participants, underwent a sleep laboratory-based experimental partial sleep deprivation (PSD) protocol including adaptation, an uninterrupted night of sleep, sleep deprivation (sleep restricted to 03:00-07:00), and recovery. Blood samples were obtained each morning to measure toll-like receptor-4 activation of monocyte intracellular production of the inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Partial sleep deprivation induced a significant increase in the production of IL-6 and/or TNF-α that persisted after a night of recovery sleep (F(2,121.2) = 3.8, P < 0.05). Age moderated the effects of sleep loss, such that younger adults had an increase in inflammatory cytokine production that was not present in older adults (F(2,121.2) = 4.0, P < 0.05). Older adults exhibit reduced toll-like receptor 4 stimulated cellular inflammation that, unlike in younger adults, is not activated after a night of partial sleep loss. Whereas sleep loss increases cellular inflammation in younger adults and may contribute to inflammatory disorders, blunted toll-like receptor activation in older adults may increase the risk of infectious disease seen with aging. © 2015 Associated Professional Sleep Societies, LLC.

Synthesis and characterization of iodinated tetrahydroquinolines targeting the G protein-coupled estrogen receptor GPR30.

PubMed

Ramesh, Chinnasamy; Nayak, Tapan K; Burai, Ritwik; Dennis, Megan K; Hathaway, Helen J; Sklar, Larry A; Prossnitz, Eric R; Arterburn, Jeffrey B

2010-02-11

A series of iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines was synthesized as potential targeted imaging agents for the G protein-coupled estrogen receptor GPR30. The affinity and specificity of binding to GPR30 versus the classical estrogen receptors ER alpha/beta and functional responses associated with ligand-binding were determined. Selected iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines exhibited IC(50) values lower than 20 nM in competitive binding studies with GPR30-expressing human endometrial cancer cells. These compounds functioned as antagonists of GPR30 and blocked estrogen-induced PI3K activation and calcium mobilization. The tributylstannyl precursors of selected compounds were radiolabeled with (125)I using the iodogen method. In vivo biodistribution studies in female ovariectomized athymic (NCr) nu/nu mice bearing GPR30-expressing human endometrial tumors revealed GPR30-mediated uptake of the radiotracer ligands in tumor, adrenal, and reproductive organs. Biodistribution and quantitative SPECT/CT studies revealed structurally related differences in the pharmacokinetic profiles, target tissue uptake, and metabolism of the radiolabeled compounds as well as differences in susceptibility to deiodination. The high lipophilicity of the compounds adversely affects the in vivo biodistribution and clearance of these radioligands and suggests that further optimization of this parameter may lead to improved targeting characteristics.

Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations

PubMed Central

Xing, Shu; Grol, Matthew W.; Grutter, Peter H.; Dixon, S. Jeffrey; Komarova, Svetlana V.

2016-01-01

Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP. PMID:27468270

In vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a selective brain penetrant small molecule antagonist of the neuropeptide Y Y(2) receptor.

PubMed

Shoblock, James R; Welty, Natalie; Nepomuceno, Diane; Lord, Brian; Aluisio, Leah; Fraser, Ian; Motley, S Timothy; Sutton, Steve W; Morton, Kirsten; Galici, Ruggero; Atack, John R; Dvorak, Lisa; Swanson, Devin M; Carruthers, Nicholas I; Dvorak, Curt; Lovenberg, Timothy W; Bonaventure, Pascal

2010-02-01

The lack of potent, selective, brain penetrant Y(2) receptor antagonists has hampered in vivo functional studies of this receptor. Here, we report the in vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a novel Y(2) receptor antagonist. The affinity of JNJ-31020028 was determined by inhibition of the PYY binding to human Y(2) receptors in KAN-Ts cells and rat Y(2) receptors in rat hippocampus. The functional activity was determined by inhibition of PYY-stimulated calcium responses in KAN-Ts cells expressing a chimeric G protein Gqi5 and in the rat vas deferens (a prototypical Y(2) bioassay). Ex vivo receptor occupancy was revealed by receptor autoradiography. JNJ-31020028 was tested in vivo with microdialysis, in anxiety models, and on corticosterone release. JNJ-31020028 bound with high affinity (pIC(50) = 8.07 +/- 0.05, human, and pIC(50) = 8.22 +/- 0.06, rat) and was >100-fold selective versus human Y(1), Y(4), and Y(5) receptors. JNJ-31020028 was demonstrated to be an antagonist (pK(B) = 8.04 +/- 0.13) in functional assays. JNJ-31020028 occupied Y(2) receptor binding sites (approximately 90% at 10 mg/kg) after subcutaneous administration in rats. JNJ-31020028 increased norepinephrine release in the hypothalamus, consistent with the colocalization of norepinephrine and neuropeptide Y. In a variety of anxiety models, JNJ-31020028 was found to be ineffective, although it did block stress-induced elevations in plasma corticosterone, without altering basal levels, and normalized food intake in stressed animals without affecting basal food intake. These results suggest that Y(2) receptors may not be critical for acute behaviors in rodents but may serve modulatory roles that can only be elucidated under specific situational conditions.

Oleuropein and hydroxytyrosol activate GPER/ GPR30-dependent pathways leading to apoptosis of ER-negative SKBR3 breast cancer cells.

PubMed

Chimento, Adele; Casaburi, Ivan; Rosano, Camillo; Avena, Paola; De Luca, Arianna; Campana, Carmela; Martire, Emilia; Santolla, Maria Francesca; Maggiolini, Marcello; Pezzi, Vincenzo; Sirianni, Rosa

2014-03-01

We have previously demonstrated that oleuropein (OL) and hydroxytyrosol (HT) reduce 17β-estradiol-mediated proliferation in MCF-7 breast cancer (BC) cells without affecting the classical genomic action of estrogen receptor (ER), but activating instead the ERK1/2 pathway. Here, we hypothesized that this inhibition could be mediated by a G-protein-coupled receptor named GPER/GPR30. Using the ER-negative and GPER-positive SKBR3 BC cells as experimental model, we investigated the effects of OL and HT on GPER-mediated activation of downstream pathways. Docking simulations and ligand-binding studies evidenced that OL and HT are able to bind GPER. MTT cell proliferation assays revealed that both phenols reduced SKBR3 cell growth; this effect was abolished silencing GPER. Focusing on OL and HT GPER-mediated pathways, using Western blot analysis we showed a sustained ERK1/2 activation triggering an intrinsic apoptotic pathway. Showing that OL and HT work as GPER inverse agonists in ER-negative and GPER-positive SKBR3 BC cells, we provide novel insights into the potential of these two molecules as tools in the therapy of this subtype of BC. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heteromerization of G2A and OGR1 enhances proton sensitivity and proton-induced calcium signals.

PubMed

Huang, Ya-Han; Su, Yeu-Shiuan; Chang, Chung-Jen; Sun, Wei-Hsin

2016-12-01

Proton-sensing G-protein-coupled receptors (GPCRs; OGR1, GPR4, G2A, TDAG8), with full activation at pH 6.4 ∼ 6.8, are important to pH homeostasis, immune responses and acid-induced pain. Although G2A mediates the G13-Rho pathway in response to acid, whether G2A activates Gs, Gi or Gq proteins remains debated. In this study, we examined the response of this fluorescence protein-tagged OGR1 family to acid stimulation in HEK293T cells. G2A did not generate detectable intracellular calcium or cAMP signals or show apparent receptor redistribution with moderate acid (pH ≥ 6.0) stimulation but reduced cAMP accumulation under strong acid stimulation (pH ≤ 5.5). Surprisingly, coexpression of OGR1- and G2A-enhanced proton sensitivity and proton-induced calcium signals. This alteration is attributed to oligomerization of OGR1 and G2A. The oligomeric potential locates receptors at a specific site, which leads to enhanced proton-induced calcium signals through channels.

Identification of Determinants Required for Agonistic and Inverse Agonistic Ligand Properties at the ADP Receptor P2Y12

PubMed Central

Schmidt, Philipp; Ritscher, Lars; Dong, Elizabeth N.; Hermsdorf, Thomas; Cöster, Maxi; Wittkopf, Doreen; Meiler, Jens

2013-01-01

The ADP receptor P2Y12 belongs to the superfamily of G protein–coupled receptors (GPCRs), and its activation triggers platelet aggregation. Therefore, potent antagonists, such as clopidogrel, are of high clinical relevance in prophylaxis and treatment of thromboembolic events. P2Y12 displays an elevated basal activity in vitro, and as such, inverse agonists may be therapeutically beneficial compared with antagonists. Only a few inverse agonists of P2Y12 have been described. To expand this limited chemical space and improve understanding of structural determinants of inverse agonist-receptor interaction, this study screened a purine compound library for lead structures using wild-type (WT) human P2Y12 and 28 constitutively active mutants. Results showed that ATP and ATP derivatives are agonists at P2Y12. The potency at P2Y12 was 2-(methylthio)-ADP > 2-(methylthio)-ATP > ADP > ATP. Determinants required for agonistic ligand activity were identified. Molecular docking studies revealed a binding pocket for the ATP derivatives that is bordered by transmembrane helices 3, 5, 6, and 7 in human P2Y12, with Y105, E188, R256, Y259, and K280 playing a particularly important role in ligand interaction. N-Methyl-anthraniloyl modification at the 3′-OH of the 2′-deoxyribose leads to ligands (mant-deoxy-ATP [dATP], mant-deoxy-ADP) with inverse agonist activity. Inverse agonist activity of mant-dATP was found at the WT human P2Y12 and half of the constitutive active P2Y12 mutants. This study showed that, in addition to ADP and ATP, other ATP derivatives are not only ligands of P2Y12 but also agonists. Modification of the ribose within ATP can result in inverse activity of ATP-derived ligands. PMID:23093496

Deletion of Gpr55 Results in Subtle Effects on Energy Metabolism, Motor Activity and Thermal Pain Sensation

PubMed Central

Ryberg, Erik; Wu, Tingting; Greasley, Peter J.; Bohlooly-Y, Mohammad; Hjorth, Stephan

2016-01-01

The G-protein coupled receptor 55 (GPR55) is activated by cannabinoids and non-cannabinoid molecules and has been speculated to play a modulatory role in a large variety of physiological and pathological processes, including in metabolically perturbed states. We therefore generated male mice deficient in the gene coding for the cannabinoid/lysophosphatidylinositol (LPI) receptor Gpr55 and characterized them under normal dietary conditions as well as during high energy dense diet feeding followed by challenge with the CB1 receptor antagonist/GPR55 agonist rimonabant. Gpr55 deficient male mice (Gpr55 KO) were phenotypically indistinguishable from their wild type (WT) siblings for the most part. However, Gpr55 KO animals displayed an intriguing nocturnal pattern of motor activity and energy expenditure (EE). During the initial 6 hours of the night, motor activity was significantly elevated without any significant effect observed in EE. Interestingly, during the last 6 hours of the night motor activity was similar but EE was significantly decreased in the Gpr55 KO mice. No significant difference in motor activity was detected during daytime, but EE was lower in the Gpr55 KO compared to WT mice. The aforementioned patterns were not associated with alterations in energy intake, daytime core body temperature, body weight (BW) or composition, although a non-significant tendency to increased adiposity was seen in Gpr55 KO compared to WT mice. Detailed analyses of daytime activity in the Open Field paradigm unveiled lower horizontal activity and rearing time for the Gpr55 KO mice. Moreover, the Gpr55 KO mice displayed significantly faster reaction time in the tail flick test, indicative of thermal hyperalgesia. The BW-decreasing effect of rimonabant in mice on long-term cafeteria diet did not differ between Gpr55 KO and WT mice. In conclusion, Gpr55 deficiency is associated with subtle effects on diurnal/nocturnal EE and motor activity behaviours but does not appear per se critically required for overall metabolism or behaviours. PMID:27941994

G protein-coupled receptor 30 contributes to improved remyelination after cuprizone-induced demyelination.

PubMed

Hirahara, Yukie; Matsuda, Ken Ichi; Yamada, Hisao; Saitou, Akira; Morisaki, Shinsuke; Takanami, Keiko; Boggs, Joan M; Kawata, Mitsuhiro

2013-03-01

Estrogen exerts neuroprotective and promyelinating actions. The therapeutic effect has been shown in animal models of multiple sclerosis, in which the myelin sheath is specifically destroyed in the central nervous system. However, it remains unproven whether estrogen is directly involved in remyelination via the myelin producing cells, oligodendrocytes, or which estrogen receptors are involved. In this study, we found that the membrane-associated estrogen receptor, the G protein-coupled receptor 30 (GPR30), also known as GPER, was expressed in oligodendrocytes in rat spinal cord and corpus callosum. Moreover, GPR30 was expressed throughout oligodendrocyte differentiation and promyelinating stages in primary oligodendrocyte cultures derived from rat spinal cords and brains. To evaluate the role of signaling via GPR30 in promyelination, a specific agonist for GPR30, G1, was administered to a rat model of demyelination induced by cuprizone treatment. Histological examination of the corpus callosum with oligodendrocyte differentiation stage-specific markers showed that G1 enhanced oligodendrocyte maturation in corpus callosum of cuprizone-treated animals. It also enhanced oligodendrocyte ensheathment of dorsal root ganglion (DRG) neurons in co-culture and myelination in cuprizone-treated animals. This study is the first evidence that GPR30 signaling promotes remyelination by oligodendrocytes after demyelination. GPR30 ligands may provide a novel therapy for the treatment of multiple sclerosis. Copyright © 2012 Wiley Periodicals, Inc.

Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages

PubMed Central

Moreira-Souza, Aline Cristina Abreu; Marinho, Ygor; Correa, Gladys; Santoro, Giani França; Coutinho, Claudia Mara Lara Melo; Vommaro, Rossiane Claudia; Coutinho-Silva, Robson

2015-01-01

Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane – subdivided into P2Y and P2X subfamilies - whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection. PMID:26192447

Role of G-protein-coupled estrogen receptor (GPER/GPR30) in maintenance of meiotic arrest in fish oocytes.

PubMed

Thomas, Peter

2017-03-01

An essential role for GPER (formerly known as GPR30) in regulating mammalian reproduction has not been identified to date, although it has shown to be involved in the regulation a broad range of other estrogen-dependent functions. In contrast, an important reproductive role for GPER in the maintenance of oocyte meiotic arrest has been identified in teleost fishes, which is briefly reviewed here. Recent studies have clearly shown that ovarian follicle production of estradiol-17β (E 2 ) maintains meiotic arrest in several teleost species through activation of GPER coupled to a stimulatory G protein (G s ) on oocyte plasma membranes resulting in stimulation of cAMP production and maintenance of elevated cAMP levels. Studies with denuded zebrafish oocytes and with microinjection of GPER antisense oligonucleotides into oocytes have demonstrated the requirement for both ovarian follicle production of estrogens and expression of GPER on the oocyte surface for maintenance of meiotic arrest. This inhibitory action of E 2 on the resumption of meiosis is mimicked by the GPER-selective agonist G-1, by the GPER agonists and nuclear ER antagonists, ICI 182,780 and tamoxifen, and also by the xenoestrogen bisphenol-A (BPA) and related alkylphenols. GPER also maintains meiotic arrest of zebrafish oocytes through estrogen- and BPA-dependent GPER activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) signaling. Interestingly, progesterone receptor component 1 (PGRMC1) is also involved in estrogen maintenance of meiotic arrest through regulation of EGFR expression on the oocyte plasma membrane. The preovulatory surge in LH secretion induces the ovarian synthesis of progestin hormones that activate a membrane progestin receptor alpha (mPRα)/inhibitory G protein (Gi) pathway. It also increases ovarian synthesis of the catecholestrogen, 2-hydroxy-estradiol-17β (2-OHE 2 ) which inhibits the GPER/Gs/adenylyl cyclase pathway. Both of these LH actions cause declines in oocyte cAMP levels resulting in the resumption of meiosis. GPER is also present on murine oocytes but there are no reports of studies investigating its possible involvement in maintaining meiotic arrest in mammals. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reprint of "Role of G protein-coupled estrogen receptor (GPER/GPR30) in maintenance of meiotic arrest in fish oocytes".

PubMed

Thomas, Peter

2018-02-01

An essential role for GPER (formerly known as GPR30) in regulating mammalian reproduction has not been identified to date, although it has shown to be involved in the regulation a broad range of other estrogen-dependent functions. In contrast, an important reproductive role for GPER in the maintenance of oocyte meiotic arrest has been identified in teleost fishes, which is briefly reviewed here. Recent studies have clearly shown that ovarian follicle production of estradiol-17β (E 2 ) maintains meiotic arrest in several teleost species through activation of GPER coupled to a stimulatory G protein (G s ) on oocyte plasma membranes, resulting in stimulation of cAMP production and maintenance of elevated cAMP levels. Studies with denuded zebrafish oocytes and with microinjection of GPER antisense oligonucleotides into oocytes have demonstrated the requirement for both ovarian follicle production of estrogens and expression of GPER on the oocyte surface for maintenance of meiotic arrest. This inhibitory action of E 2 on the resumption of meiosis is mimicked by the GPER-selective agonist G-1, by the GPER agonists and nuclear ER antagonists, ICI 182,780 and tamoxifen, and also by the xenoestrogen bisphenol-A (BPA) and related alkylphenols. GPER also maintains meiotic arrest of zebrafish oocytes through estrogen- and BPA-dependent GPER activation of epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) signaling. Interestingly, progesterone receptor component 1 (PGRMC1) is also involved in estrogen maintenance of meiotic arrest through regulation of EGFR expression on the oocyte plasma membrane. The preovulatory surge in LH secretion induces the ovarian synthesis of progestin hormones that activate a membrane progestin receptor alpha (mPRα)/inhibitory G protein (Gi) pathway. It also increases ovarian synthesis of the catecholestrogen, 2-hydroxy-estradiol-17β (2-OHE 2 ) which inhibits the GPER/Gs/adenylyl cyclase pathway. Both of these LH actions cause declines in oocyte cAMP levels resulting in the resumption of meiosis. GPER is also present on murine oocytes but there are no reports of studies investigating its possible involvement in maintaining meiotic arrest in mammals. Copyright © 2017 Elsevier Ltd. All rights reserved.

Activation of G-protein-coupled receptor 30 increases T-type calcium currents in trigeminal ganglion neurons via the cholera toxin-sensitive protein kinase A pathway.

PubMed

Yue, Jingxia; Zhang, Yi; Li, Xuemin; Gong, Shan; Tao, Jin; Jiang, Xinghong

2014-11-01

G protein-coupled receptor 30 (GPR30) is a seven transmembrane domain G protein coupled receptor. In our study, GPR30 expression was found in trigeminal ganglia (TG) in mice, detected by RT-PCR and western blotting. We examined the effects of GPR30 activation on T-type calcium channels using GPR30-specific compound 1 (G-1), a GPR30-selective agonist, in TG neurons and demonstrated that G-1 induced an increase in T-type calcium channel currents (T-currents) in TGs. Intracellular infusion of GDP-β-S and pre-treatment of the neurons with cholera toxin (CTX) blocked the effects of G-1, suggesting that the G(s)-protein was involved. Intracellular application of the protein kinase A (PKA) inhibitor PKI 6-22 or pretreatment of the neurons with H89 abolished G-1 -induced enhancement of T-currents in TG neurons. However, incubation with PKC inhibitor elicited no such effects. In conclusion, our study shows that activation of GPR30 by G-1 increases T-currents via the CTX-sensitive and PKA-dependent pathway.

G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions.

PubMed

Bouchard, Caroline; Pagé, Julie; Bédard, Andréanne; Tremblay, Pierrot; Vallières, Luc

2007-06-01

G protein-coupled receptor 84 (GPR84) is a recently discovered member of the seven transmembrane receptor superfamily whose function and regulation are unknown. Here, we report that in mice suffering from endotoxemia, microglia express GPR84 in a strong and sustained manner. This property is shared by subpopulations of peripheral macrophages and, to a much lesser extent, monocytes. The induction of GPR84 expression by endotoxin is mediated, at least in part, by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1), because mice lacking either one or both of these molecules have fewer GPR84-expressing cells in their cerebral cortex than wild-type mice during the early phase of endotoxemia. Moreover, when injected intracerebrally or added to microglial cultures, recombinant TNF stimulates GPR84 expression through a dexamethasone-insensitive mechanism. Finally, we show that microglia produce GPR84 not only during endotoxemia, but also during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In conclusion, this study reports the identification of a new sensitive marker of microglial activation, which may play an important regulatory role in neuroimmunological processes, acting downstream to the effects of proinflammatory mediators.

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

PubMed

Bae, Jae Hyun; Lew, Erin Denise; Yuzawa, Satoru; Tomé, Francisco; Lax, Irit; Schlessinger, Joseph

2009-08-07

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

Orphan receptor GPR179 forms macromolecular complexes with components of metabotropic signaling cascade in retina ON-bipolar neurons.

PubMed

Orlandi, Cesare; Cao, Yan; Martemyanov, Kirill A

2013-10-29

In the mammalian retina, synaptic transmission between light-excited rod photoreceptors and downstream ON-bipolar neurons is indispensable for dim vision, and disruption of this process leads to congenital stationary night blindness in human patients. The ON-bipolar neurons use the metabotropic signaling cascade, initiated by the mGluR6 receptor, to generate depolarizing responses to light-induced changes in neurotransmitter glutamate release from the photoreceptor axonal terminals. Evidence for the identity of the components involved in transducing these signals is growing rapidly. Recently, the orphan receptor, GPR179, a member of the G protein-coupled receptor (GPCR) superfamily, has been shown to be indispensable for the synaptic responses of ON-bipolar cells. In our study, we investigated the interaction of GPR179 with principle components of the signal transduction cascade. We used immunoprecipitation and proximity ligation assays in transfected cells and native retinas to characterize the protein-protein interactions involving GPR179. The influence of cascade components on GPR179 localization was examined through immunohistochemical staining of the retinas from genetic mouse models. We demonstrated that, in mouse retinas, GPR179 forms physical complexes with the main components of the metabotropic cascade, recruiting mGluR6, TRPM1, and the RGS proteins. Elimination of mGluR6 or RGS proteins, but not TRPM1, detrimentally affects postsynaptic targeting or GPR179 expression. These observations suggest that the mGluR6 signaling cascade is scaffolded as a macromolecular complex in which the interactions between the components ensure the optimal spatiotemporal characteristics of signal transduction.

Targeting Renal Purinergic Signalling for the Treatment of Lithium-induced Nephrogenic Diabetes Insipidus

PubMed Central

Kishore, B. K.; Carlson, N. G.; Ecelbarger, C. M.; Kohan, D. E.; Müller, C. E.; Nelson, R. D.; Peti-Peterdi, J.; Zhang, Y.

2015-01-01

Lithium still retains its critical position in the treatment of bipolar disorder by virtue of its ability to prevent suicidal tendencies. However, chronic use of lithium is often limited by the development nephrogenic diabetes insipidus (NDI), a debilitating condition. Lithium-induced NDI is due to resistance of the kidney to arginine vasopressin (AVP), leading to polyuria, natriuresis and kaliuresis. Purinergic signalling mediated by extracellular nucleotides (ATP/UTP), acting via P2Y receptors, opposes the action of AVP on renal collecting duct (CD) by decreasing the cellular cAMP and thus AQP2 protein levels. Taking a cue from this phenomenon, we discovered the potential involvement of ATP/UTP-activated P2Y2 receptor in lithium-induced NDI in rats, and showed that P2Y2 receptor knockout mice are significantly resistant to Li-induced polyuria, natriuresis and kaliuresis. Extension of these studies revealed that ADP-activated P2Y12 receptor is expressed in the kidney, and its irreversible blockade by the administration of clopidogrel bisulfate (Plavix®) ameliorates Li-induced NDI in rodents. Parallel in vitro studies showed that P2Y12 receptor blockade by the reversible antagonist PSB-0739 sensitizes CD to the action of AVP. Thus, our studies unraveled the potential beneficial effects of targeting P2Y2 or P2Y12 receptors to counter AVP resistance in lithium-induced NDI. If established in further studies, our findings may pave the way for the development of better and safer methods for the treatment of NDI by bringing a paradigm shift in the approach from the current therapies that predominantly counter the anti-AVP effects to those that enhance the sensitivity of the kidney to AVP action. PMID:25877068

K(Ca)3.1 channels facilitate K+ secretion or Na+ absorption depending on apical or basolateral P2Y receptor stimulation.

PubMed

Palmer, Melissa L; Peitzman, Elizabeth R; Maniak, Peter J; Sieck, Gary C; Prakash, Y S; O'Grady, Scott M

2011-07-15

Human mammary epithelial (HME) cells express several P2Y receptor subtypes located in both apical and basolateral membranes. Apical UTP or ATP-γ-S stimulation of monolayers mounted in Ussing chambers evoked a rapid, but transient decrease in short circuit current (I(sc)), consistent with activation of an apical K+ conductance. In contrast, basolateral P2Y receptor stimulation activated basolateral K+ channels and increased transepithelial Na+ absorption. Chelating intracellular Ca2+ using the membrane-permeable compound BAPTA-AM, abolished the effects of purinoceptor activation on I(sc). Apical pretreatment with charybdotoxin also blocked the I(sc) decrease by >90% and similar magnitudes of inhibition were observed with clotrimazole and TRAM-34. In contrast, iberiotoxin and apamin did not block the effects of apical P2Y receptor stimulation. Silencing the expression of K(Ca)3.1 produced ∼70% inhibition of mRNA expression and a similar reduction in the effects of apical purinoceptor agonists on I(sc). In addition, silencing P2Y2 receptors reduced the level of P2Y2 mRNA by 75% and blocked the effects of ATP-γ-S by 65%. These results suggest that P2Y2 receptors mediate the effects of purinoceptor agonists on K+ secretion by regulating the activity of K(Ca)3.1 channels expressed in the apical membrane of HME cells. The results also indicate that release of ATP or UTP across the apical or basolateral membrane elicits qualitatively different effects on ion transport that may ultimately determine the [Na+]/[K+] composition of fluid within the mammary ductal network.

β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum

PubMed Central

Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Xia, Yun; Zou, Fei; Qu, Meihua; Needleman, Bradley J.; Mikami, Dean J.

2015-01-01

Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions. PMID:25813057

The association of GPR85 with PSD-95-neuroligin complex and autism spectrum disorder: a molecular analysis.

PubMed

Fujita-Jimbo, Eriko; Tanabe, Yuko; Yu, Zhiling; Kojima, Karin; Mori, Masato; Li, Hong; Iwamoto, Sadahiko; Yamagata, Takanori; Momoi, Mariko Y; Momoi, Takashi

2015-01-01

Autism spectrum disorder (ASD) has a complex genetic etiology. Some symptoms and mutated genes, including neuroligin (NLGN), neurexin (NRXN), and SH3 and multiple ankyrin repeat domains protein (SHANK), are shared by schizophrenia and ASD. Little is known about the molecular pathogenesis of ASD. One of the possible molecular pathogenesis is an imbalance of excitatory and inhibitory receptors linked with the NLGN-PSD-95-SHANK complex via postsynaptic density protein/Drosophila disc large tumor suppressor/zonula occludens-1 protein (PDZ) binding. In the present study, we focused on GPR85 as a candidate gene for ASD because the C-terminal amino acid sequence of GPR85 [Thr-Cys-Val-Ile (YCVI)] is classified as a type II PDZ-binding motif, and GPR85 is a risk factor for schizophrenia. GPR85 is an orphan receptor that regulates neural and synaptic plasticity and modulates diverse behaviors, including learning and memory. While searching for molecules that associate with GPR85, we found that GPR85 was associated with postsynaptic density protein (PSD)-95 linked with NLGN in the brain. We examined the proteins that associate with the C-terminal sequence of GPR85 by pull-down assay and immunoblot analysis and searched for a mutation of the GPR85 gene in patients with ASD. We used immunostaining to examine the intracellular localization of mutated GPR85 and its influence on the morphology of cells and neurons. The C-terminal sequence of GPR85 interacted with PSD-95 at PDZ1, while NLGN interacted with PSD-95 at PDZ3. Two male patients with ASD from independent Japanese families possessed inherited missense mutations at conserved sites in GPR85: one had T1033C (M152T) and the other had G1239T (V221L). These mutations were located in a domain related to G protein interaction and signal transduction. In contrast to wild-type GPR85, mutated GPR85 was more preferentially accumulated, causing endoplasmic reticulum stress, and disturbed the dendrite formation of hippocampal neurons. GPR85 associated with the PSD-95 linked with NLGN, which is related to ASD. GPR85 carrying the mutations detected in ASD patients disturbed dendrite formation that could be the candidate for molecular pathogenesis of ASD through the associated NLGN-PSD-95 receptor complex.

P2Y2 Nucleotide Receptor Prompts Human Cardiac Progenitor Cell Activation by Modulating Hippo Signaling.

PubMed

Khalafalla, Farid G; Greene, Steven; Khan, Hashim; Ilves, Kelli; Monsanto, Megan M; Alvarez, Roberto; Chavarria, Monica; Nguyen, Jonathan; Norman, Benjamin; Dembitsky, Walter P; Sussman, Mark A

2017-11-10

Autologous stem cell therapy using human c-Kit + cardiac progenitor cells (hCPCs) is a promising therapeutic approach for treatment of heart failure (HF). However, hCPCs derived from aged patients with HF with genetic predispositions and comorbidities of chronic diseases exhibit poor proliferative and migratory capabilities, which impair overall reparative potential for injured myocardium. Therefore, empowering functionally compromised hCPCs with proregenerative molecules ex vivo is crucial for improving the therapeutic outcome in patients with HF. To improve hCPC proliferation and migration responses that are critical for regeneration by targeting proregenerative P2Y 2 nucleotide receptor (P2Y 2 R) activated by extracellular ATP and UTP molecules released following injury/stress. c-Kit + hCPCs were isolated from cardiac tissue of patients with HF undergoing left ventricular assist device implantation surgery. Correlations between P2 nucleotide receptor expression and hCPC growth kinetics revealed downregulation of select P2 receptors, including P2Y 2 R, in slow-growing hCPCs compared with fast growers. hCPC proliferation and migration significantly improved by overexpressing or stimulating P2Y 2 R. Mechanistically, P2Y 2 R-induced proliferation and migration were dependent on activation of YAP (yes-associated protein)-the downstream effector of Hippo signaling pathway. Proliferation and migration of functionally impaired hCPCs are enhanced by P2Y 2 R-mediated YAP activation, revealing a novel link between extracellular nucleotides released during injury/stress and Hippo signaling-a central regulator of cardiac regeneration. Functional correlations exist between hCPC phenotypic properties and P2 purinergic receptor expression. Lack of P2Y 2 R and other crucial purinergic stress detectors could compromise hCPC responsiveness to presence of extracellular stress signals. These findings set the stage for subsequent studies to assess purinergic signaling modulation as a potential strategy to improve therapeutic outcome for use of hCPCs in patients with HF. © 2017 American Heart Association, Inc.

P2Y nucleotide receptors: promise of therapeutic applications.

PubMed

Jacobson, Kenneth A; Boeynaems, Jean-Marie

2010-07-01

Extracellular nucleotides, such as ATP and UTP, have distinct signaling roles through a class of G-protein-coupled receptors, termed P2Y. The receptor ligands are typically charged molecules of low bioavailability and stability in vivo. Recent progress in the development of selective agonists and antagonists for P2Y receptors and study of knockout mice have led to new drug concepts based on these receptors. The rapidly accelerating progress in this field has already resulted in drug candidates for cystic fibrosis, dry eye disease and thrombosis. On the horizon are novel treatments for cardiovascular diseases, inflammatory diseases and neurodegeneration. Published by Elsevier Ltd.

Nutritional Signaling via Free Fatty Acid Receptors

PubMed Central

Miyamoto, Junki; Hasegawa, Sae; Kasubuchi, Mayu; Ichimura, Atsuhiko; Nakajima, Akira; Kimura, Ikuo

2016-01-01

Excess energy is stored primarily as triglycerides, which are mobilized when demand for energy arises. Dysfunction of energy balance by excess food intake leads to metabolic diseases, such as obesity and diabetes. Free fatty acids (FFAs) provided by dietary fat are not only important nutrients, but also contribute key physiological functions via FFA receptor (FFAR)-mediated signaling molecules, which depend on FFAs’ carbon chain length and the ligand specificity of the receptors. Functional analyses have revealed that FFARs are critical for metabolic functions, such as peptide hormone secretion and inflammation, and contribute to energy homeostasis. In particular, recent studies have shown that the administration of selective agonists of G protein-coupled receptor (GPR) 40 and GPR120 improved glucose metabolism and systemic metabolic disorders. Furthermore, the anti-inflammation and energy metabolism effects of short chain FAs have been linked to the activation of GPR41 and GPR43. In this review, we summarize recent progress in research on FFAs and their physiological roles in the regulation of energy metabolism. PMID:27023530

Mice Lacking Free Fatty Acid Receptor 1 (GPR40/FFAR1) are Protected Against Conjugated Linoleic Acid-Induced Fatty Liver but Develop Inflammation and Insulin Resistance in the Brain.

PubMed

Sartorius, Tina; Drescher, Andrea; Panse, Madhura; Lastovicka, Petr; Peter, Andreas; Weigert, Cora; Kostenis, Evi; Ullrich, Susanne; Häring, Hans-Ulrich

2015-01-01

Conjugated linoleic acids (CLAs) affect body fat distribution, induce insulin resistance and stimulate insulin secretion. The latter effect is mediated through the free fatty acid receptor-1 (GPR40/FFAR1). This study examines whether GPR40/FFAR1 interacts with tissue specific metabolic changes induced by CLAs. After chronic application of CLAs C57BL/6J wild type (WT) and GPR40/FFAR1 (Ffar1(-/-)) knockout mice developed insulin resistance. Although CLAs accumulated in liver up to 46-fold genotype-independently, hepatic triglycerides augmented only in WT mice. This triglyceride deposition was not associated with increased inflammation. In contrast, in brain of CLA fed Ffar1(-/-) mice mRNA levels of TNF-α were 2-fold higher than in brain of WT mice although CLAs accumulated genotype-independently in brain up to 4-fold. Concomitantly, Ffar1(-/-) mice did not respond to intracerebroventricular (i.c.v.) insulin injection with an increase in cortical activity while WT mice reacted as assessed by radiotelemetric electrocorticography (ECoG) measurements. In vitro incubation of primary murine astrocytes confirmed that CLAs stimulate neuronal inflammation independent of GPR40/FFAR1. This study discloses that GPR40/FFAR1 indirectly modulates organ-specific effects of CLAs: the expression of functional GPR40/FFAR1 counteracts CLA-induced inflammation and insulin resistance in the brain, but favors the development of fatty liver. © 2015 S. Karger AG, Basel.

Expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in control of GnRH secretion.

PubMed

Yang, Ying; Zhou, Li-bin; Liu, Shang-quan; Tang, Jing-feng; Li, Feng-yin; Li, Rong-ying; Song, Huai-dong; Chen, Ming-dao

2005-08-01

To investigate the expression of feeding-related peptide receptors mRNA in GT1-7 cell line and roles of leptin and orexins in the control of GnRH secretion. Receptors of bombesin3, cholecystokinin (CCK)-A, CCK-B, glucagon-like peptide (GLP)1, melanin-concentrating hormone (MCH)1, orexin1, orexin2, neuromedin-B, neuropeptide Y (NPY)1 and NPY5, neurotensin (NT)1, NT2, NT3, and leptin receptor long form mRNA in GT1-7 cells were detected by reversed transcriptase-polymerase chain reaction. GT1-7 cells were treated with leptin, orexin A and orexin B at a cohort of concentrations for different lengths of time, and GnRH in medium was determined by radioimmunoassay (RIA). Receptors of bombesin 3, CCK-B, GLP1, MCH1, orexin1, neuromedin-B, NPY1, NPY5, NT1, NT3, and leptin receptor long form mRNA were expressed in GT1-7 cells, of which, receptors of GLP1, neuromedin-B, NPY1, and NT3 were highly expressed. No amplified fragments of orexin2, NT2, and CCK-A receptor cDNA were generated with GT1-7 RNA, indicating that the GT1-7 cells did not express mRNA of them. Leptin induced a significant stimulation of GnRH release, the results being most significant at 0.1 nmol/L for 15 min. In contrast to other studies in hypothalamic explants, neither orexin A nor orexin B affected basal GnRH secretion over a wide range of concentrations ranging from 1 nmol/L to 500 nmol/Lat 15, 30, and 60 min. Feeding and reproductive function are closely linked. Many orexigenic and anorexigenic signals may control feeding behavior as well as alter GnRH secretion through their receptors on GnRH neurons.

Impact of Morphological Changes on the Motility of Amoeba proteus

NASA Astrophysics Data System (ADS)

Shroff N, Sunitha

2016-11-01

Bio-mechanical properties of cell membrane, actin and cytoskeleton have influence on the cell locomotion. To explore, morphological changes were induced in Amoeba proteus by depriving nutrition, also either through ATP mediated or through KCl mediated membrane depolarization. We observed that, membrane depolarization leads to complete loss/reduction of pseudopodia in a dose dependent manner, gradually A. proteus becomes globular. We also report that with depravation of its nutrition (Chilomonas) A. proteus transforms them into tube/filament like structure and this transformation is reversible with the supply of Chilomonas. Results indicate that the structural and locomotion variation of A. proteus through nucleotides may not be just a membrane phenomenon, but may involve signaling mechanisms. Further, we carried out immunostaining of A. proteus with P2X2 and P2Y2 antibodies to analyze their localization and the extent of expression. The result indicated that in normal A. proteus receptors are dispersed uniformly, whereas in filament shaped A. proteus P2X2-receptor was found to be localized, unlike P2Y2 receptor. As nucleotides are known to cause structural changes in the organism, we report corresponding changes in their locomotion. Assistant Professor, Department of Biotechnology. Mount Carmel College, Bangalore 560 052.

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

PubMed

Hu, Liang; Chang, Lin; Zhang, Yan; Zhai, Lili; Zhang, Shenghui; Qi, Zhiyong; Yan, Hongmei; Yan, Yan; Luo, Xinping; Zhang, Si; Wang, Yiping; Kunapuli, Satya P; Ye, Hongying; Ding, Zhongren

2017-08-29

Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. We measured P2Y 12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y 12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-κB pathway in regulating P2Y 12 receptor expression in megakaryocytes. Platelet P2Y 12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y 12 expression correlates with ADP-induced platelet aggregation (r=0.89, P <0.01). P2Y 12 in platelets from patients with diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y 12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, P <0.05). Using a FeCl 3 -injury mesenteric arteriole thrombosis model in rats and an arteriovenous shunt thrombosis model in rats, we found that the inverse agonist AR-C78511 has greater antithrombotic effects on GK rats with diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P <0.01). We also found that a pathway involving high glucose-reactive oxygen species-nuclear factor-κB increases platelet P2Y 12 receptor expression in diabetes mellitus. Platelet P2Y 12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus. © 2017 American Heart Association, Inc.

Strength Exercise Improves Muscle Mass and Hepatic Insulin Sensitivity in Obese Youth

PubMed Central

van der Heijden, Gert-Jan; Wang, Zhiyue J.; Chu, Zili; Toffolo, Gianna; Manesso, Erica; Sauer, Pieter J.J.; Sunehag, Agneta L.

2010-01-01

Introduction Data are limited on the metabolic effects of resistance exercise (strength training) in adolescents. Purpose The objective of this study was to determine whether a controlled resistance exercise program without dietary intervention or weight loss, reduces body fat accumulation, increases lean body mass, and improves insulin sensitivity and glucose metabolism in sedentary obese Hispanic adolescents. Methods Twelve obese adolescents (15.5±0.5y; 35.3 ±0.8kg/m2;40.8±1.5% body fat), completed a 12 wk resistance exercise program (2×1h/wk, exercising all major muscle groups). At baseline and completion of the program, body composition was measured by DXA, abdominal fat distribution by Magnetic Resonance Imaging, hepatic and intramyocellular fat by Magnetic Resonance Spectroscopy, peripheral insulin sensitivity by the Stable Labeled IV Glucose Tolerance Test and hepatic insulin sensitivity by the Hepatic Insulin Sensitivity Index =1000/(GPR*fasting insulin). Glucose production rate (GPR), gluconeogenesis and glycogenolysis were quantified using Stable Isotope-Gas Chromatography/Mass Spectrometry techniques. Results All participants were normoglycemic. The exercise program resulted in significant strength gain in both upper and lower body muscle groups. Body weight increased from 97.0±3.8 to 99.6±4.2 kg (p<0.01). The major part (~80%) was accounted for by increased lean body mass (55.7±2.8 to 57.9±3.0 kg; p≤0.01).Total, visceral, hepatic and intramyocellular fat content remained unchanged. Hepatic insulin sensitivity increased by 24±9% (p<0.05), while peripheral insulin sensitivity did not change significantly. GPR decreased by 8±1% (p<0.01) due to a 12±5% decrease in glycogenolysis (p<0.05). Conclusion We conclude that a controlled resistance exercise program without weight loss increases strength and lean body mass, improves hepatic insulin sensitivity and decreases GPR without affecting total fat mass or visceral, hepatic and intramyocellular fat content. PMID:20351587

Ocular Purine Receptors as Drug Targets in the Eye

PubMed Central

Civan, Mortimer M.

2016-01-01

Abstract Agonists and antagonists of various subtypes of G protein coupled adenosine receptors (ARs), P2Y receptors (P2YRs), and ATP-gated P2X receptor ion channels (P2XRs) are under consideration as agents for the treatment of ocular diseases, including glaucoma and dry eye. Numerous nucleoside and nonnucleoside modulators of the receptors are available as research tools and potential therapeutic molecules. Three of the 4 subtypes of ARs have been exploited with clinical candidate molecules for treatment of the eye: A1, A2A, and A3. An A1AR agonist is in clinical trials for glaucoma, A2AAR reduces neuroinflammation, A3AR protects retinal ganglion cells from apoptosis, and both A3AR agonists and antagonists had been reported to lower intraocular pressure (IOP). Extracellular concentrations of endogenous nucleotides, including dinucleoside polyphosphates, are increased in pathological states, activating P2Y and P2XRs throughout the eye. P2YR agonists, including P2Y2 and P2Y6, lower IOP. Antagonists of the P2X7R prevent the ATP-induced neuronal apoptosis in the retina. Thus, modulators of the purinome in the eye might be a source of new therapies for ocular diseases. PMID:27574786

Ocular Purine Receptors as Drug Targets in the Eye.

PubMed

Jacobson, Kenneth A; Civan, Mortimer M

2016-10-01

Agonists and antagonists of various subtypes of G protein coupled adenosine receptors (ARs), P2Y receptors (P2YRs), and ATP-gated P2X receptor ion channels (P2XRs) are under consideration as agents for the treatment of ocular diseases, including glaucoma and dry eye. Numerous nucleoside and nonnucleoside modulators of the receptors are available as research tools and potential therapeutic molecules. Three of the 4 subtypes of ARs have been exploited with clinical candidate molecules for treatment of the eye: A 1 , A 2A , and A 3 . An A 1 AR agonist is in clinical trials for glaucoma, A 2A AR reduces neuroinflammation, A 3 AR protects retinal ganglion cells from apoptosis, and both A 3 AR agonists and antagonists had been reported to lower intraocular pressure (IOP). Extracellular concentrations of endogenous nucleotides, including dinucleoside polyphosphates, are increased in pathological states, activating P2Y and P2XRs throughout the eye. P2YR agonists, including P2Y 2 and P2Y 6 , lower IOP. Antagonists of the P2X7R prevent the ATP-induced neuronal apoptosis in the retina. Thus, modulators of the purinome in the eye might be a source of new therapies for ocular diseases.

N-arachidonoyl--serine is neuroprotective after traumatic brain injury by reducing apoptosis

PubMed Central

Cohen-Yeshurun, Ayelet; Trembovler, Victoria; Alexandrovich, Alexander; Ryberg, Erik; Greasley, Peter J; Mechoulam, Raphael; Shohami, Esther; Leker, Ronen R

2011-01-01

N-arachidonoyl--serine (AraS) is a brain component structurally related to the endocannabinoid family. We investigated the neuroprotective effects of AraS following closed head injury induced by weight drop onto the exposed fronto-parietal skull and the mechanisms involved. A single injection of AraS following injury led to a significant improvement in functional outcome, and to reduced edema and lesion volume compared with vehicle. Specific antagonists to CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) or large conductance calcium-activated potassium (BK) channels reversed these effects. Specific binding assays did not indicate binding of AraS to the GPR55 cannabinoid receptor. N-arachidonoyl--serine blocked the attenuation in phosphorylated extracellular-signal-regulated kinase 1/2 (ERK) levels and led to an increase in pAkt in both the ipsilateral and contralateral cortices. Increased levels of the prosurvival factor Bcl-xL were evident 24 hours after injury in AraS-treated mice, followed by a 30% reduction in caspase-3 activity, measured 3 days after injury. Treatment with a CB2 antagonist, but not with a CB1 antagonist, reversed this effect. Our results suggest that administration of AraS leads to neuroprotection via ERK and Akt phosphorylation and induction of their downstream antiapoptotic pathways. These protective effects are related mostly to indirect signaling via the CB2R and TRPV1 channels but not through CB1 or GPR55 receptors. PMID:21505478

N-arachidonoyl-L-serine is neuroprotective after traumatic brain injury by reducing apoptosis.

PubMed

Cohen-Yeshurun, Ayelet; Trembovler, Victoria; Alexandrovich, Alexander; Ryberg, Erik; Greasley, Peter J; Mechoulam, Raphael; Shohami, Esther; Leker, Ronen R

2011-08-01

N-arachidonoyl-L-serine (AraS) is a brain component structurally related to the endocannabinoid family. We investigated the neuroprotective effects of AraS following closed head injury induced by weight drop onto the exposed fronto-parietal skull and the mechanisms involved. A single injection of AraS following injury led to a significant improvement in functional outcome, and to reduced edema and lesion volume compared with vehicle. Specific antagonists to CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) or large conductance calcium-activated potassium (BK) channels reversed these effects. Specific binding assays did not indicate binding of AraS to the GPR55 cannabinoid receptor. N-arachidonoyl-L-serine blocked the attenuation in phosphorylated extracellular-signal-regulated kinase 1/2 (ERK) levels and led to an increase in pAkt in both the ipsilateral and contralateral cortices. Increased levels of the prosurvival factor Bcl-xL were evident 24 hours after injury in AraS-treated mice, followed by a 30% reduction in caspase-3 activity, measured 3 days after injury. Treatment with a CB2 antagonist, but not with a CB1 antagonist, reversed this effect. Our results suggest that administration of AraS leads to neuroprotection via ERK and Akt phosphorylation and induction of their downstream antiapoptotic pathways. These protective effects are related mostly to indirect signaling via the CB2R and TRPV1 channels but not through CB1 or GPR55 receptors.

P2 receptor signaling in neurons and glial cells of the central nervous system.

PubMed

Köles, Laszlo; Leichsenring, Anna; Rubini, Patrizia; Illes, Peter

2011-01-01

Purine and pyrimidine nucleotides are extracellular signaling molecules in the central nervous system (CNS) leaving the intracellular space of various CNS cell types via nonexocytotic mechanisms. In addition, ATP is a neuro-and gliotransmitter released by exocytosis from neurons and neuroglia. These nucleotides activate P2 receptors of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) types. In mammalians, seven P2X and eight P2Y receptor subunits occur; three P2X subtypes form homomeric or heteromeric P2X receptors. P2Y subtypes may also hetero-oligomerize with each other as well as with other G protein-coupled receptors. P2X receptors are able to physically associate with various types of ligand-gated ion channels and thereby to interact with them. The P2 receptor homomers or heteromers exhibit specific sensitivities against pharmacological ligands and have preferential functional roles. They may be situated at both presynaptic (nerve terminals) and postsynaptic (somatodendritic) sites of neurons, where they modulate either transmitter release or the postsynaptic sensitivity to neurotransmitters. P2 receptors exist at neuroglia (e.g., astrocytes, oligodendrocytes) and microglia in the CNS. The neuroglial P2 receptors subserve the neuron-glia cross talk especially via their end-feets projecting to neighboring synapses. In addition, glial networks are able to communicate through coordinated oscillations of their intracellular Ca(2+) over considerable distances. P2 receptors are involved in the physiological regulation of CNS functions as well as in its pathophysiological dysregulation. Normal (motivation, reward, embryonic and postnatal development, neuroregeneration) and abnormal regulatory mechanisms (pain, neuroinflammation, neurodegeneration, epilepsy) are important examples for the significance of P2 receptor-mediated/modulated processes. Copyright © 2011 Elsevier Inc. All rights reserved.

G protein-coupled estrogen receptor inhibits the P2Y receptor-mediated Ca(2+) signaling pathway in human airway epithelia.

PubMed

Hao, Yuan; Chow, Alison W; Yip, Wallace C; Li, Chi H; Wan, Tai F; Tong, Benjamin C; Cheung, King H; Chan, Wood Y; Chen, Yangchao; Cheng, Christopher H; Ko, Wing H

2016-08-01

P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (E2) receptor, may play an anti-inflammatory role in this process. We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca(2+) signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca(2+)]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5'-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca(2+) were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca(2+) mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.

PubMed

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-05-01

The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding

PubMed Central

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-01-01

Background and Purpose: The P2X7 receptor exhibits complex pharmacological properties. In this study, binding of a [3H]-labelled P2X7 receptor antagonist to human P2X7 receptors has been examined to further understand ligand interactions with this receptor. Experimental Approach: The P2X7 receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X7 receptors. Key Results: Binding of [3H]-compound-17 was higher in membranes prepared from cells expressing P2X7 receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X7 receptors. Binding was reversible, saturable and modulated by P2X7 receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. Conclusions: These data demonstrate that human P2X7 receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X7 receptor complex enhances subsequent binding to other P2X7 subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X7 receptor. PMID:17339830

Characterization of a novel function-blocking antibody targeted against the platelet P2Y1 receptor.

PubMed

Karim, Zubair A; Vemana, Hari Priya; Alshbool, Fatima Z; Lin, Olivia A; Alshehri, Abdullah M; Javaherizadeh, Payam; Paez Espinosa, Enma V; Khasawneh, Fadi T

2015-03-01

Platelet hyperactivity is associated with vascular disease and contributes to the genesis of thrombotic disorders. ADP plays an important role in platelet activation and activates platelets through 2 G-protein-coupled receptors, the Gq-coupled P2Y1 receptor (P2Y1R), and the Gi-coupled P2Y12 receptor. Although the involvement of the P2Y1R in thrombogenesis is well established, there are no antagonists that are currently available for clinical use. Our goal is to determine whether a novel antibody targeting the ligand-binding domain, ie, second extracellular loop (EL2) of the P2Y1R (EL2Ab) could inhibit platelet function and protect against thrombogenesis. Our results revealed that the EL2Ab does indeed inhibit ADP-induced platelet aggregation, in a dose-dependent manner. Furthermore, EL2Ab was found to inhibit integrin GPIIb-IIIa activation, dense and α granule secretion, and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl3-induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose-dependent displacement of the radiolabeled P2Y1R antagonist [(3)H]MRS2500 from its ligand-binding site by EL2Ab. Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y1R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y1R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies. © 2015 American Heart Association, Inc.

Potent and Orally Bioavailable GPR142 Agonists as Novel Insulin Secretagogues for the Treatment of Type 2 Diabetes

PubMed Central

2013-01-01

GPR142 is a G protein-coupled receptor that is predominantly expressed in pancreatic β-cells. GPR142 agonists stimulate insulin secretion in the presence of high glucose concentration, so that they could be novel insulin secretagogues with reduced or no risk of hypoglycemia. We report here the optimization of HTS hit compound 1 toward a proof of concept compound 33, which showed potent glucose lowering effects during an oral glucose tolerance test in mice and monkeys. PMID:24900747

Metal-organic frameworks assembled from lanthanide and 2,5-pyridinedicaboxylate with cubane-like [Ln4(OH)4] building units

NASA Astrophysics Data System (ADS)

Abdelbaky, Mohammed S. M.; Amghouz, Zakariae; Fernández-Zapico, Eva; García-Granda, Santiago; García, José R.

2015-09-01

Lanthanide-organic frameworks based on 2,5-pyridinedicaboxylate (25p) ligand, formulated as [Yb4(OH)4(25p)4(H2O)3]·H2O (25pYb), [Y4(OH)4(25p)4(H2O)3]·H2O (25pY-1) and [Y6(OH)8(25p)5(H2O)2] (25pY-2), have been obtained as single phases under hydrothermal conditions. 25pYb and 25pY-1 are isostructural, and crystallize in the triclinic space group, P-1, with a=8.6075(5) Å, b=14.8478(7) Å, c=15.9164(9) Å, α=86.277(4)°, β=80.196(5)°, γ=81.785(4)°, and a=8.7166(6) Å, b=14.966(1) Å, c=15.966(1) Å, α=86.260(6)°, β=80.036(6)°, γ=81.599(6)°, respectively. 25pY-2 crystallizes in the monoclinic space group, P21/c, with a=24.9117(17) Å, b=13.7340(8) Å, c=14.3385(10) Å, β=100.551(7)°. 25pYb and 25pY-2 have been structurally characterized by single-crystal X-ray diffraction. The 25pYb structure is based on tetranuclear cubane-like [Yb4(OH)4]8+ clusters, which are interconnected to eight neighbouring clusters through teen surrounding 25p ligands leading to neutral 3D framework, while the structure of 25pY-2 is based on two independent cuban-like [Y4(OH)4]8+ clusters, which are joined together through Y1 cation leading to the formation of hexanuclear [Y6(OH)8]10+ clusters, which in turn are joined via Y2 cation resulting in infinite inorganic chain extending along c-axis, and each chain is interconnected to six adjacent chains through 25p ligands leading finally to 3D framework. The luminescence properties of Eu3+ and Tb3+ doped 25pY-1 and 25pY-2 compounds have also been investigated. All materials has been characterized by powder X-ray diffraction, thermal analyses (TG-SDTA-MS), FTIR spectroscopy, C-H-N elemental analysis, scanning electron microscopy (SEM-EDX), and powder X-ray thermodiffraction.

Selective stimulation of catecholamine release from bovine adrenal chromaffin cells by an ionotropic purinergic receptor sensitive to 2-methylthio ATP.

PubMed

Tomé, Angelo R; Castro, Enrique; Santos, Rosa M; Rosário, Luís M

2007-06-20

2-Methylthioadenosine 5'-triphosphate (2-MeSATP), formerly regarded as a specific P2Y (metabotropic) purinergic receptor agonist, stimulates Ca2+ influx and evokes catecholamine release from adrenal chromaffin cells. These cells express P2Y and P2X (ionotropic) purinoceptors, with the latter providing an important Ca2+ influx pathway. Using single cell calcium imaging techniques, we have determined whether 2-MeSATP might be a specific P2X receptor agonist in bovine chromaffin cells and assessed the relative role of P2X and P2Y receptors on catecholamine secretion from these cells. ATP raised the [Ca2+]i in ~50% of the cells. Removing extracellular Ca2+ suppressed the [Ca2+]i-raising ability of 2-MeSATP, observed in ~40% of the ATP-sensitive cells. This indicates that 2-MeSATP behaves as a specific ionotropic purinoceptor agonist in bovine chromaffin cells. The 2-MeSATP-induced [Ca2+]i-rises were suppressed by PPADS. UTP raised the [Ca2+]i in ~40% of the ATP-sensitive cells, indicating that these expressed Ca2+-mobilizing P2Y receptors. UTP-sensitive receptors may not be the only P2Y receptors present, as suggested by the observation that ~20% of the ATP-sensitive pool did not respond to either 2-MeSATP or UTP. The average sizes of the ATP- and 2-MeSATP-evoked [Ca2+]i responses were identical in UTP-insensitive cells. 2-MeSATP stimulated Ca2+ influx and evoked catecholamine release, whereas UTP elicited Ca2+ release from intracellular stores but did not evoke secretion. 2-MeSATP-induced secretion was strongly inhibited by Cd2+ and suppressed by extracellular Ca2+ or Na+ removal. TTX inhibited 2-MeSATP-evoked secretion by ~20%. 2-MeSATP is a specific P2X purinoceptor agonist and a potent secretagogue in bovine chromaffin cells. Activation of 2-MeSATP-sensitive receptors stimulates Ca2+ influx mainly via voltage-sensitive Ca2+ channels. For the most part, these are activated by the depolarization brought about by Na+ influx across P2X receptor pores.

INS365 suppresses loss of corneal epithelial integrity by secretion of mucin-like glycoprotein in a rabbit short-term dry eye model.

PubMed

Fujihara, Tsutomu; Murakami, Tadahiro; Nagano, Takashi; Nakamura, Masatsugu; Nakata, Katsuhiko

2002-08-01

P2Y2 receptor agonists, like UTP and ATP, stimulate mucin secretion from goblet cells in vitro. Therefore, mucin stimulants could be good candidates for the treatment of dry eye syndrome because mucin increases the tear film stability and protects against desiccation of ocular surface. INS365 is a more stable P2Y2 receptor agonist than UTP. In the present study, we evaluated, in normal rabbit eyes, its effectiveness to release mucin from goblet cells and to protect the corneal damage induced by desiccation. For mucin secretion, impression cytology was performed following the instillation of INS365 solution or saline into the conjunctival sac. The specimens were stained with periodic acid and Schiff (PAS) reagent, and then the staining area was calculated using computer software. INS365 dose-dependently decreased the PAS staining area of conjunctival goblet cells from 2 to 15 min post-application. Furthermore, we utilized the rabbit short-term dry eye model to evaluate if INS365 eyedrops could protect against any of the damage produced by blockage of blinking with ocular speculum. INS365 significantly suppressed corneal damage at concentrations of more than 0.1% w/v. These results suggest that this P2Y2 agonist is a good candidate for the treatment of dry eye disease.

Receptors and effects of gut hormones in three osteoblastic cell lines.

PubMed

Pacheco-Pantoja, Elda L; Ranganath, Lakshminarayan R; Gallagher, James A; Wilson, Peter J M; Fraser, William D

2011-07-29

In recent years the interest on the relationship of gut hormones to bone processes has increased and represents one of the most interesting aspects in skeletal research. The proportion of bone mass to soft tissue is a relationship that seems to be controlled by delicate and subtle regulations that imply "cross-talks" between the nutrient intake and tissues like fat. Thus, recognition of the mechanisms that integrate a gastrointestinal-fat-bone axis and its application to several aspects of human health is vital for improving treatments related to bone diseases. This work analysed the effects of gut hormones in cell cultures of three osteoblastic cell lines which represent different stages in osteoblastic development. Also, this is the first time that there is a report on the direct effects of glucagon-like peptide 2, and obestatin on osteoblast-like cells. mRNA expression levels of five gut hormone receptors (glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide 1 [GLP-1], glucagon-like peptide 2 [GLP-2], ghrelin [GHR] and obestatin [OB]) were analysed in three osteoblastic cell lines (Saos-2, TE-85 and MG-63) showing different stages of osteoblast development using reverse transcription and real time polymerase chain reaction. The responses to the gut peptides were studied using assays for cell viability, and biochemical bone markers: alkaline phosphatase (ALP), procollagen type 1 amino-terminal propeptides (P1NP), and osteocalcin production. The gut hormone receptor mRNA displayed the highest levels for GIP in Saos-2 and the lowest levels in MG-63, whereas GHR and GPR39 (the putative obestatin receptor) expression was higher in TE-85 and MG-63 and lower in Saos-2. GLP-1 and GLP-2 were expressed only in MG-63 and TE-85. Treatment of gut hormones to cell lines showed differential responses: higher levels in cell viability in Saos-2 after GIP, in TE-85 and MG-63 after GLP-1, GLP-2, ghrelin and obestatin. ALP showed higher levels in Saos-2 after GIP, GHR and OB and in TE-85 after GHR. P1NP showed higher levels after GIP and OB in Saos-2. Decreased levels of P1NP were observed in TE-85 and MG-63 after GLP-1, GLP-2 and OB. MG-63 showed opposite responses in osteocalcin levels after GLP-2. These results suggest that osteoblast activity modulation varies according to different development stage under different nutrition related-peptides.

Insights into unbound-bound states of GPR142 receptor in a membrane-aqueous system using molecular dynamics simulations.

PubMed

Kaushik, Aman Chandra; Sahi, Shakti

2018-05-01

G protein coupled receptors (GPCRs) are source machinery in signal transduction pathways and being one of the major therapeutic targets play a significant in drug discovery. GPR142, an orphan GPCR, has been implicated in the regulation of insulin, thereby having a crucial role in Type II diabetes management. Deciphering of the structures of orphan, GPCRs (O-GPCRs) offer better prospects for advancements in research in ion translocation and transduction of extracellular signals. As the crystallographic structure of GPR142 is not available in PDB, therefore, threading and ab initio-based approaches were used for 3D modeling of GPR142. Molecular dynamic simulations (900 ns) were performed on the 3D model of GPR142 and complexes of GPR142 with top five hits, obtained through virtual screening, embedded in lipid bilayer with aqueous system using OPLS force field. Compound 1, 3, and 4 may act as scaffolds for designing potential lead agonists for GPR142. The finding of GPR142 MD simulation study provides more comprehensive representation of the functional properties. The concern for Type II diabetes is increasing worldwide and successful treatment of this disease demands novel drugs with better efficacy.

Uridine Adenosine Tetraphosphate-Induced Coronary Relaxation Is Blunted in Swine With Pressure Overload: A Role for Vasoconstrictor Prostanoids

PubMed Central

Zhou, Zhichao; Lankhuizen, Inge M.; van Beusekom, Heleen M.; Cheng, Caroline; Duncker, Dirk J.; Merkus, Daphne

2018-01-01

Plasma levels of the vasoactive substance uridine adenosine tetraphosphate (Up4A) are elevated in hypertensive patients and Up4A-induced vascular contraction is exacerbated in various arteries isolated from hypertensive animals, suggesting a potential role of Up4A in development of hypertension. We previously demonstrated that Up4A produced potent and partially endothelium-dependent relaxation in the porcine coronary microvasculature. Since pressure-overload is accompanied by structural abnormalities in the coronary microvasculature as well as by endothelial dysfunction, we hypothesized that pressure-overload blunts the coronary vasodilator response to Up4A, and that the involvement of purinergic receptors and endothelium-derived factors is altered. The effects of Up4A were investigated using wire-myography in isolated coronary small arteries from Sham-operated swine and swine with prolonged (8 weeks) pressure overload of the left ventricle induced by aortic banding (AoB). Expression of purinergic receptors and endothelium-derived factors was assessed in isolated coronary small arteries using real-time PCR. Up4A (10-9 to 10-5 M) failed to produce contraction in isolated coronary small arteries from either Sham or AoB swine, but produced relaxation in preconstricted arteries, which was significantly blunted in AoB compared to Sham. Blockade of purinergic P1, and P2 receptors attenuated Up4A-induced coronary relaxation more, while the effect of P2X1-blockade was similar and the effects of A2A- and P2Y1-blockade were reduced in AoB as compared to Sham. mRNA expression of neither A1, A2, A3, nor P2X1, P2X7, P2Y1, P2Y2, nor P2Y6-receptors was altered in AoB as compared to Sham, while P2Y12 expression was higher in AoB. eNOS inhibition attenuated Up4A-induced coronary relaxation in both Sham and AoB. Additional blockade of cyclooxygenase enhanced Up4A-induced coronary relaxation in AoB but not Sham swine, suggesting the involvement of vasoconstrictor prostanoids. In endothelium-denuded coronary small arteries from normal swine, thromboxane synthase (TxS) inhibition enhanced relaxation to Up4A compared to endothelium-intact arteries, to a similar extent as P2Y12 inhibition, while the combination inhibition of P2Y12 and TxS had no additional effect. In conclusion, Up4A-induced coronary relaxation is blunted in swine with AoB, which appears to be due to the production of a vasoconstrictor prostanoid, likely thromboxane A2. PMID:29632487

Uridine Adenosine Tetraphosphate-Induced Coronary Relaxation Is Blunted in Swine With Pressure Overload: A Role for Vasoconstrictor Prostanoids.

PubMed

Zhou, Zhichao; Lankhuizen, Inge M; van Beusekom, Heleen M; Cheng, Caroline; Duncker, Dirk J; Merkus, Daphne

2018-01-01

Plasma levels of the vasoactive substance uridine adenosine tetraphosphate (Up 4 A) are elevated in hypertensive patients and Up 4 A-induced vascular contraction is exacerbated in various arteries isolated from hypertensive animals, suggesting a potential role of Up 4 A in development of hypertension. We previously demonstrated that Up 4 A produced potent and partially endothelium-dependent relaxation in the porcine coronary microvasculature. Since pressure-overload is accompanied by structural abnormalities in the coronary microvasculature as well as by endothelial dysfunction, we hypothesized that pressure-overload blunts the coronary vasodilator response to Up 4 A, and that the involvement of purinergic receptors and endothelium-derived factors is altered. The effects of Up 4 A were investigated using wire-myography in isolated coronary small arteries from Sham-operated swine and swine with prolonged (8 weeks) pressure overload of the left ventricle induced by aortic banding (AoB). Expression of purinergic receptors and endothelium-derived factors was assessed in isolated coronary small arteries using real-time PCR. Up 4 A (10 -9 to 10 -5 M) failed to produce contraction in isolated coronary small arteries from either Sham or AoB swine, but produced relaxation in preconstricted arteries, which was significantly blunted in AoB compared to Sham. Blockade of purinergic P1, and P2 receptors attenuated Up 4 A-induced coronary relaxation more, while the effect of P2X 1 -blockade was similar and the effects of A 2A - and P2Y 1 -blockade were reduced in AoB as compared to Sham. mRNA expression of neither A1, A2, A3, nor P2X 1 , P2X 7 , P2Y 1 , P2Y 2 , nor P2Y 6 -receptors was altered in AoB as compared to Sham, while P2Y 12 expression was higher in AoB. eNOS inhibition attenuated Up 4 A-induced coronary relaxation in both Sham and AoB. Additional blockade of cyclooxygenase enhanced Up 4 A-induced coronary relaxation in AoB but not Sham swine, suggesting the involvement of vasoconstrictor prostanoids. In endothelium-denuded coronary small arteries from normal swine, thromboxane synthase (TxS) inhibition enhanced relaxation to Up 4 A compared to endothelium-intact arteries, to a similar extent as P2Y 12 inhibition, while the combination inhibition of P2Y 12 and TxS had no additional effect. In conclusion, Up 4 A-induced coronary relaxation is blunted in swine with AoB, which appears to be due to the production of a vasoconstrictor prostanoid, likely thromboxane A 2 .

Physically disconnected non-diffusible cell-to-cell communication between neuroblastoma SH-SY5Y and DRG primary sensory neurons.

PubMed

Chaban, Victor V; Cho, Taehoon; Reid, Christopher B; Norris, Keith C

2013-01-01

Cell-cell communication occurs via a variety of mechanisms, including long distances (hormonal), short distances (paracrine and synaptic) or direct coupling via gap junctions, antigen presentation, or ligand-receptor interactions. We evaluated the possibility of neuro-hormonal independent, non-diffusible, physically disconnected pathways for cell-cell communication using dorsal root ganglion (DRG) neurons. We assessed intracellular calcium ([Ca(2+)]) in primary culture DRG neurons that express ATP-sensitive P2X3, capsaicinsensitive TRPV1 receptors modulated by estradiol. Physically disconnected (dish-in-dish system; inner chamber enclosed) mouse DRG were cultured for 12 hours near: a) media alone (control 1), b) mouse DRG (control 2), c) human neuroblastoma SHSY-5Y cells (cancer intervention), or d) mouse DRG treated with KCl (apoptosis intervention). Chemosensitive receptors [Ca(2+)](i) signaling did not differ between control 1 and 2. ATP (10 μM) and capsaicin (100nM) increased [Ca(2+)](i) transients to 425.86 + 49.5 nM, and 399.21 ± 44.5 nM, respectively. 17β-estradiol (100 nM) exposure reduced ATP (171.17 ± 48.9 nM) and capsaicin (175.01±34.8 nM) [Ca(2+)](i) transients. The presence of cancer cells reduced ATP- and capsaicin-induced [Ca(2+)](i) by >50% (p<0.05) and abolished the 17β-estradiol effect. By contrast, apoptotic DRG cells increased initial ATP-induced [Ca(2+)](i), flux four fold and abolished subsequent [Ca(2+)](i), responses to ATP stimulation (p<0.001). Capsaicin (100nM) induced [Ca(2+)](i) responses were totally abolished. The local presence of apoptotic DRG or human neuroblastoma cells induced differing abnormal ATP and capsaicin-mediated [Ca(2+)](i) fluxes in normal DRG. These findings support physically disconnected, non-diffusible cell-to-cell signaling. Further studies are needed to delineate the mechanism(s) of and model(s) of communication.

GPR84 and TREM-1 Signaling Contribute to the Pathogenesis of Reflux Esophagitis

PubMed Central

Abdel-Aziz, Heba; Schneider, Mathias; Neuhuber, Winfried; Kassem, Abdel Meguid; Khailah, Saleem; Müller, Jürgen; Eldeen, Hadeel Gamal; Khairy, Ahmed; Khayyal, Mohamed T; Shcherbakova, Anastasiia; Efferth, Thomas; Ulrich-Merzenich, Gudrun

2015-01-01

Gastro-esophageal reflux disease (GERD) is one of the most common disorders in gastroenterology. Patients present with or without increased acid exposure indicating a nonuniform etiology. Thus, the common treatment with proton pump inhibitors (PPIs) fails to control symptoms in up to 40% of patients. To further elucidate the pathophysiology of the condition and explore new treatment targets, transcriptomics, proteomics and histological methods were applied to a surgically induced subchronic reflux esophagitis model in Wistar rats after treatment with either omeprazole (PPI) or STW5, a herbal preparation shown to ameliorate esophagitis without affecting refluxate pH. The normal human esophageal squamous cell line HET-1A and human endoscopic biopsies were used to confirm our findings to the G-protein–coupled receptor (GPR) 84 in human tissue. Both treatments reduced reflux-induced macroscopic and microscopic lesions of the esophagi as well as known proinflammatory cytokines. Proteomic and transcriptomic analyses identified CINC1–3, MIP-1/3α, MIG, RANTES and interleukin (IL)-1β as prominent mediators in GERD. Most regulated cyto-/chemokines are linked to the TREM-1 signaling pathway. The fatty acid receptor GPR84 was upregulated in esophagitis but significantly decreased in treated groups, a finding supported by Western blot and immunohistochemistry in both rat tissue and HET-1A cells. GPR84 was also found to be significantly upregulated in patients with grade B reflux esophagitis. The expression of GPR84 in esophageal tissue and its potential involvement in GERD are reported for the first time. IL-8 (CINC1–3) and the TREM-1 signaling pathway are proposed, besides GPR84, to play an important role in the pathogenesis of GERD.org PMID:26650186

GPR84 and TREM-1 signaling contribute to the pathogenesis of reflux esophagitis.

PubMed

Abdel-Aziz, Heba; Schneider, Mathias; Neuhuber, Winfried; Kassem, Abdel Meguid; Khailah, Saleem; Müller, Jürgen; Gamaleldeen, Hadeel; Khairy, Ahmed; Khayyal, Mohamed T; Shcherbakova, Anastasiia; Efferth, Thomas; Ulrich-Merzenich, Gudrun

2015-11-24

Gastro-esophageal reflux disease (GERD) is one of the most common disorders in gastroenterology. Patients present with or without increased acid exposure indicating a non-uniform etiology. Thus the common treatment with proton pump inhibitors (PPIs) fails to control symptoms in up to 40% of patients.To further elucidate the pathophysiology of the condition and explore new treatment targets, transcriptomics, proteomics and histological methods were applied to a surgically induced sub-chronic reflux esophagitis model in Wistar rats after treatment with either omeprazole (PPI) or STW5, a herbal preparation shown to ameliorate esophagitis without affecting refluxate pH. The normal human esophageal squamous cellline HET-1A and human endoscopic biopsies were used to confirm our findings to the G-protein coupled receptor (GPR) 84 in human tissue.Both treatments reduced reflux-induced macroscopic and microscopic lesions of the esophagi as well as known pro-inflammatory cytokines. Proteomic and transcriptomic analyses identified CINC1-3, MIP-1/3α, MIG, RANTES and IL-1β as prominent mediators in GERD. Most regulated cyto-/chemokines are linked to the TREM-1 signaling pathway. The fatty acid receptor GPR84 was up-regulated in esophagitis but significantly decreased in treated groups, a finding supported by Western blot and immunohistochemistry in both rat tissue and HET-1A cells. GPR84 was also found to be significantly up-regulated in patients with grade B reflux esophagitis.The expression of GPR84 in esophageal tissue and its potential involvement in GERD are reported for the first time. IL-8 (CINC1-3) and the TREM-1 signaling pathway are proposed, besides GPR84, to play an important role in the pathogenesis of GERD.

Neuropharmacology of Purinergic Receptors in Human Submucous Plexus: Involvement of P2X1, P2X2, P2X3 Channels, P2Y and A3 Metabotropic Receptors in Neurotransmission

PubMed Central

Liñán-Rico, A.; Wunderlich, JE.; Enneking, JT.; Tso, DR.; Grants, I.; Williams, KC.; Otey, A.; Michel, K.; Schemann, M.; Needleman, B.; Harzman, A.; Christofi, FL.

2015-01-01

Rationale The role of purinergic signaling in the human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. Experimental Approach LSCM-Fluo-4-(Ca2+)-imaging of postsynaptic Ca2+ transients (PSCaTs) was used as a reporter of neural activity. Synaptic transmission was evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons from 234 separate ganglia 107 patients; immunochemical labeling for P2XRs of neurons in ganglia from 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging was used to test effects of adenosine on fast excitatory synaptic potentials (fEPSPs). Results Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines. Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50=25μM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50=111nM) in neurons without stimulatory ADPβS responses (EC50=960nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca2+ transients or Ca2+ oscillations (EC50=400μM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20nM) or high (5μM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-ir follow the order P2X2>P2X1≫P2X3; P2X1+ P2X2 and P2X3+P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7,P2Y1,2,12-14R. Responsive neurons were also identified by HuC/D-ir. Conclusions Purines are critical regulators of neurotransmission in the human enteric nervous system. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1+P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology. PMID:25724083

Modulation of CaV1.2 calcium channel by neuropeptide W regulates vascular myogenic tone via G protein-coupled receptor 7.

PubMed

Ji, Li; Zhu, Huayuan; Chen, Hong; Fan, Wenyong; Chen, Junjie; Chen, Jing; Zhu, Guoqing; Wang, Juejin

2015-12-01

Neuropeptide W (NPW), an endogenous ligand for the G protein-coupled receptor 7 (GPR7), was first found to make important roles in central nerve system. In periphery, NPW was also present and regulated intracellular calcium homeostasis by L-type calcium channels. This study was designed to discover the effects of NPW-GPR7 on the function of CaV1.2 calcium channels in the vascular smooth muscle cells (VSMCs) and vasotone of arterial vessels. By whole-cell patch clamp, we studied the effects of NPW-23, the active form of NPW, on the CaV1.2 channels in the heterologously transfected human embryonic kidney 293 cells and VSMCs isolated from rat. Living system was used to explore the physiological function of NPW-23 in arterial myogenic tone. To investigate the pathological relevance, NPW mRNA level of mesenteric arteries was measured in the hypertensive and normotensive rats. NPW's receptor GPR7 was coexpressed with CaV1.2 channels in arterial smooth muscle. NPW-23 increased the ICa,L in transfected human embryonic kidney 293 cells and VSMCs via GPR7, which could be abrogated by phospholipase C (PLC)/protein kinase C (PKC) inhibitors, not protein kinase A or protein kinase G inhibitor. After NPW-23 application, the expression of pan phospho-PKC was increased; moreover, intracellular diacylglycerol level, the second messenger catalyzed by PLC, was increased 1.5-2-fold. Application with NPW-23 increased pressure-induced vasotone of the rat mesenteric arteries. Importantly, the expression of NPW was decreased in the hypertensive rats. NPW-23 regulates ICa,L via GPR7, which is mediated by PLC/PKC signaling, and such a mechanism plays a role in modulating vascular myogenic tone, which may involve in the development of vascular hypertension.

Lauric Acid Stimulates Mammary Gland Development of Pubertal Mice through Activation of GPR84 and PI3K/Akt Signaling Pathway.

PubMed

Meng, Yingying; Zhang, Jing; Zhang, Fenglin; Ai, Wei; Zhu, Xiaotong; Shu, Gang; Wang, Lina; Gao, Ping; Xi, Qianyun; Zhang, Yongliang; Liang, Xingwei; Jiang, Qingyan; Wang, Songbo

2017-01-11

It has been demonstrated that dietary fat affects pubertal mammary gland development. However, the role of lauric acid (LA) in this process remains unclear. Thus, this study aimed to investigate the effects of LA on mammary gland development in pubertal mice and to explore the underlying mechanism. In vitro, 100 μM LA significantly promoted proliferation of mouse mammary epithelial cell line HC11 by regulating expression of proliferative markers (cyclin D1/3, p21, PCNA). Meanwhile, LA activated the G protein-coupled receptor 84 (GPR84) and PI3K/Akt signaling pathway. In agreement, dietary 1% LA enhanced mammary duct development, increased the expression of GPR84 and cyclin D1, and activated PI3K/Akt in mammary gland of pubertal mice. Furthermore, knockdown of GPR84 or inhibition of PI3K/Akt totally abolished the promotion of HC11 proliferation induced by LA. These results showed that LA stimulated mammary gland development of pubertal mice through activation of GPR84 and PI3K/Akt signaling pathway.

Monitoring platelet inhibition after clopidogrel with the VerifyNow-P2Y12(R) rapid analyzer: the VERIfy Thrombosis risk ASsessment (VERITAS) study.

PubMed

Malinin, Alex; Pokov, Alex; Spergling, Malcolm; Defranco, Anthony; Schwartz, Kenneth; Schwartz, Dianne; Mahmud, Ehtisham; Atar, Dan; Serebruany, Victor

2007-01-01

Clopidogrel inhibits platelet P2Y12 ADP receptors, while ADP, as an inductor of aggregation, stimulates both P2Y12 and P2Y1 platelet receptors. Despite a clinical loading dose routine with clopidogrel, some patients still experience coronary stent thrombosis suggesting persistent platelet activation. The VerifyNow-P2Y12 is a rapid assay that test platelet activity over 3 min and uses of the combination of ADP and prostaglandin E1 (PGE1) to directly measure the effects of clopidogrel on the P2Y12 receptor. ADP is used to maximally activate the platelets by binding to the P2Y1 and P2Y12 platelet receptors, while PGE1 is used to suppress the ADP-induced P2Y1-mediated increase in intracellular calcium levels. The VERIfy Thrombosis risk ASsessment (VERITAS) was a prospective study designed to measure platelet response to clopidogrel therapy in subjects with multiple risk factors or history of vascular disease using this novel point-of-care assay. 166 participants were enrolled in 4 participating sites. Data from 147 participants were analyzed after exclusion of 19 patients due to protocol violations. Platelets were assessed twice at baseline (before clopidogrel) and at 24 h post-loading 450 mg (110 participants) or 7 days after chronic clopidogrel treatment (75 mg/day) (37 patients). All participants received aspirin 81-325 mg for at least 2 days before the study enrollment. Results from the VerifyNow-P2Y12 assay are reported in P2Y12 reaction units (PRU). Clopidogrel therapy resulted in a mean 64.0+/-25.3% PRU reduction. No participant reached PRU inhibition below 10% of baseline. Distribution of PRU values for the VerifyNow-P2Y12 assay shows a separation from baseline to post-clopidogrel assay values with some overlap due to high inter-individual variations in response. VerifyNow-P2Y12 is a reliable, fast and sensitive device suitable for monitoring of platelet inhibition during clopidogrel therapy.

Activation of GPR55 Receptors Exacerbates oxLDL-Induced Lipid Accumulation and Inflammatory Responses, while Reducing Cholesterol Efflux from Human Macrophages.

PubMed

Lanuti, Mirko; Talamonti, Emanuela; Maccarrone, Mauro; Chiurchiù, Valerio

2015-01-01

The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases.