Sample records for ah receptor ligands

  1. Use of natural AhR ligands as potential therapeutic modalities against inflammatory disorders

    PubMed Central

    Busbee, Philip B; Rouse, Michael; Nagarkatti, Mitzi; Nagarkatti, Prakash S

    2014-01-01

    The aim of this review is to discuss research involving ligands for the aryl hydrocarbon receptor (AhR) and their role in immunomodulation. While activation of the AhR is well known for its ability to regulate the biochemical and toxic effects of environmental chemicals, more recently an exciting discovery has been made indicating that AhR ligation can also regulate T-cell differentiation, specifically through activation of Foxp3+ regulatory T cells (Tregs) and downregulation of the proinflammatory Th17 cells. Such findings have opened new avenues of research on the possibility of targeting the AhR to treat inflammatory and autoimmune diseases. Specifically, this review will discuss the current research involving natural and dietary AhR ligands. In addition, evidence indicating the potential use of these ligands in regulating inflammation in various diseases will be highlighted. The importance of the AhR in immunological processes can be illustrated by expression of this receptor on a majority of immune cell types. In addition, AhR signaling pathways have been reported to influence a number of genes responsible for mediating inflammation and other immune responses. As interest in the AhR and its ligands increases, it seems prudent to consolidate current research on the contributions of these ligands to immune regulation during the course of inflammatory diseases. PMID:23731446

  2. Regulation of subcellular localization of the Aryl Hydrocarbon Receptor (AhR)

    USGS Publications Warehouse

    Richter, Catherine A.; Tillitt, Donald E.; Hannink, Mark

    2001-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxin and other xenobiotics. In the absence of exogenous ligand, AhR is cytosolic. We investigated how AhR is retained in the cytosol and how dioxin induces AhR to move to the nucleus. Disruption of nuclear export of AhR by the nuclear export inhibitor leptomycin B (LMB) or by mutation of the AhR nuclear export signal resulted in nuclear accumulation of AhR in the absence of exogenous ligand. Mutation of the AhR nuclear localization signal resulted in defects in nuclear import of AhR in both the presence and the absence of exogenous ligand. Dioxin treatment caused a more rapid accumulation of AhR in the nucleus than LMB treatment. In the presence of both dioxin and LMB, nuclear accumulation of AhR was more rapid than in the presence of dioxin alone. Our results show that AhR shuttles between the nucleus and the cytosol in the absence of exogenous ligand. Binding of ligand induces an increase in the rate of nuclear import of AhR but does not eliminate nuclear export of AhR.

  3. Mixed-ligand copper(II) complexes activate aryl hydrocarbon receptor AhR and induce CYP1A genes expression in human hepatocytes and human cell lines.

    PubMed

    Kubešová, Kateřina; Dořičáková, Aneta; Trávníček, Zdeněk; Dvořák, Zdeněk

    2016-07-25

    The effects of four copper(II) mixed-ligand complexes [Cu(qui1)(L)]NO3·H2O (1-3) and [Cu(qui2)(phen)]NO3 (4), where qui1=2-phenyl-3-hydroxy-4(1H)-quinolinone, Hqui2=2-(4-amino-3,5-dichlorophenyl)-N-propyl-3-hydroxy-4(1H)-quinolinone-7-carboxamide, L=1,10-phenanthroline (phen) (1), 5-methyl-1,10-phenanthroline (mphen) (2), bathophenanthroline (bphen) (3), on transcriptional activities of steroid receptors, nuclear receptors and xenoreceptors have been studied. The complexes (1-4) did not influence basal or ligand-inducible activities of glucocorticoid receptor, androgen receptor, thyroid receptor, pregnane X receptor and vitamin D receptor, as revealed by gene reporter assays. The complexes 1 and 2 dose-dependently induced luciferase activity in stable gene reporter AZ-AhR cell line, and this induction was reverted by resveratrol, indicating involvement of aryl hydrocarbon receptor (AhR) in the process. The complexes 1, 2 and 3 induced CYP1A1 mRNA in LS180 cells and CYP1A1/CYP1A2 in human hepatocytes through AhR. Electrophoretic mobility shift assay EMSA showed that the complexes 1 and 2 transformed AhR in its DNA-binding form. Collectively, we demonstrate that the complexes 1 and 2 activate AhR and induce AhR-dependent genes in human hepatocytes and cancer cell lines. In conclusion, the data presented here might be of toxicological importance, regarding the multiple roles of AhR in human physiology and pathophysiology. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Ligand-dependent interactions of the Ah receptor with coactivators in a mammalian two-hybrid assay

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

    Zhang Shu; Rowlands, Craig; Safe, Stephen

    2008-03-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a high affinity ligand for the aryl hydrocarbon receptor (AhR). In this study, we investigated structure-dependent differences in activation of the AhR by a series of halogenated aromatic hydrocarbons. TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB126) induced CYP1A1-dependent activities in HEK293 human embryonic kidney, Panc1 pancreatic cancer, and Hepa1c1c7 mouse hepatoma cell lines. There was a structure-dependent difference in the efficacy of TCDF and PCB126 in HEK293 and Panc1 cells since induced CYP1A1 mRNA levels were lower than observed for the other congeners. A mammalian two-hybrid assay in cells transfected with GAL4-coactivator and AhR-VP16more » chimeras was used to investigate structure-dependent interactions of these chimeras in Panc1, HEK293, and Hepa1c1c7 cells. The reporter construct pGAL4-luc contains five tandem GAL4 response elements linked to the luciferase gene and the GAL4-coactivator chimeras express several coactivators including steroid receptor coactivator 1 (SRC-1), SRC-2 and SRC-3, the mediator coactivator TRAP220, coactivator associated arginine methyl transferase 1 (CARM-1), and peroxisome proliferator-activated receptor {gamma} coactivator 1 (PGC-1). Results of the mammalian two-hybrid studies clearly demonstrate that activation of pGAL4-luc in cells transfected with VP-AhR and GAL4-coactivator chimeras is dependent on the structure of the HAH congener, cell context, and coactivator, suggesting that the prototypical HAH congeners used in this study exhibit selective AhR modulator activity.« less

  5. Binding Mode and Structure-Activity Relationships of ITE as an Aryl Hydrocarbon Receptor (AhR) Agonist.

    PubMed

    Dolciami, Daniela; Gargaro, Marco; Cerra, Bruno; Scalisi, Giulia; Bagnoli, Luana; Servillo, Giuseppe; Fazia, Maria Agnese Della; Puccetti, Paolo; Quintana, Francisco J; Fallarino, Francesca; Macchiarulo, Antonio

    2018-02-06

    Discovered as a modulator of the toxic response to environmental pollutants, aryl hydrocarbon receptor (AhR) has recently gained attention for its involvement in various physiological and pathological pathways. AhR is a ligand-dependent transcription factor activated by a large array of chemical compounds, which include metabolites of l-tryptophan (l-Trp) catabolism as endogenous ligands of the receptor. Among these, 2-(1'H-indole-3'-carbonyl)thiazole-4-carboxylic acid methyl ester (ITE) has attracted interest in the scientific community, being endowed with nontoxic, immunomodulatory, and anticancer AhR-mediated functions. So far, no information about the binding mode and interactions of ITE with AhR is available. In this study, we used docking and molecular dynamics to propose a putative binding mode of ITE into the ligand binding pocket of AhR. Mutagenesis studies were then instrumental in validating the proposed binding mode, identifying His 285 and Tyr 316 as important key residues for ligand-dependent receptor activation. Finally, a set of ITE analogues was synthesized and tested to further probe molecular interactions of ITE to AhR and characterize the relevance of specific functional groups in the chemical structure for receptor activity. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Newspapers and Newspaper Ink Contain Agonists for the Ah Receptor

    PubMed Central

    Bohonowych, Jessica E. S.; Zhao, Bin; Timme-Laragy, Alicia; Jung, Dawoon; Di Giulio, Richard T.; Denison, Michael S.

    2010-01-01

    Ligand-dependent activation of the aryl hydrocarbon receptor (AhR) pathway leads to a diverse array of biological and toxicological effects. The best-studied ligands for the AhR include polycyclic and halogenated aromatic hydrocarbons, the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, as new AhR ligands are identified and characterized, their structural and physiochemical diversity continues to expand. Our identification of AhR agonists in crude extracts from diverse materials raises questions as to the magnitude and extent of human exposure to AhR ligands through normal daily activities. We have found that solvent extracts of newspapers from countries around the world stimulate the AhR signaling pathway. AhR agonist activity was observed for dimethyl sulfoxide (DMSO), ethanol, and water extracts of printed newspaper, unprinted virgin paper, and black printing ink, where activation of luciferase reporter gene expression was transient, suggesting that the AhR active chemical(s) was metabolically labile. DMSO and ethanol extracts also stimulated AhR transformation and DNA binding, and also competed with [3H]TCDD for binding to the AhR. In addition, DMSO extracts of printed newspaper induced cytochrome P450 1A associated 7-ethoxyresorufin-O-deethylase activity in zebrafish embryos in vivo. Although the responsible bioactive chemical(s) remain to be identified, our results demonstrate that newspapers and printing ink contain relatively potent metabolically labile agonists of the AhR. Given the large amount of recycling and reprocessing of newspapers throughout the world, release of these easily extractable AhR agonists into the environment should be examined and their potential effects on aquatic organisms assessed. PMID:18203687

  7. Interaction of Diuron and Related Substituted Phenylureas with the Ah Receptor Pathway

    PubMed Central

    Zhao, Bin; Baston, David S.; Hammock, Bruce; Denison, Michael S.

    2011-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many of the biological and toxicological actions of structurally diverse chemicals, including the ubiquitous environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin. Here, we have examined the ability of diuron, a widely used herbicide, and several structurally related substituted phenylureas to bind to and activate/inhibit the AhR and AhR signal transduction. Diuron induced CYP1A1 mRNA levels in mouse hepatoma (Hepa1c1c7) cells and AhR-dependent luciferase reporter gene expression in stably transfected mouse, rat, guinea pig, and human cell lines. In addition, ligand binding and gel retardation analysis demonstrated the ability of diuron to competitively bind to and stimulate AhR transformation and DNA binding in vitro and in intact cells. Several structurally related substituted phenylureas competitively bound to the guinea pig hepatic cytosolic AhR, inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced AhR-dependent luciferase reporter gene expression in a species-specific manner and stimulated AhR transformation and DNA binding, consistent with their role as partial AhR agonists. These results demonstrate not only that diuron and related substituted phenylureas are AhR ligands but also that exposure to these chemicals could induce/inhibit AhR-dependent biological effects. PMID:16788953

  8. The Aryl Hydrocarbon Receptor (AhR) as a Drug Target for Cancer Chemotherapy.

    PubMed

    Safe, Stephen; Cheng, Yating; Jin, Un-Ho

    2017-02-01

    The aryl hydrocarbon receptor (AhR) is overexpressed in some patients with different tumor types, and the receptor can be a negative or positive prognostic factor. There is also evidence from both in vivo and in vitro cell culture models that the AhR can exhibit tumor-specific pro-oncogenic and tumor suppressor-like functions and therefore can be treated with AhR antagonists or agonists, respectively. Successful clinical applications of AhR ligands will require the synthesis and development of selective AhR modulators (SAhRMs) with tumor-specific AhR agonist or antagonist activity, and some currently available compounds such as indole-3-carbinol and diindolylmethane-(DIM) and synthetic AhR antagonists are potential drug candidates. There is also evidence that some AhR-active pharmaceuticals, including tranilast, flutamide, hydroxytamoxifen and omeprazole or their derivatives, may be effective AhR-dependent anticancer agents for single or combination cancer chemotherapies for treatment of breast and pancreatic cancers.

  9. An endogenous aryl hydrocarbon receptor (AhR) ligand, ITE induces regulatory T cells (Tregs) and ameliorates experimental colitis.

    PubMed

    Abron, Jessicca D; Singh, Narendra P; Mishra, Manoj K; Price, Robert L; Nagarkatti, Mitzi; Nagarkatti, Prakash S; Singh, Udai P

    2018-04-19

    Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that affects millions of people with high morbidity and health-care cost. The precise etiology of IBD is unknown, but clear evidence suggests that intestinal inflammation is caused by an excessive immune response to mucosal antigens. Recent studies have shown that activation of the aryl hydrocarbon receptor (AhR) induces regulatory T cells (Tregs) and suppresses autoimmune diseases. In the current study, we investigated if nontoxic ligand of AhR, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), can attenuate dextran sodium sulphate (DSS)-induced colitis. Our studies demonstrated that in mice that received ITE treatment, in-vivo colitis pathogenesis, including a decrease in body weight, was significantly reversed along with the systemic and intestinal inflammatory cytokines. ITE increased the expression of Tregs in spleen, mesenteric lymph nodes (MLNs) and colon lamina propria lymphocytes (cLPL) of mice with colitis when compared to controls. This induction of Tregs was reversed by AhR antagonist treatment in-vitro. ITE treatment also increased dendritic cells (DCs; CD11c+) and decreased F4/80+ (macrophage) from the spleen, MLNs and cLPL in mice with colitis. ITE also reversed the systemic and intestinal frequency of CD4+T cells during colitis and suppressed inflammatory cytokines including IFN-γ, TNF-α, IL-17, IL-6 and IL-1 as well as induced IL-10 levels. These findings suggest that ITE attenuates colitis through induction of Tregs and reduction in inflammatory CD4+ T cells and cytokines. Thus, our work demonstrates that the nontoxic endogenous AhR ligand ITE, may serve as a therapeutic modality to treat IBD.

  10. The tertiary structures of porcine AhR and ARNT proteins and molecular interactions within the TCDD/AhR/ARNT complex.

    PubMed

    Orlowska, Karina; Molcan, Tomasz; Swigonska, Sylwia; Sadowska, Agnieszka; Jablonska, Monika; Nynca, Anna; Jastrzebski, Jan P; Ciereszko, Renata E

    2016-06-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activated by structurally diverse synthetic and natural chemicals, including toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In the present study, homology models of the porcine AhR-ligand binding domain (LBD) and the porcine aryl hydrocarbon receptor nuclear translocator-ligand binding domain (ARNT-LBD) were created on the basis of structures of closely related respective proteins i.e., human Hif-2α and ARNT. Molecular docking of TCDD to the porcine AhR-LBD model revealed high binding affinity (-8.8kcal/mol) between TCDD and the receptor. Moreover, formation of the TCDD/AhR-LBD complex was confirmed experimentally with the use of electrophoretic mobility shift assay (EMSA). It was found that TCDD (10nM, 2h of incubation) not only bound to the AhR in the porcine granulosa cells but also activated the receptor. The current study provides a framework for examining the key events involved in the ligand-dependent activation of the AhR. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Novel cell-based assay reveals associations of circulating serum AhR-ligands with metabolic syndrome and mitochondrial dysfunction.

    PubMed

    Park, Wook-Ha; Jun, Dae Won; Kim, Jin Taek; Jeong, Jae Hoon; Park, Hyokeun; Chang, Yoon-Seok; Park, Kyong Soo; Lee, Hong Kyu; Pak, Youngmi Kim

    2013-01-01

    Serum concentrations of environmental pollutants have been positively correlated with diabetes and metabolic syndrome in epidemiologic studies. In turn, abnormal mitochondrial function has been associated with the diseases. The relationships between these variables, however, have not been studied. We developed novel cell-based aryl hydrocarbon receptor (AhR) agonist bioassay system without solvent extraction process and analyzed whether low-dose circulating AhR ligands in human serum are associated with parameters of metabolic syndrome and mitochondrial function. Serum AhR ligand activities were measured as serum 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent (sTCDDeq) in pM using 10 μL human sera from 97 Korean participants (47 with glucose intolerance and 50 matched controls, average age of 46.6 ± 9.9 years, 53 male and 45 female). sTCDDeq were higher in participants with glucose intolerance than normal controls and were positively associated (P < 0.01) with obesity, blood pressure, serum triglyceride, and fasting glucose, but not with HDL-cholesterol. Body mass index was in a positive linear relationship with serum AhR ligands in healthy participants. When myoblast cells were incubated with human sera, ATP generating power of mitochondria became impaired in an AhR ligand concentration-dependent manner. Our results support that circulating AhR ligands may directly reduce mitochondrial function in tissues, leading to weight gain, glucose intolerance, and metabolic syndrome. Our rapid cell-based assay using minute volume of human serum may provide one of the best monitoring systems for circulating AhR ligands, good clinical biomarkers for the progress of disease and therapeutic efficacy. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  12. The Nuclear Receptor AhR Controls Bone Homeostasis by Regulating Osteoclast Differentiation via the RANK/c-Fos Signaling Axis

    PubMed Central

    Izawa, Takashi; Arakaki, Rieko; Mori, Hiroki; Tsunematsu, Takaaki; Kudo, Yasusei; Tanaka, Eiji

    2016-01-01

    The aryl hydrocarbon receptor (AhR) pathway plays a key role in receptor activator of NF-κB ligand (RANKL)–mediated osteoclastogenesis. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow–derived osteoclasts was upregulated by RANKL at an earlier stage than was the expression of signature osteoclast genes such as those encoding cathepsin K and NFAT, cytoplasmic, calcineurin-dependent 1. In response to RANKL, bone marrow macrophages isolated from AhR−/− mice exhibited impaired phosphorylation of Akt and MAPK as well as NF-κB, whereas their response to M-CSF remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in an RANKL/c-Fos–dependent manner. Furthermore, ligand activation of AhR by the smoke toxin benzo[a]pyrene accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR−/− mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest that the RANKL/AhR/c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone. PMID:27849171

  13. Bidirectional communication between the Aryl hydrocarbon Receptor (AhR) and the microbiome tunes host metabolism.

    PubMed

    Korecka, Agata; Dona, Anthony; Lahiri, Shawon; Tett, Adrian James; Al-Asmakh, Maha; Braniste, Viorica; D'Arienzo, Rossana; Abbaspour, Afrouz; Reichardt, Nicole; Fujii-Kuriyama, Yoshiaki; Rafter, Joseph; Narbad, Arjan; Holmes, Elaine; Nicholson, Jeremy; Arulampalam, Velmurugesan; Pettersson, Sven

    2016-01-01

    The ligand-induced transcription factor, aryl hydrocarbon receptor (AhR) is known for its capacity to tune adaptive immunity and xenobiotic metabolism-biological properties subject to regulation by the indigenous microbiome. The objective of this study was to probe the postulated microbiome-AhR crosstalk and whether such an axis could influence metabolic homeostasis of the host. Utilising a systems-biology approach combining in-depth 1 H-NMR-based metabonomics (plasma, liver and skeletal muscle) with microbiome profiling (small intestine, colon and faeces) of AhR knockout (AhR -/- ) and wild-type (AhR +/+ ) mice, we assessed AhR function in host metabolism. Microbiome metabolites such as short-chain fatty acids were found to regulate AhR and its target genes in liver and intestine. The AhR signalling pathway, in turn, was able to influence microbiome composition in the small intestine as evident from microbiota profiling of the AhR +/+ and AhR -/- mice fed with diet enriched with a specific AhR ligand or diet depleted of any known AhR ligands. The AhR -/- mice also displayed increased levels of corticosterol and alanine in serum. In addition, activation of gluconeogenic genes in the AhR -/- mice was indicative of on-going metabolic stress. Reduced levels of ketone bodies and reduced expression of genes involved in fatty acid metabolism in the liver further underscored this observation. Interestingly, exposing AhR -/- mice to a high-fat diet showed resilience to glucose intolerance. Our data suggest the existence of a bidirectional AhR-microbiome axis, which influences host metabolic pathways.

  14. The Nuclear Receptor AhR Controls Bone Homeostasis by Regulating Osteoclast Differentiation via the RANK/c-Fos Signaling Axis.

    PubMed

    Izawa, Takashi; Arakaki, Rieko; Mori, Hiroki; Tsunematsu, Takaaki; Kudo, Yasusei; Tanaka, Eiji; Ishimaru, Naozumi

    2016-12-15

    The aryl hydrocarbon receptor (AhR) pathway plays a key role in receptor activator of NF-κB ligand (RANKL)-mediated osteoclastogenesis. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow-derived osteoclasts was upregulated by RANKL at an earlier stage than was the expression of signature osteoclast genes such as those encoding cathepsin K and NFAT, cytoplasmic, calcineurin-dependent 1. In response to RANKL, bone marrow macrophages isolated from AhR -/- mice exhibited impaired phosphorylation of Akt and MAPK as well as NF-κB, whereas their response to M-CSF remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in an RANKL/c-Fos-dependent manner. Furthermore, ligand activation of AhR by the smoke toxin benzo[a]pyrene accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR -/- mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest that the RANKL/AhR/c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone. Copyright © 2016 by The American Association of Immunologists, Inc.

  15. Bidirectional communication between the Aryl hydrocarbon Receptor (AhR) and the microbiome tunes host metabolism

    PubMed Central

    Korecka, Agata; Dona, Anthony; Lahiri, Shawon; Tett, Adrian James; Al-Asmakh, Maha; Braniste, Viorica; D’Arienzo, Rossana; Abbaspour, Afrouz; Reichardt, Nicole; Fujii-Kuriyama, Yoshiaki; Rafter, Joseph; Narbad, Arjan; Holmes, Elaine; Nicholson, Jeremy; Arulampalam, Velmurugesan; Pettersson, Sven

    2016-01-01

    The ligand-induced transcription factor, aryl hydrocarbon receptor (AhR) is known for its capacity to tune adaptive immunity and xenobiotic metabolism—biological properties subject to regulation by the indigenous microbiome. The objective of this study was to probe the postulated microbiome-AhR crosstalk and whether such an axis could influence metabolic homeostasis of the host. Utilising a systems-biology approach combining in-depth 1H-NMR-based metabonomics (plasma, liver and skeletal muscle) with microbiome profiling (small intestine, colon and faeces) of AhR knockout (AhR−/−) and wild-type (AhR+/+) mice, we assessed AhR function in host metabolism. Microbiome metabolites such as short-chain fatty acids were found to regulate AhR and its target genes in liver and intestine. The AhR signalling pathway, in turn, was able to influence microbiome composition in the small intestine as evident from microbiota profiling of the AhR+/+ and AhR−/− mice fed with diet enriched with a specific AhR ligand or diet depleted of any known AhR ligands. The AhR−/− mice also displayed increased levels of corticosterol and alanine in serum. In addition, activation of gluconeogenic genes in the AhR−/− mice was indicative of on-going metabolic stress. Reduced levels of ketone bodies and reduced expression of genes involved in fatty acid metabolism in the liver further underscored this observation. Interestingly, exposing AhR−/− mice to a high-fat diet showed resilience to glucose intolerance. Our data suggest the existence of a bidirectional AhR-microbiome axis, which influences host metabolic pathways. PMID:28721249

  16. Simultaneous inhibition of aryl hydrocarbon receptor (AhR) and Src abolishes androgen receptor signaling.

    PubMed

    Ghotbaddini, Maryam; Cisse, Keyana; Carey, Alexis; Powell, Joann B

    2017-01-01

    Altered c-Src activity has been strongly implicated in the development, growth, progression, and metastasis of human cancers including prostate cancer. Src is known to regulate several biological functions of tumor cells, including proliferation. There are several Src inhibitors under evaluation for clinical effectiveness but have shown little activity in monotherapy trials of solid tumors. Combination studies are being explored by in vitro analysis and in clinical trials. Here we investigate the effect of simultaneous inhibition of the aryl hydrocarbon receptor (AhR) and Src on androgen receptor (AR) signaling in prostate cancer cells. AhR has also been reported to interact with the Src signaling pathway during prostate development. c-Src protein kinase is associated with the AhR complex in the cytosol and upon ligand binding to AhR, c-Src is activated and released from the complex. AhR has also been shown to regulate AR signaling which remains functionally important in the development and progression of prostate cancer. We provide evidence that co-inhibition of AhR and Src abolish AR activity. Evaluation of total protein and cellular fractions revealed decreased pAR expression and AR nuclear localization. Assays utilizing an androgen responsive element (ARE) and qRT-PCR analysis of AR genes revealed decreased AR promoter activity and transcriptional activity in the presence of both AhR and Src inhibitors. Furthermore, co-inhibition of AhR and Src reduced the growth of prostate cancer cells compared to individual treatments. Several studies have revealed that AhR and Src individually inhibit cellular proliferation. However, this study is the first to suggest simultaneous inhibition of AhR and Src to inhibit AR signaling and prostate cancer cell growth.

  17. Transcript variations, phylogenetic tree and chromosomal localization of porcine aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) genes.

    PubMed

    Sadowska, Agnieszka; Paukszto, Lukasz; Nynca, Anna; Szczerbal, Izabela; Orlowska, Karina; Swigonska, Sylwia; Ruszkowska, Monika; Molcan, Tomasz; Jastrzebski, Jan P; Panasiewicz, Grzegorz; Ciereszko, Renata E

    2017-03-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor best known for mediating xenobiotic-induced toxicity. AhR requires aryl hydrocarbon receptor nuclear translocator (ARNT) to form an active transcription complex and promote the activation of genes which have dioxin responsive element in their regulatory regions. The present study was performed to determine the complete cDNA sequences of porcine AhR and ARNT genes and their chromosomal localization. Total RNA from porcine livers were used to obtain the sequence of the entire porcine transcriptome by next-generation sequencing (NGS; lllumina HiSeq2500). In addition, both, in silico analysis and fluorescence in situ hybridization (FISH) were used to determine chromosomal localization of porcine AhR and ARNT genes. In silico analysis of nucleotide sequences showed that there were two transcript variants of AhR and ARNT genes in the pig. In addition, computer analysis revealed that AhR gene in the pig is located on chromosome 9 and ARNT on chromosome 4. The results of FISH experiment confirmed the localization of porcine AhR and ARNT genes. In the present study, for the first time, the full cDNAs of AhR and ARNT were demonstrated in the pig. In future, it would be interesting to determine the tissue distribution of AhR and ARNT transcript variants in the pig and to test whether these variants are associated with different biological functions and/or different activation pathways.

  18. The aryl hydrocarbon receptor (AhR) mediates resistance to apoptosis induced in breast cancer cells.

    PubMed

    Bekki, Kanae; Vogel, Helena; Li, Wen; Ito, Tomohiro; Sweeney, Colleen; Haarmann-Stemmann, Thomas; Matsumura, Fumio; Vogel, Christoph F A

    2015-05-01

    The aryl hydrocarbon receptor (AhR) is well known as a ligand binding transcription factor regulating various biological effects. Previously we have shown that long-term exposure to estrogen in breast cancer cells caused not only down regulation of estrogen receptor (ER) but also overexpression of AhR. The AhR interacts with several cell signaling pathways associated with induction of tyrosine kinases, cytokines and growth factors which may support the survival roles of AhR escaping from apoptosis elicited by a variety of apoptosis inducing agents in breast cancer. In this study, we studied the anti-apoptotic role of AhR in different breast cancer cells when apoptosis was induced by exposure to UV light and chemotherapeutic agents. Activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in AhR overexpressing breast cancer cells effectively suppressed the apoptotic response induced by UV-irradiation, doxorubicin, lapatinib and paclitaxel. The anti-apoptotic response of TCDD was uniformly antagonized by the treatment with 3'methoxy-4'nitroflavone (MNF), a specific antagonist of AhR. TCDD's survival action of apoptosis was accompanied with the induction of well-known inflammatory genes, such as cyclooxygenase-2 (COX-2) and NF-κB subunit RelB. Moreover, TCDD increased the activity of the immunosuppressive enzyme indoleamine 2, 3-dioxygenase (IDO), which metabolizes tryptophan to kynurenine (Kyn) and mediates tumor immunity. Kyn also acts as an AhR ligand like TCDD, and kyn induced an anti-apoptotic response in breast cancer cells. Accordingly, our present study suggests that AhR plays a pivotal role in the development of breast cancer via the suppression of apoptosis, and provides an idea that the use of AhR antagonists with chemotherapeutic agents may effectively synergize the elimination of breast cancer cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells

    PubMed Central

    Jiang, Yi-Zhou; Dai, Cai-Feng; Patankar, Manish S.; Song, Jia-Sheng; Zheng, Jing

    2013-01-01

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor mediates many biological processes. Herein, we investigated if 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, an endogenous AhR ligand) regulated proliferation and migration of human ovarian cancer cells via AhR. We found that AhR was widely present in many histotypes of ovarian cancer tissues. ITE suppressed OVCAR-3 cell proliferation and SKOV-3 cell migration in vitro, which were blocked by AhR knockdown. ITE also suppressed OVCAR-3 cell growth in mice. These data suggest that the ITE might potentially be used for therapeutic intervention for at least a subset of human ovarian cancer. PMID:23851185

  20. Identification of the Ah-Receptor Structural Determinants for Ligand Preferences

    PubMed Central

    Xing, Yongna

    2012-01-01

    The aryl hydrocarbon receptor (AHR) is a transcription factor that responds to diverse ligands and plays a critical role in toxicology, immune function, and cardiovascular physiology. The structural basis of the AHR for ligand promiscuity and preferences is critical for understanding AHR function. Based on the structure of a closely related protein HIF2α, we modeled the AHR ligand binding domain (LBD) bound to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene (BaP) and identified residues that control ligand preferences by shape and H-bond potential. Mutations to these residues, particularly Q377 and G298, resulted in robust and opposite changes in the potency of TCDD and BaP and up to a 20-fold change in the ratio of TCDD/BaP efficacy. The model also revealed a flexible “belt” structure; molecular dynamic (MD) simulation suggested that the “belt” and several other structural elements in the AHR-LBD are more flexible than HIF2α and likely contribute to ligand promiscuity. Molecular docking of TCDD congeners to a model of human AHR-LBD ranks their binding affinity similar to experimental ranking of their toxicity. Our study reveals key structural basis for prediction of toxicity and understanding the AHR signaling through diverse ligands. PMID:22659362

  1. Aryl Hydrocarbon Receptor (AhR) Deletion in Cerebellar Granule Neuron Precursors Impairs Neurogenesis

    PubMed Central

    Dever, Daniel P.; Adham, Zachariah O.; Thompson, Bryan; Genestine, Matthieu; Cherry, Jonathan; Olschowka, John A.; DiCicco-Bloom, Emanuel; Opanashuk, Lisa A.

    2015-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix (bHLH)/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell autonomous manner, we created a GNP-specific AhR deletion mouse, AhRfx/fx/Math1CRE/+ (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ~25% reductions in thymidine (in vitro) and BrdU (in vivo) incorporation. Furthermore, total granule neuron numbers in the IGL at PND21 and PND60 were diminished in AhR CKO mice compared to controls. On the other hand, differentiation was enhanced, including ~40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis, and may have important implications for the effects of environmental factors in cerebellar dysgenesis. PMID:26243376

  2. An endogenous aryl hydrocarbon receptor ligand inhibits proliferation and migration of human ovarian cancer cells.

    PubMed

    Wang, Kai; Li, Yan; Jiang, Yi-Zhou; Dai, Cai-Feng; Patankar, Manish S; Song, Jia-Sheng; Zheng, Jing

    2013-10-28

    The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor mediates many biological processes. Herein, we investigated if 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, an endogenous AhR ligand) regulated proliferation and migration of human ovarian cancer cells via AhR. We found that AhR was widely present in many histotypes of ovarian cancer tissues. ITE suppressed OVCAR-3 cell proliferation and SKOV-3 cell migration in vitro, which were blocked by AhR knockdown. ITE also suppressed OVCAR-3 cell growth in mice. These data suggest that the ITE might potentially be used for therapeutic intervention for at least a subset of human ovarian cancer. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  3. The AhR Ligand, TCDD, Regulates Androgen Receptor Activity Differently in Androgen-Sensitive versus Castration-Resistant Human Prostate Cancer Cells.

    PubMed

    Ghotbaddini, Maryam; Powell, Joann B

    2015-07-06

    The reported biological effects of TCDD include induction of drug metabolizing enzymes, wasting syndrome and tumor promotion. TCDD elicits most of its effects through binding the aryl hydrocarbon receptor (AhR). TCDD induced degradation of AhR has been widely reported and requires ubiquitination of the protein. The rapid depletion of AhR following TCDD activation serves as a mechanism to modulate AhR mediated gene induction. In addition to inducing AhR degradation, TCDD has been reported to induce degradation of hormone receptors. The studies reported here, evaluate the effect of TCDD exposure on androgen receptor (AR) expression and activity in androgen-sensitive LNCaP and castration-resistant C4-2 prostate cancer cells. Our results show that TCDD exposure does not induce AhR or AR degradation in C4-2 cells. However, both AhR and AR are degraded in LNCaP cells following TCDD exposure. In addition, TCDD enhances AR phosphorylation and induces expression of AR responsive genes in LNCaP cells. Our data reveals that TCDD effect on AR expression and activity differs in androgen-sensitive and castration-resistant prostate cancer cell models.

  4. Screening a mouse liver gene expression Compendium Identifies Effectors of the Aryl Hydrocarbon receptor (AhR)

    EPA Science Inventory

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxic effects of 2,3, 7 ,8-tetrachlorodibenzo-p-dioxin {TCDD), dioxin-like compounds (DLC) as well as some drugs and endogenous tryptophan metabolites. Short-term act...

  5. TCDD and a putative endogenous AhR ligand, ITE, elicit the same immediate changes in gene expression in mouse lung fibroblasts.

    PubMed

    Henry, Ellen C; Welle, Stephen L; Gasiewicz, Thomas A

    2010-03-01

    The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, mediates toxicity of several classes of xenobiotics and also has important physiological roles in differentiation, reproduction, and immunity, although the endogenous ligand(s) mediating these functions is/are as yet unidentified. One candidate endogenous ligand, 2-(1'H-indolo-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), is a potent AhR agonist in vitro, activates the murine AhR in vivo, but does not induce toxicity. We hypothesized that ITE and the toxic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may modify transcription of different sets of genes to account for their different toxicity. To test this hypothesis, primary mouse lung fibroblasts were exposed to 0.5muM ITE, 0.2nM TCDD, or vehicle for 4 h, and total gene expression was evaluated using microarrays. After this short-term and low-dose treatment, several hundred genes were changed significantly, and the response to ITE and TCDD was remarkably similar, both qualitatively and quantitatively. Induced gene sets included the expected battery of AhR-dependent xenobiotic-metabolizing enzymes, as well as several sets that reflect the inflammatory role of lung fibroblasts. Real time quantitative RT-qPCR assay of several selected genes confirmed these microarray data and further suggested that there may be kinetic differences in expression between ligands. These data suggest that ITE and TCDD elicit an analogous change in AhR conformation such that the initial transcription response is the same. Furthermore, if the difference in toxicity between TCDD and ITE is mediated by differences in gene expression, then it is likely that secondary changes enabled by the persistent TCDD, but not by the shorter lived ITE, are responsible.

  6. TCDD and a Putative Endogenous AhR Ligand, ITE, Elicit the Same Immediate Changes in Gene Expression in Mouse Lung Fibroblasts

    PubMed Central

    Henry, Ellen C.; Welle, Stephen L.; Gasiewicz, Thomas A.

    2010-01-01

    The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, mediates toxicity of several classes of xenobiotics and also has important physiological roles in differentiation, reproduction, and immunity, although the endogenous ligand(s) mediating these functions is/are as yet unidentified. One candidate endogenous ligand, 2-(1′H-indolo-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), is a potent AhR agonist in vitro, activates the murine AhR in vivo, but does not induce toxicity. We hypothesized that ITE and the toxic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may modify transcription of different sets of genes to account for their different toxicity. To test this hypothesis, primary mouse lung fibroblasts were exposed to 0.5μM ITE, 0.2nM TCDD, or vehicle for 4 h, and total gene expression was evaluated using microarrays. After this short-term and low-dose treatment, several hundred genes were changed significantly, and the response to ITE and TCDD was remarkably similar, both qualitatively and quantitatively. Induced gene sets included the expected battery of AhR-dependent xenobiotic-metabolizing enzymes, as well as several sets that reflect the inflammatory role of lung fibroblasts. Real time quantitative RT-qPCR assay of several selected genes confirmed these microarray data and further suggested that there may be kinetic differences in expression between ligands. These data suggest that ITE and TCDD elicit an analogous change in AhR conformation such that the initial transcription response is the same. Furthermore, if the difference in toxicity between TCDD and ITE is mediated by differences in gene expression, then it is likely that secondary changes enabled by the persistent TCDD, but not by the shorter lived ITE, are responsible. PMID:19933214

  7. Adaptive changes in global gene expression profile of lung carcinoma A549 cells acutely exposed to distinct types of AhR ligands.

    PubMed

    Procházková, Jiřina; Strapáčová, Simona; Svržková, Lucie; Andrysík, Zdeněk; Hýžďalová, Martina; Hrubá, Eva; Pěnčíková, Kateřina; Líbalová, Helena; Topinka, Jan; Kléma, Jiří; Espinosa, Joaquín M; Vondráček, Jan; Machala, Miroslav

    2018-08-01

    Exposure to persistent ligands of aryl hydrocarbon receptor (AhR) has been found to cause lung cancer in experimental animals, and lung adenocarcinomas are often associated with enhanced AhR expression and aberrant AhR activation. In order to better understand the action of toxic AhR ligands in lung epithelial cells, we performed global gene expression profiling and analyze TCDD-induced changes in A549 transcriptome, both sensitive and non-sensitive to CH223191 co-treatment. Comparison of our data with results from previously reported microarray and ChIP-seq experiments enabled us to identify candidate genes, which expression status reflects exposure of lung cancer cells to TCDD, and to predict processes, pathways (e.g. ER stress, Wnt/β-cat, IFNɣ, EGFR/Erbb1), putative TFs (e.g. STAT, AP1, E2F1, TCF4), which may be implicated in adaptive response of lung cells to TCDD-induced AhR activation. Importantly, TCDD-like expression fingerprint of selected genes was observed also in A549 cells exposed acutely to both toxic (benzo[a]pyrene, benzo[k]fluoranthene) and endogenous AhR ligands (2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester and 6-formylindolo[3,2-b]carbazole). Overall, our results suggest novel cellular candidates, which could help to improve monitoring of AhR-dependent transcriptional activity during acute exposure of lung cells to distinct types of environmental pollutants. Copyright © 2018 Elsevier B.V. All rights reserved.

  8. Crucial Role of the Aryl Hydrocarbon Receptor (AhR) in Indoxyl Sulfate-Induced Vascular Inflammation.

    PubMed

    Ito, Shunsuke; Osaka, Mizuko; Edamatsu, Takeo; Itoh, Yoshiharu; Yoshida, Masayuki

    2016-08-01

    The aryl hydrocarbon receptor (AhR), a ligand-inducible transcription factor mediating toxic effects of dioxins and uremic toxins, has recently emerged as a pathophysiological regulator of immune-inflammatory conditions. Indoxyl sulfate, a uremic toxin, is associated with cardiovascular disease in patients with chronic kidney disease and has been shown to be a ligand for AhR. The aim of this study was to investigate the potential role of AhR in indoxyl sulfate-induced leukocyte-endothelial interactions. Endothelial cell-specific AhR knockout (eAhR KO) mice were produced by crossing AhR floxed mice with Tie2 Cre mice. Indoxyl sulfate was administered for 2 weeks, followed by injection of TNF-α. Leukocyte recruitment to the femoral artery was assessed by intravital microscopy. Vascular endothelial cells were transfected with siRNA specific to AhR (siAhR) and treated with indoxyl sulfate, followed by stimulation with TNF-α. Indoxyl sulfate dramatically enhanced TNF-α-induced leukocyte recruitment to the vascular wall in control animals but not in eAhR KO mice. In endothelial cells, siAhR significantly reduced indoxyl sulfate-enhanced leukocyte adhesion as well as E-selectin expression, whereas the activation of JNK and nuclear factor-κB was not affected. A luciferase assay revealed that the region between -153 and -146 bps in the E-selectin promoter was responsible for indoxyl sulfate activity via AhR. Mutational analysis of this region revealed that activator protein-1 (AP-1) is responsible for indoxyl sulfate-triggered E-selectin expression via AhR. AhR mediates indoxyl sulfate-enhanced leukocyte-endothelial interactions through AP-1 transcriptional activity, which may constitute a new mechanism of vascular inflammation in patients with renal disease.

  9. Aryl hydrocarbon receptor (AhR)-dependent regulation of pulmonary miRNA by chronic cigarette smoke exposure.

    PubMed

    Rogers, Sarah; de Souza, Angela Rico; Zago, Michela; Iu, Matthew; Guerrina, Necola; Gomez, Alvin; Matthews, Jason; Baglole, Carolyn J

    2017-01-12

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor historically known for its toxic responses to man-made pollutants such as dioxin. More recently, the AhR has emerged as a suppressor of inflammation, oxidative stress and apoptosis from cigarette smoke by mechanisms that may involve the regulation of microRNA. However, little is known about the AhR regulation of miRNA expression in the lung in response to inhaled toxicants. Therefore, we exposed Ahr -/- and Ahr +/- mice to cigarette smoke for 4 weeks and evaluated lung miRNA expression by PCR array. There was a dramatic regulation of lung miRNA by the AhR in the absence of exogenous ligand. In response to cigarette smoke, there were more up-regulated miRNA in Ahr -/- mice compared to Ahr +/- mice, including the cancer-associated miRNA miR-96. There was no significant change in the expression of the AhR regulated proteins HuR and cyclooxygenase-2 (COX-2). There were significant increases in the anti-oxidant gene sulfiredoxin 1 (Srxn1) and FOXO3a- predicted targets of miR-96. Collectively, these data support a prominent role for the AhR in regulating lung miRNA expression. Further studies to elucidate a role for these miRNA may further uncover novel biological function for the AhR in respiratory health and disease.

  10. Aryl hydrocarbon receptor (AhR)-dependent regulation of pulmonary miRNA by chronic cigarette smoke exposure

    PubMed Central

    Rogers, Sarah; de Souza, Angela Rico; Zago, Michela; Iu, Matthew; Guerrina, Necola; Gomez, Alvin; Matthews, Jason; Baglole, Carolyn J.

    2017-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor historically known for its toxic responses to man-made pollutants such as dioxin. More recently, the AhR has emerged as a suppressor of inflammation, oxidative stress and apoptosis from cigarette smoke by mechanisms that may involve the regulation of microRNA. However, little is known about the AhR regulation of miRNA expression in the lung in response to inhaled toxicants. Therefore, we exposed Ahr−/− and Ahr+/− mice to cigarette smoke for 4 weeks and evaluated lung miRNA expression by PCR array. There was a dramatic regulation of lung miRNA by the AhR in the absence of exogenous ligand. In response to cigarette smoke, there were more up-regulated miRNA in Ahr−/− mice compared to Ahr+/− mice, including the cancer-associated miRNA miR-96. There was no significant change in the expression of the AhR regulated proteins HuR and cyclooxygenase-2 (COX-2). There were significant increases in the anti-oxidant gene sulfiredoxin 1 (Srxn1) and FOXO3a- predicted targets of miR-96. Collectively, these data support a prominent role for the AhR in regulating lung miRNA expression. Further studies to elucidate a role for these miRNA may further uncover novel biological function for the AhR in respiratory health and disease. PMID:28079158

  11. Cell and region specificity of Aryl hydrocarbon Receptor (AhR) system in the testis and the epididymis.

    PubMed

    Wajda, A; Łapczuk, J; Grabowska, M; Pius-Sadowska, E; Słojewski, M; Laszczynska, M; Urasinska, E; Machalinski, B; Drozdzik, M

    2017-04-01

    Aryl hydrocarbon receptor (AhR) plays multiple important functions in adaptive responses. Exposure to AhR ligands may produce an altered metabolic activity controlled by the AhR pathways, and consequently affect drug/toxin responses, hormonal status and cellular homeostasis. This research revealed species-, cell- and region-specific pattern of the AhR system expression in the rat and human testis and epididymis, complementing the existing knowledge, especially within the epididymal segments. The study showed that AhR level in the rat and human epididymis is higher than in the testis. The downregulation of AhR expression after TCDD treatment was revealed in the spermatogenic cells at different stages and the epididymal epithelial cells, but not in the Sertoli and Leydig cells. Hence, this basic research provides information about the AhR function in the testis and epididymis, which may provide an insight into deleterious effects of drugs, hormones and environmental pollutants on male fertility. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Ligand independent aryl hydrocarbon receptor inhibits lung cancer cell invasion by degradation of Smad4.

    PubMed

    Lee, Chen-Chen; Yang, Wen-Hao; Li, Ching-Hao; Cheng, Yu-Wen; Tsai, Chi-Hao; Kang, Jaw-Jou

    2016-07-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that regulates the metabolism of xenobiotic and endogenous compounds. Although AhR plays a crucial role in air toxicant-induced carcinogenesis, AhR expression was shown to negatively regulate tumorigenesis. Therefore, in the present study, we investigated the effect of AhR without ligand treatment on cancer invasion in lung cancer cell lines. Lung cancer cells expressing lower levels of AhR showed higher invasion ability (H1299 cells) compared with cells expressing higher levels of AhR (A549 cells). Overexpression of AhR in H1299 cells inhibited the invasion ability. We found that vimentin expression was inhibited in AhR-overexpressing H1299 cells. Additionally, the expression of EMT-related transcriptional factors Snail and ID-1 decreased. Interestingly, we found that Smad4 degradation was induced in AhR-overexpressing H1299 cells. Our data showed that AhR could interact with Jun-activation domain binding protein (Jab1) and Smad4, which may cause degradation of Smad4 by the proteasome. Our data suggest that AhR affects the transforming growth factor-β signaling pathway by inducing Smad4 degradation by the proteasome and suppressing tumor metastasis via epithelial to mesenchymal transition reduction in lung cancer cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. The regulation mechanisms of AhR by molecular chaperone complex.

    PubMed

    Kudo, Ikuru; Hosaka, Miki; Haga, Asami; Tsuji, Noriko; Nagata, Yuhtaroh; Okada, Hirotaka; Fukuda, Kana; Kakizaki, Yuka; Okamoto, Tomoya; Grave, Ewa; Itoh, Hideaki

    2018-03-01

    The AhR, so called the dioxin receptor, is a member of the nuclear receptor superfamily. The ligand-free AhR forms a cytosolic protein complex with the molecular chaperone HSP90, co-chaperone p23, and XAP2 in the cytoplasm. Following ligand binding like 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the AhR translocates into the nucleus. Although it has been reported that HSP90 regulates the translocation of the AhR to the nucleus, the precise activation mechanisms of the AhR have not yet been fully understood. AhR consists of the N-terminal bHLH domain containing NLS and NES, the middle PAS domain and the C-terminal transactivation domain. The PAS domain is familiar as a ligand and HSP90 binding domain. In this study, we focused on the bHLH domain that was thought to be a HSP90 binding domain. We investigated the binding properties of bHLH to HSP90. We analyzed the direct interaction of bHLH with HSP90, p23 and XAP2 using purified proteins. We found that not only the PAS domain but also the bHLH domain bound to HSP90. The bHLH domain forms complex with HSP90, p23 and XAP2. We also determined the bHLH binding domain was HSP90 N-domain. The bHLH domain makes a complex with HSP90, p23 and XAP2 via the HSP90 N-domain. Although the NLS is closed in the absence of a ligand, the structure of AhR will be changed in the presence of a ligand, which leads to NLS open, result in the nuclear translocation of AhR.

  14. Divergent Ah Receptor Ligand Selectivity during Hominin Evolution

    PubMed Central

    Hubbard, Troy D.; Murray, Iain A.; Bisson, William H.; Sullivan, Alexis P.; Sebastian, Aswathy; Perry, George H.; Jablonski, Nina G.; Perdew, Gary H.

    2016-01-01

    We have identified a fixed nonsynonymous sequence difference between humans (Val381; derived variant) and Neandertals (Ala381; ancestral variant) in the ligand-binding domain of the aryl hydrocarbon receptor (AHR) gene. In an exome sequence analysis of four Neandertal and Denisovan individuals compared with nine modern humans, there are only 90 total nucleotide sites genome-wide for which archaic hominins are fixed for the ancestral nonsynonymous variant and the modern humans are fixed for the derived variant. Of those sites, only 27, including Val381 in the AHR, also have no reported variability in the human dbSNP database, further suggesting that this highly conserved functional variant is a rare event. Functional analysis of the amino acid variant Ala381 within the AHR carried by Neandertals and nonhuman primates indicate enhanced polycyclic aromatic hydrocarbon (PAH) binding, DNA binding capacity, and AHR mediated transcriptional activity compared with the human AHR. Also relative to human AHR, the Neandertal AHR exhibited 150–1000 times greater sensitivity to induction of Cyp1a1 and Cyp1b1 expression by PAHs (e.g., benzo(a)pyrene). The resulting CYP1A1/CYP1B1 enzymes are responsible for PAH first pass metabolism, which can result in the generation of toxic intermediates and perhaps AHR-associated toxicities. In contrast, the human AHR retains the ancestral sensitivity observed in primates to nontoxic endogenous AHR ligands (e.g., indole, indoxyl sulfate). Our findings reveal that a functionally significant change in the AHR occurred uniquely in humans, relative to other primates, that would attenuate the response to many environmental pollutants, including chemicals present in smoke from fire use during cooking. PMID:27486223

  15. TCDD, FICZ, and Other High Affinity AhR Ligands Dose-Dependently Determine the Fate of CD4+ T Cell Differentiation.

    PubMed

    Ehrlich, Allison K; Pennington, Jamie M; Bisson, William H; Kolluri, Siva K; Kerkvliet, Nancy I

    2018-02-01

    FICZ and TCDD, two high-affinity AhR ligands, are reported to have opposite effects on T cell differentiation with TCDD inducing regulatory T cells and FICZ inducing Th17 cells. This dichotomy has been attributed to ligand-intrinsic differences in AhR activation, although differences in sensitivity to metabolism complicate the issue. TCDD is resistant to AhR-induced metabolism and produces sustained AhR activation following a single dose in the μg/kg range, whereas FICZ is rapidly metabolized and AhR activation is transient. Nonetheless, prior studies comparing FICZ with TCDD have generally used the same 10-50 μg/kg dose range, and thus the two ligands would not equivalently activate AhR. We hypothesized that high-affinity AhR ligands can promote CD4+ T cell differentiation into both Th17 cells and Tregs, with fate depending on the extent and duration of AhR activation. We compared the immunosuppressive effects of TCDD and FICZ, along with two other rapidly metabolized ligands (ITE and 11-Cl-BBQ) in an acute alloresponse mouse model. The dose and timing of administration of each ligand was optimized for TCDD-equivalent Cyp1a1 induction. When optimized, all of the ligands suppressed the alloresponse in conjunction with the induction of Foxp3- Tr1 cells on day 2 and the expansion of natural Foxp3+ Tregs on day 10. In contrast, a low dose of FICZ induced transient expression of Cyp1a1 and did not induce Tregs or suppress the alloresponse but enhanced IL-17 production. Interestingly, low doses of the other ligands, including TCDD, also increased IL-17 production on day 10. These findings support the conclusion that the dose and the duration of AhR activation by high-affinity AhR ligands are the primary factors driving the fate of T cell differentiation. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Characterizing the role of endothelin-1 in the progression of cardiac hypertrophy in aryl hydrocarbon receptor (AhR) null mice

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

    Lund, Amie K.; Goens, M. Beth; Nunez, Bethany A.

    2006-04-15

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor characterized to play a role in detection and adaptation to environmental stimuli. Genetic deletion of AhR results in hypertension, and cardiac hypertrophy and fibrosis, associated with elevated plasma angiotensin II (Ang II) and endothelin-1 (ET-1), thus AhR appears to contribute to cardiovascular homeostasis. In these studies, we tested the hypothesis that ET-1 mediates cardiovascular pathology in AhR null mice via ET{sub A} receptor activation. First, we determine the time courses of cardiac hypertrophy, and of plasma and tissue ET-1 expression in AhR wildtype and null mice. AhR null mice exhibitedmore » increases in heart-to-body weight ratio and age-related expression of cardiac hypertrophy markers, {beta}-myosin heavy chain ({beta}-MHC), and atrial natriuretic factor (ANF), which were significant at 2 months. Similarly, plasma and tissue ET-1 expression was significantly elevated at 2 months and increased further with age. Second, AhR null mice were treated with ET{sub A} receptor antagonist, BQ-123 (100 nmol/kg/day), for 7, 28, or 58 days and blood pressure, cardiac fibrosis, and cardiac hypertrophy assessed, respectively. BQ-123 for 7 days significantly reduced mean arterial pressure in conscious, catheterized mice. BQ-123 for 28 days significantly reduced the histological appearance of cardiac fibrosis. Treatment for 58 days significantly reduced cardiac mass, assessed by heart weight, echocardiography, and {beta}-MHC and ANF expression; and reduced cardiac fibrosis as determined by osteopontin and collagen I mRNA expression. These findings establish ET-1 and the ET{sub A} receptor as primary determinants of hypertension and cardiac pathology in AhR null mice.« less

  17. Inhibitory Ah Receptor-Androgen Receptor Crosstalk in Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    Balk,S.P. Selection for androgen receptor mutations in prostate cancers treated with androgen antagonist. Cancer Res. 59:2511-2515, 1999. 5. Ris...expression, 24-hydroxylase activity, and inhibition of growth hydrocarbon receptor modulators ( SARMs ) for treatment of breast by lca,25-dihydroxyvitamin D3...Safe, A. McDougal, M.S. Gupta, K. Ramamoorthy, Selective Ah [20] D.M. Peehl, R.J. Skowronski, G.K. Leung, S.T. Wong, T.A. Stamey, receptor modulators

  18. Molecular modeling of the AhR structure and interactions can shed light on ligand-dependent activation and transformation mechanisms.

    PubMed

    Bonati, Laura; Corrada, Dario; Tagliabue, Sara Giani; Motta, Stefano

    2017-02-01

    Molecular modeling has given important contributions to elucidation of the main stages in the AhR signal transduction pathway. Despite the lack of experimentally determined structures of the AhR functional domains, information derived from homologous systems has been exploited for modeling their structure and interactions. Homology models of the AhR PASB domain have provided information on the binding cavity and contributed to elucidate species-specific differences in ligand binding. Molecular Docking simulations of the ligand binding process have given insights into differences in binding of diverse agonists, antagonists, and selective AhR modulators, and their application to virtual screening of large databases of compounds have allowed identification of novel AhR ligands. Recently available structural information on protein-protein and protein-DNA complexes of other bHLH-PAS systems has opened the way for modeling the AhR:ARNT dimer structure and investigating the mechanisms of AhR transformation and DNA binding. Future research directions should include simulation of the protein dynamics to obtain a more reliable description of intermolecular interactions involved in signal transmission.

  19. Phytomonitoring and phytoremediation of agrochemicals and related compounds based on recombinant cytochrome P450s and aryl hydrocarbon receptors (AhRs).

    PubMed

    Shimazu, Sayuri; Inui, Hideyuki; Ohkawa, Hideo

    2011-04-13

    Molecular mechanisms of metabolism and modes of actions of agrochemicals and related compounds are important for understanding selective toxicity, biodegradability, and monitoring of biological effects on nontarget organisms. It is well-known that in mammals, cytochrome P450 (P450 or CYP) monooxygenases metabolize lipophilic foreign compounds. These P450 species are inducible, and both CYP1A1 and CYP1A2 are induced by aryl hydrocarbon receptor (AhR) combined with a ligand. Gene engineering of P450 and NADPH cytochrome P450 oxidoreductase (P450 reductase) was established for bioconversion. Also, gene modification of AhRs was developed for recombinant AhR-mediated β-glucronidase (GUS) reporter assay of AhR ligands. Recombinant P450 genes were transformed into plants for phytoremediation, and recombinant AhR-mediated GUS reporter gene expression systems were each transformed into plants for phytomonitoring. Transgenic rice plants carrying CYP2B6 metabolized the herbicide metolachlor and remarkably reduced the residues in the plants and soils under paddy field conditions. Transgenic Arabidopsis plants carrying recombinant guinea pig (g) AhR-mediated GUS reporter genes detected PCB126 at the level of 10 ng/g soils in the presence of biosurfactants MEL-B. Both phytomonitoring and phytoremediation plants were each evaluated from the standpoint of practical uses.

  20. Regulatory effects of dioxin-like and non-dioxin-like PCBs and other AhR ligands on the antioxidant enzymes paraoxonase 1/2/3.

    PubMed

    Shen, Hua; Robertson, Larry W; Ludewig, Gabriele

    2016-02-01

    Paraoxonase 1 (PON1), an antioxidant enzyme, is believed to play a critical role in many diseases, including cancer. PCBs are widespread environmental contaminants known to induce oxidative stress and cancer and to produce changes in gene expression of various pro-oxidant and antioxidant enzymes. Thus, it appeared of interest to explore whether PCBs may modulate the activity and/or gene expression of PON1 as well. In this study, we compared the effects of dioxin-like and non-dioxin-like PCBs and of various aryl hydrocarbon receptor (AhR) ligands on PON1 regulation and activity in male and female Sprague-Dawley rats. Our results demonstrate that (i) the non-dioxin-like PCB154, PCB155, and PCB184 significantly reduced liver and serum PON1 activities, but only in male rats; (ii) the non-dioxin-like PCB153, the most abundant PCB in many matrices, did not affect PON1 messenger RNA (mRNA) level in the liver but significantly decreased serum PON1 activity in male rats; (iii) PCB126, an AhR ligand and dioxin-like PCB, increased both PON1 activities and gene expression; and (iv) even though three tested AhR ligands induced CYP1A in several tissues to a similar extent, they displayed differential effects on the three PONs and AhR, i.e., PCB126 was an efficacious inducer of PON1, PON2, PON3, and AhR in the liver, while 3-methylcholantrene induced liver AhR and lung PON3, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent AhR agonist, increased only PON3 in the lung, at the doses and exposure times used in these studies. These results show that PCBs may have an effect on the antioxidant protection by paraoxonases in exposed populations and that regulation of gene expression through AhR is highly diverse.

  1. Is chronic AhR activation by rapidly metabolized ligands safe for the treatment of immune-mediated diseases?

    PubMed

    Ehrlich, Allison K; Kerkvliet, Nancy I

    2017-02-01

    There is a long standing perception that AhR ligands are automatically disqualified from pharmaceutical development due to their induction of Cyp1a1 as well as their potential for causing "dioxin-like" toxicities. However, recent discoveries of new AhR ligands with potential therapeutic applications have been reported, inviting reconsideration of this policy. One area of exploration is focused on the activation of AhR to promote the generation of regulatory T cells, which control the intensity and duration of immune responses. Rapidly metabolized AhR ligands (RMAhRLs), which do not bioaccumulate in the same manner as 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) have been discovered that induce Tregs and display impressive therapeutic efficacy in a broad range of preclinical models of immune-mediated diseases. Given the promise of these RMAhRLs, is the bias against AhR activators still valid? Can RMAhRLs be given chronically to maintain therapeutic levels of AhR activation without producing the same toxicity profile as dioxin-like compounds? Based on our review of the data, there is little evidence to support the indiscriminate exclusion of AhR activators/Cyp1a1 inducers from early drug developmental pipelines. We also found no evidence that short-term treatment with RMAhRLs produce "dioxin-like toxicity" and, in fact, were well tolerated. However, safety testing of individual RMAhRLs under therapeutic conditions, as performed with all promising new drugs, will be needed to reveal whether or not chronic activation of AhR leads to unacceptable adverse outcomes.

  2. The aryl hydrocarbon receptor repressor - More than a simple feedback inhibitor of AhR signaling: Clues for its role in inflammation and cancer.

    PubMed

    Vogel, Christoph F A; Haarmann-Stemmann, Thomas

    2017-02-01

    The aryl hydrocarbon receptor repressor (AhRR) was first described as a specific competitive repressor of aryl hydrocarbon receptor (AhR) activity based on its ability to dimerize with the AhR nuclear translocator (ARNT) and through direct competition of AhR/ARNT and AhRR/ARNT complexes for binding to dioxin-responsive elements (DREs). Like AhR, AhRR belongs to the basic Helix-Loop-Helix/Per-ARNT-Sim (bHLH/PAS) protein family but lacks functional ligand-binding and transactivation domains. Transient transfection experiments with ARNT and AhRR mutants examining the inhibitory mechanism of AhRR suggested a more complex mechanism than the simple mechanism of negative feedback through sequestration of ARNT to regulate AhR signaling. Recently, AhRR has been shown to act as a tumor suppressor gene in several types of cancer cells. Furthermore, epidemiological studies have found epigenetic changes and silencing of AhRR associated with exposure to cigarette smoke and cancer development. Additional studies from our laboratories have demonstrated that AhRR represses other signaling pathways including NF-κB and is capable of regulating inflammatory responses. A better understanding of the regulatory mechanisms of AhRR in AhR signaling and adverse outcome pathways leading to deregulated inflammatory responses contributing to tumor promotion and other adverse health effects is expected from future studies. This review article summarizes the characteristics of AhRR as an inhibitor of AhR activity and highlights more recent findings pointing out the role of AhRR in inflammation and tumorigenesis.

  3. Kynurenine 3-monooxygenase mediates inhibition of Th17 differentiation via catabolism of endogenous aryl hydrocarbon receptor ligands.

    PubMed

    Stephens, Geoffrey L; Wang, Qun; Swerdlow, Bonnie; Bhat, Geetha; Kolbeck, Roland; Fung, Michael

    2013-07-01

    The aryl hydrocarbon receptor (AhR) is a key transcriptional regulator of Th17-cell differentiation. Although endogenous ligands have yet to be identified, evidence suggests that tryptophan metabolites can act as agonists for the AhR. Tryptophan metabolites are abundant in circulation, so we hypothesized that cell intrinsic factors might exist to regulate the exposure of Th17 cells to AhR-dependent activities. Here, we find that Th17 cells preferentially express kynurenine 3-monooxygenase (KMO), which is an enzyme involved in catabolism of the tryptophan metabolite kynurenine. KMO inhibition, either with a specific inhibitor or via siRNA-mediated silencing, markedly increased IL-17 production in vitro, whereas IFN-γ production by Th1 cells was unaffected. Inhibition of KMO significantly exacerbated disease in a Th17-driven model of autoimmune gastritis, suggesting that expression of KMO by Th17 cells serves to limit their continuous exposure to physiological levels of endogenous AhR ligands in vivo. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The crystal structure of the AhRR-ARNT heterodimer reveals the structural basis of the repression of AhR-mediated transcription.

    PubMed

    Sakurai, Shunya; Shimizu, Toshiyuki; Ohto, Umeharu

    2017-10-27

    2,3,7,8-Tetrachlorodibenzo- p -dioxin and related compounds are extraordinarily potent environmental toxic pollutants. Most of the 2,3,7,8-tetrachlorodibenzo- p -dioxin toxicities are mediated by aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor belonging to the basic helix-loop-helix (bHLH) Per-ARNT-Sim (PAS) family. Upon ligand binding, AhR forms a heterodimer with AhR nuclear translocator (ARNT) and induces the expression of genes involved in various biological responses. One of the genes induced by AhR encodes AhR repressor (AhRR), which also forms a heterodimer with ARNT and represses the activation of AhR-dependent transcription. The control of AhR activation is critical for managing AhR-mediated diseases, but the mechanisms by which AhRR represses AhR activation remain poorly understood, because of the lack of structural information. Here, we determined the structure of the AhRR-ARNT heterodimer by X-ray crystallography, which revealed an asymmetric intertwined domain organization presenting structural features that are both conserved and distinct among bHLH-PAS family members. The structures of AhRR-ARNT and AhR-ARNT were similar in the bHLH-PAS-A region, whereas the PAS-B of ARNT in the AhRR-ARNT complex exhibited a different domain arrangement in this family reported so far. The structure clearly disclosed that AhRR competitively represses AhR binding to ARNT and target DNA and further suggested the existence of an AhRR-ARNT-specific repression mechanism. This study provides a structural basis for understanding the mechanism by which AhRR represses AhR-mediated gene transcription. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Ablating the aryl hydrocarbon receptor (AhR) in CD11c+ cells perturbs intestinal epithelium development and intestinal immunity.

    PubMed

    Chng, Song Hui; Kundu, Parag; Dominguez-Brauer, Carmen; Teo, Wei Ling; Kawajiri, Kaname; Fujii-Kuriyama, Yoshiaki; Mak, Tak Wah; Pettersson, Sven

    2016-04-12

    Diet and microbiome derived indole derivatives are known to activate the ligand induced transcription factor, the Aryl hydrocarbon Receptor (AhR). While the current understanding of AhR biology has confirmed its role in mucosal lymphocytes, its function in intestinal antigen presenting cells (APCs) is poorly understood. Here, we report that Cre-mediated deletion of AhR in CD11c-expressing cells in C57/BL6 mice is associated with altered intestinal epithelial morphogenesis in vivo. Moreover, when co-cultured with AhR-deficient DCs ex vivo, intestinal organoids showed reduced SRY (sex determining region Y)-box 9 and increased Mucin 2 expression, which correlates with reduced Paneth cells and increased goblet cell differentiation, similar to the data obtained in vivo. Further, characterization of intestinal APC subsets, devoid of AhR, revealed an expression pattern associated with aberrant intrinsic Wnt pathway regulation. At a functional level, the loss of AhR in APCs resulted in a dysfunctional epithelial barrier, associated with a more aggressive chemically induced colitis compared to wild type animals. Our results are consistent with a model whereby the AhR signalling pathway may participate in the regulation of innate immunity through intestinal epithelium development and mucosal immunity.

  6. Ligand-biased ensemble receptor docking (LigBEnD): a hybrid ligand/receptor structure-based approach

    NASA Astrophysics Data System (ADS)

    Lam, Polo C.-H.; Abagyan, Ruben; Totrov, Maxim

    2018-01-01

    Ligand docking to flexible protein molecules can be efficiently carried out through ensemble docking to multiple protein conformations, either from experimental X-ray structures or from in silico simulations. The success of ensemble docking often requires the careful selection of complementary protein conformations, through docking and scoring of known co-crystallized ligands. False positives, in which a ligand in a wrong pose achieves a better docking score than that of native pose, arise as additional protein conformations are added. In the current study, we developed a new ligand-biased ensemble receptor docking method and composite scoring function which combine the use of ligand-based atomic property field (APF) method with receptor structure-based docking. This method helps us to correctly dock 30 out of 36 ligands presented by the D3R docking challenge. For the six mis-docked ligands, the cognate receptor structures prove to be too different from the 40 available experimental Pocketome conformations used for docking and could be identified only by receptor sampling beyond experimentally explored conformational subspace.

  7. A constitutively active dioxin/aryl hydrocarbon receptor induces stomach tumors

    NASA Astrophysics Data System (ADS)

    Andersson, Patrik; McGuire, Jacqueline; Rubio, Carlos; Gradin, Katarina; Whitelaw, Murray L.; Pettersson, Sven; Hanberg, Annika; Poellinger, Lorenz

    2002-07-01

    The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding xenobiotic metabolizing enzymes. Known receptor ligands are environmental pollutants including polycyclic aromatic hydrocarbons and polychlorinated dioxins. Loss-of-function (gene-disruption) studies in mice have demonstrated that the AhR is involved in toxic effects of dioxins but have not yielded unequivocal results concerning the physiological function of the receptor. Gain-of-function studies therefore were performed to unravel the biological functions of the AhR. A constitutively active AhR expressed in transgenic mice reduced the life span of the mice and induced tumors in the glandular part of the stomach, demonstrating the oncogenic potential of the AhR and implicating the receptor in regulation of cell proliferation.

  8. Identification of a Raloxifene Analog That Promotes AhR-Mediated Apoptosis in Cancer Cells.

    PubMed

    Jang, Hyo Sang; Pearce, Martin; O'Donnell, Edmond F; Nguyen, Bach Duc; Truong, Lisa; Mueller, Monica J; Bisson, William H; Kerkvliet, Nancy I; Tanguay, Robert L; Kolluri, Siva Kumar

    2017-12-01

    We previously reported that raloxifene, an estrogen receptor modulator, is also a ligand for the aryl hydrocarbon receptor (AhR). Raloxifene induces apoptosis in estrogen receptor-negative human cancer cells through the AhR. We performed structure-activity studies with seven raloxifene analogs to better understand the structural requirements of raloxifene for induction of AhR-mediated transcriptional activity and apoptosis. We identified Y134 as a raloxifene analog that activates AhR-mediated transcriptional activity and induces apoptosis in MDA-MB-231 human triple negative breast cancer cells. Suppression of AhR expression strongly reduced apoptosis induced by Y134, indicating the requirement of AhR for Y134-induced apoptosis. Y134 also induced apoptosis in hepatoma cells without having an effect on cell cycle regulation. Toxicity testing on zebrafish embryos revealed that Y134 has a significantly better safety profile than raloxifene. Our studies also identified an analog of raloxifene that acts as a partial antagonist of the AhR, and is capable of inhibiting AhR agonist-induced transcriptional activity. We conclude that Y134 is a promising raloxifene analog for further optimization as an anti-cancer agent targeting the AhR.

  9. Pityriazepin and other potent AhR ligands isolated from Malassezia furfur yeast

    PubMed Central

    Mexia, Nikitia; Gaitanis, George; Velegraki, Aristea; Soshilov, Anatoly; Denison, Michael S.; Magiatis, Prokopios

    2015-01-01

    Malassezia furfur yeast strains isolated from diseased human skin preferentially biosynthesize indole alkaloids which can be detected in human skin and are highly potent activators of the aryl hydrocarbon receptor (AhR) and AhR-dependent gene expression. Chemical analysis of an EtOAc extract of a M. furfur strain obtained from diseased human skin and grown on L-tryptophan agar revealed several known AhR active tryptophan metabolites along with a previously unidentified compound, pityriazepin. While its structure resembled that of the known alkaloid pityriacitrin, the comprised pyridine ring had been transformed into an azepinone. The indoloazepinone scaffold of pityriazepin is extremely rare in nature and has only been reported once previously. Pityriazepin, like the other isolated compounds, was found to be a potent activator of the AhR-dependent reporter gene assays in recombinant cell lines derived from four different species, although significant species differences in relative potency was observed. The ability of pityriazepin to competitively bind to the AhR and directly stimulate AhR DNA binding classified it as a new naturally-occurring potent AhR agonist. Malassezia furfur produces an expanded collection of extremely potent naturally occurring AhR agonists, which produce their biological effects in a species-specific manner.1 PMID:25721496

  10. Malassezia yeasts produce a collection of exceptionally potent activators of the Ah (dioxin) receptor detected in diseased human skin

    PubMed Central

    Magiatis, Prokopios; Pappas, Periklis; Gaitanis, George; Mexia, Nikitia; Melliou, Eleni; Galanou, Maria; Vlachos, Christophoros; Stathopoulou, Konstantina; Skaltsounis, Alexios Leandros; Marselos, Marios; Velegraki, Aristea; Denison, Michael S.; Bassukas, Ioannis D.

    2013-01-01

    Malassezia yeasts are commensal microorganisms which under insufficiently understood conditions can become pathogenic. We have previously shown that specific strains isolated from diseased human skin can preferentially produce agonists of the aryl hydrocarbon receptor (AhR), whose activation has been linked to certain skin diseases. Investigation of skin scale extracts from patients with Malassezia associated diseases demonstrated 10–1000 fold higher AhR activating capacity than control skin extracts. LC/MS/MS analysis of the patients’ extracts revealed the presence of indirubin, 6-formylindolo[3,2-b]carbazole (FICZ), indolo[3,2-b]carbazole (ICZ), malassezin, and pityriacitrin. The same compounds were also identified in 9/12 Malassezia species culture extracts tested, connecting their presence in skin scales with this yeast. Studying the activity of the Malassezia culture-extracts and pure metabolites in HaCaT cells by Reverse Transcriptase Real-Time PCR revealed significant alterations in mRNA levels of the endogenous AhR-responsive genes Cyp1A1, Cyp1B1 and AhRR. Indirubin and FICZ activated AhR in HaCaT and human HepG2 cells with significantly higher, yet transient, potency as compared to the prototypical AhR ligand, dioxin. In loco synthesis of these highly potent AhR inducers by Malassezia yeasts could have a significant impact on skin homeostatic mechanisms and disease development. PMID:23448877

  11. Malassezia yeasts produce a collection of exceptionally potent activators of the Ah (dioxin) receptor detected in diseased human skin.

    PubMed

    Magiatis, Prokopios; Pappas, Periklis; Gaitanis, George; Mexia, Nikitia; Melliou, Eleni; Galanou, Maria; Vlachos, Christophoros; Stathopoulou, Konstantina; Skaltsounis, Alexios Leandros; Marselos, Marios; Velegraki, Aristea; Denison, Michael S; Bassukas, Ioannis D

    2013-08-01

    Malassezia yeasts are commensal microorganisms, which under insufficiently understood conditions can become pathogenic. We have previously shown that specific strains isolated from diseased human skin can preferentially produce agonists of the aryl hydrocarbon receptor (AhR), whose activation has been linked to certain skin diseases. Investigation of skin scale extracts from patients with Malassezia-associated diseases demonstrated 10- to 1,000-fold higher AhR-activating capacity than control skin extracts. Liquid chromatography-tandem mass spectrometry analysis of the patients' extracts revealed the presence of indirubin, 6-formylindolo[3,2-b]carbazole (FICZ), indolo[3,2-b]carbazole (ICZ), malassezin, and pityriacitrin. The same compounds were also identified in 9 out of 12 Malassezia species culture extracts tested, connecting their presence in skin scales with this yeast. Studying the activity of the Malassezia culture extracts and pure metabolites in HaCaT cells by reverse transcriptase real-time PCR revealed significant alterations in mRNA levels of the endogenous AhR-responsive genes Cyp1A1, Cyp1B1, and AhRR. Indirubin- and FICZ-activated AhR in HaCaT and human HepG2 cells with significantly higher, yet transient, potency as compared with the prototypical AhR ligand, dioxin. In loco synthesis of these highly potent AhR inducers by Malassezia yeasts could have a significant impact on skin homeostatic mechanisms and disease development.

  12. Increased Accuracy of Ligand Sensing by Receptor Internalization and Lateral Receptor Diffusion

    NASA Astrophysics Data System (ADS)

    Aquino, Gerardo; Endres, Robert

    2010-03-01

    Many types of cells can sense external ligand concentrations with cell-surface receptors at extremely high accuracy. Interestingly, ligand-bound receptors are often internalized, a process also known as receptor-mediated endocytosis. While internalization is involved in a vast number of important functions for the life of a cell, it was recently also suggested to increase the accuracy of sensing ligand as overcounting of the same ligand molecules is reduced. A similar role may be played by receptor diffusion om the cell membrane. Fast, lateral receptor diffusion is known to be relevant in neurotransmission initiated by release of neurotransmitter glutamate in the synaptic cleft between neurons. By binding ligand and removal by diffusion from the region of release of the neurotransmitter, diffusing receptors can be reasonably expected to reduce the local overcounting of the same ligand molecules in the region of signaling. By extending simple ligand-receptor models to out-of-equilibrium thermodynamics, we show that both receptor internalization and lateral diffusion increase the accuracy with which cells can measure ligand concentrations in the external environment. We confirm this with our model and give quantitative predictions for experimental parameters values. We give quantitative predictions, which compare favorably to experimental data of real receptors.

  13. A potential endogenous ligand for the aryl hydrocarbon receptor has potent agonist activity in vitro and in vivo.

    PubMed

    Henry, E C; Bemis, J C; Henry, O; Kende, A S; Gasiewicz, T A

    2006-06-01

    The aryl hydrocarbon receptor (AhR) is best known as a mediator of toxicity of a diverse family of xenobiotic chemicals such as dioxins and PCBs. However, many naturally occurring compounds also activate AhR. One such compound, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), was isolated from tissue and found to be potent in preliminary tests [J. Song, M. Clagett-Dame, R.E. Peterson, M.E. Hahn, W.M. Westler, R.R. Sicinski, H.F. DeLuca, Proc. Natl. Acad. Sci. USA 99 (2002) 14694-14699]. We have synthesized ITE and [(3)H]ITE and further evaluated its AhR activity in several in vitro and in vivo assays in comparison with the toxic ligand, TCDD. AhR in Hepa1c1c7 cell cytosol bound [(3)H]ITE with high affinity and the AhR.ITE complex formed in vitro bound dioxin response element (DRE) oligonucleotide as potently as TCDD.AhR. In cells treated with ITE, nuclear translocation of AhR, and induction of CYP1A1 protein and of a DRE-dependent luciferase reporter gene were observed. ITE administered to pregnant DRE-LacZ transgenic mice activated fetal AhR, observed as X-gal staining in the same sites as in TCDD-treated mice. However, unlike TCDD, ITE did not induce cleft palate or hydronephrosis. TCDD but not ITE induced thymic atrophy in young adult mice, but both ITE and TCDD caused similar loss of cells and alterations of cell profiles in cultured fetal thymi. These data demonstrate that ITE is a potent AhR agonist in cell extracts, cultured cells, and intact animals, but does not cause the toxicity associated with the more stable xenobiotic ligand, TCDD.

  14. Sensing multiple ligands with single receptor

    NASA Astrophysics Data System (ADS)

    Singh, Vijay; Nemenman, Ilya

    2015-03-01

    Cells use surface receptors to measure concentrations of external ligand molecules. Limits on the accuracy of such sensing are well-known for the scenario where concentration of one molecular species is being determined by one receptor [Endres]. However, in more realistic scenarios, a cognate (high-affinity) ligand competes with many non-cognate (low-affinity) ligands for binding to the receptor. We analyze effects of this competition on the accuracy of sensing. We show that maximum-likelihood statistical inference allows determination of concentrations of multiple ligands, cognate and non-cognate, by the same receptor concurrently. While it is unclear if traditional biochemical circuitry downstream of the receptor can implement such inference exactly, we show that an approximate inference can be performed by coupling the receptor to a kinetic proofreading cascade. We characterize the accuracy of such kinetic proofreading sensing in comparison to the exact maximum-likelihood approach. We acknowledge the support from the James S. McDonnell Foundation and the Human Frontier Science Program.

  15. Immuno-detection of dioxins using a recombinant protein of aryl hydrocarbon receptor (AhR) fused with sfGFP.

    PubMed

    Faiad, Walaa; Hanano, Abdulsamie; Kabakibi, Mohamed Maher; Abbady, Abdul Qader

    2016-06-21

    Dioxins are one of the most toxic groups of persistent organic pollutants. Their bioaccumulation through the food chain constitutes a potential risk for human health. Upon cell entry, dioxins bind specifically and firmly to the aryl hydrocarbon receptor (AhR), leading to the stimulation of several enzymes responsible for its detoxification. Dioxin/AhR interaction could be exploited as an affordable alternative to a variety of analytical methods for detecting dioxin contamination in the environment. In this work, the ligand binding domain (LBD) of the AhR was cloned downstream a superfolder form of the green fluorescent protein (sfGFP), resulting in the construct pRSET-sfGFP-AhR. High level of expressed sfGFP-AhR fusion protein (50 kDa) was recovered from the inclusion bodies of E. coli by simple solubilization with the Arginine, and purified by affinity chromatography via its N-terminal 6 × His tag. Its purity was confirmed by SDS-PAGE analysis and immunoblotting with anti-His or anti-GFP antibodies. Indirect ELISA revealed the ability of the sfGFP-AhR, but not the sfGFP, to bind to the immobilized dioxin with the possibility to detect such interaction by both its 6 × His and GFP tags,Competitive ELISA showed that anti-dioxin antibody was more sensitive to low dioxin concentrations than sfGFP-AhR. Nevertheless,the detection range of sfGFP-AhR fusion was much wider and the detection limit was of about 10 ppt (parts per trillion) of free dioxin in the tested artificial samples. this highly expressed and functional sfGFP-AhR fusion protein provides a promising molecular tool for detecting and quantifying different congeners of dioxins.

  16. Engineering death receptor ligands for cancer therapy.

    PubMed

    Wajant, Harald; Gerspach, Jeannette; Pfizenmaier, Klaus

    2013-05-28

    CD95, TNFR1, TRAILR1 and TRAILR2 belong to a subgroup of TNF receptors which is characterized by a conserved cell death-inducing protein domain that connects these receptors to the apoptotic machinery of the cell. Activation of death receptors in malignant cells attracts increasing attention as a principle to fight cancer. Besides agonistic antibodies the major way to stimulate death receptors is the use of their naturally occurring "death ligands" CD95L, TNF and TRAIL. However, dependent from the concept followed to develop a death ligand-based therapy various limiting aspects have to be taken into consideration on the way to a "bedside" usable drug. Problems arise in particular from the cell associated transmembrane nature of the death ligands, the poor serum half life of the soluble fragments derived from the transmembrane ligands, the ubiquitous expression of the death receptors and the existence of additional non-death receptors of the death ligands. Here, we summarize strategies how these limitations can be overcome by genetic engineering. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  17. ITE Suppresses Angiogenic Responses in Human Artery and Vein Endothelial Cells: Differential Roles of AhR.

    PubMed

    Li, Yan; Wang, Kai; Zou, Qing-Yun; Jiang, Yi-Zhou; Zhou, Chi; Zheng, Jing

    2017-12-01

    Aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor is involved in regulation of many essential biological processes including vascular development and angiogenesis. 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an AhR ligand, which regulates immune responses and cancer cell growth. However, the roles of the ITE/AhR pathway in mediating placental angiogenesis remains elusive. Here, we determined if ITE affected placental angiogenic responses via AhR in human umbilical vein (HUVECs) and artery endothelial (HUAECs) cells in vitro. We observed that ITE dose- and time-dependently inhibited proliferation and viability of HUAECs and HUVECs, whereas it inhibited migration of HUAECs, but not HUVECs. While AhR siRNA significantly suppressed AhR protein expression in HUVECs and HUAECs, it attenuated the ITE-inhibited angiogenic responses of HUAECs, but not HUVECs. Collectively, ITE suppressed angiogenic responses of HUAECs and HUVECs, dependent and independent of AhR, respectively. These data suggest that ITE may regulate placental angiogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. The aryl hydrocarbon receptor ligand ITE inhibits TGFβ1-induced human myofibroblast differentiation.

    PubMed

    Lehmann, Geniece M; Xi, Xia; Kulkarni, Ajit A; Olsen, Keith C; Pollock, Stephen J; Baglole, Carolyn J; Gupta, Shikha; Casey, Ann E; Huxlin, Krystel R; Sime, Patricia J; Feldon, Steven E; Phipps, Richard P

    2011-04-01

    Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  19. The Aryl Hydrocarbon Receptor Ligand ITE Inhibits TGFβ1-Induced Human Myofibroblast Differentiation

    PubMed Central

    Lehmann, Geniece M.; Xi, Xia; Kulkarni, Ajit A.; Olsen, Keith C.; Pollock, Stephen J.; Baglole, Carolyn J.; Gupta, Shikha; Casey, Ann E.; Huxlin, Krystel R.; Sime, Patricia J.; Feldon, Steven E.; Phipps, Richard P.

    2011-01-01

    Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR−/− fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent. PMID:21406171

  20. Predicting receptor-ligand pairs through kernel learning

    PubMed Central

    2011-01-01

    Background Regulation of cellular events is, often, initiated via extracellular signaling. Extracellular signaling occurs when a circulating ligand interacts with one or more membrane-bound receptors. Identification of receptor-ligand pairs is thus an important and specific form of PPI prediction. Results Given a set of disparate data sources (expression data, domain content, and phylogenetic profile) we seek to predict new receptor-ligand pairs. We create a combined kernel classifier and assess its performance with respect to the Database of Ligand-Receptor Partners (DLRP) 'golden standard' as well as the method proposed by Gertz et al. Among our findings, we discover that our predictions for the tgfβ family accurately reconstruct over 76% of the supported edges (0.76 recall and 0.67 precision) of the receptor-ligand bipartite graph defined by the DLRP "golden standard". In addition, for the tgfβ family, the combined kernel classifier is able to relatively improve upon the Gertz et al. work by a factor of approximately 1.5 when considering that our method has an F-measure of 0.71 while that of Gertz et al. has a value of 0.48. Conclusions The prediction of receptor-ligand pairings is a difficult and complex task. We have demonstrated that using kernel learning on multiple data sources provides a stronger alternative to the existing method in solving this task. PMID:21834994

  1. New ligands for melanocortin receptors.

    PubMed

    Kaelin, C B; Candille, S I; Yu, B; Jackson, P; Thompson, D A; Nix, M A; Binkley, J; Millhauser, G L; Barsh, G S

    2008-12-01

    Named originally for their effects on peripheral end organs, the melanocortin system controls a diverse set of physiological processes through a series of five G-protein-coupled receptors and several sets of small peptide ligands. The central melanocortin system plays an essential role in homeostatic regulation of body weight, in which two alternative ligands, alpha-melanocyte-stimulating hormone and agouti-related protein, stimulate and inhibit receptor signaling in several key brain regions that ultimately affect food intake and energy expenditure. Much of what we know about the relationship between central melanocortin signaling and body weight regulation stems from genetic studies. Comparative genomic studies indicate that melanocortin receptors used for controlling pigmentation and body weight regulation existed more than 500 million years ago in primitive vertebrates, but that fine-grained control of melanocortin receptors through neuropeptides and endogenous antagonists developed more recently. Recent studies based on dog coat-color genetics revealed a new class of melanocortin ligands, the beta-defensins, which reveal the potential for cross talk between the melanocortin and the immune systems.

  2. AIP mutations impair AhR signaling in pituitary adenoma patients fibroblasts and in GH3 cells.

    PubMed

    Lecoq, Anne-Lise; Viengchareun, Say; Hage, Mirella; Bouligand, Jérôme; Young, Jacques; Boutron, Audrey; Zizzari, Philippe; Lombès, Marc; Chanson, Philippe; Kamenický, Peter

    2016-05-01

    Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas through unknown molecular mechanisms. The best-known interacting partner of AIP is the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the effects of xenobiotics implicated in carcinogenesis. As 75% of AIP mutations disrupt the physical and/or functional interaction with AhR, we postulated that the tumorigenic potential of AIP mutations might result from altered AhR signaling. We evaluated the impact of AIP mutations on the AhR signaling pathway, first in fibroblasts from AIP-mutated patients with pituitary adenomas, by comparison with fibroblasts from healthy subjects, then in transfected pituitary GH3 cells. The AIP protein level in mutated fibroblasts was about half of that in cells from healthy subjects, but AhR expression was unaffected. Gene expression analyses showed significant modifications in the expression of the AhR target genes CYP1B1 and AHRR in AIP-mutated fibroblasts, both before and after stimulation with the endogenous AhR ligand kynurenine. Kynurenine increased Cyp1b1 expression to a greater extent in GH3 cells overexpressing wild type compared with cells expressing mutant AIP Knockdown of endogenous Aip in these cells attenuated Cyp1b1 induction by the AhR ligand. Both mutant AIP expression and knockdown of endogenous Aip affected the kynurenine-dependent GH secretion of GH3 cells. This study of human fibroblasts bearing endogenous heterozygous AIP mutations and transfected pituitary GH3 cells shows that AIP mutations affect the AIP protein level and alter AhR transcriptional activity in a gene- and tissue-dependent manner. © 2016 Society for Endocrinology.

  3. [Functional selectivity of opioid receptors ligands].

    PubMed

    Audet, Nicolas; Archer-Lahlou, Elodie; Richard-Lalonde, Mélissa; Piñeyro-Filpo, Graciela

    2010-01-01

    Opiates are the most effective analgesics available for the treatment of severe pain. However, their clinical use is restricted by unwanted side effects such as tolerance, physical dependence and respiratory depression. The strategy to develop new opiates with reduced side effects has mainly focused on the study and production of ligands that specifically bind to different opiate receptors subtypes. However, this strategy has not allowed the production of novel therapeutic ligands with a better side effects profile. Thus, other research strategies need to be explored. One which is receiving increasing attention is the possibility of exploiting ligand ability to stabilize different receptor conformations with distinct signalling profiles. This newly described property, termed functional selectivity, provides a potential means of directing the stimulus generated by an activated receptor towards a specific cellular response. Here we summarize evidence supporting the existence of ligand-specific active conformations for two opioid receptors subtypes (delta and mu), and analyze how functional selectivity may contribute in the production of longer lasting, better tolerated opiate analgesics. double dagger.

  4. Characterization and validation of fluorescent receptor ligands: a case study of the ionotropic serotonin receptor.

    PubMed

    Hovius, Ruud

    2013-01-01

    The application of fluorescent receptor ligands has become widespread, incited by two important reasons. "Seeing is believing"-it is possible to visualize in real time in live cells ligand-receptor interactions, and to locate the receptors with subcellular precision allowing one to follow, e.g., internalization of the ligand-receptor complex. The high sensitivity of photon detection permits observation of on the one hand receptor-ligand interactions on cells with low, native receptor abundance, and on the other of individual fluorophores unveiling the stochastic properties of single ligand-receptor complexes.The major bottlenecks that impede extensive use of fluorescent ligands are due to possible dramatic changes of the pharmacological properties of a ligand upon chemical modification and fluorophore conjugation, aggravated by the observation that different fluorophores can provoke very dissimilar effects. This makes it virtually impossible to predict beforehand which labelling strategy to use to produce a fluorescent ligand with the desired qualities.Here, we focus on the design, synthesis, and evaluation of a high-affinity fluorescent antagonist for the ionotropic serotonin type-3 receptor.

  5. Therapeutic androgen receptor ligands

    PubMed Central

    Allan, George F.; Sui, Zhihua

    2003-01-01

    In the past several years, the concept of tissue-selective nuclear receptor ligands has emerged. This concept has come to fruition with estrogens, with the successful marketing of drugs such as raloxifene. The discovery of raloxifene and other selective estrogen receptor modulators (SERMs) has raised the possibility of generating selective compounds for other pathways, including androgens (that is, selective androgen receptor modulators, or SARMs). PMID:16604181

  6. Excessive activation of AhR signaling disrupts neuronal migration in the hippocampal CA1 region in the developing mouse.

    PubMed

    Kimura, Eiki; Kubo, Ken-Ichiro; Endo, Toshihiro; Nakajima, Kazunori; Kakeyama, Masaki; Tohyama, Chiharu

    2017-01-01

    The aryl hydrocarbon receptor (AhR) avidly binds dioxin, a ubiquitous environmental contaminant. Disruption of downstream AhR signaling has been reported to alter neuronal development, and rodent offspring exposed to dioxin during gestation and lactation showed abnormalities in learning and memory, emotion, and social behavior. However, the mechanism behind the disrupted AhR signaling and developmental neurotoxicity induced by xenobiotic ligands remains elusive. Therefore, we studied how excessive AhR activation affects neuronal migration in the hippocampal CA1 region of the developing mouse brain. We transfected constitutively active (CA)-AhR, AhR, or control vector plasmids into neurons via in utero electroporation on gestational day 14 and analyzed neuronal positioning in the hippocampal CA1 region of offspring on postnatal day 14. CA-AhR transfection affected neuronal positioning, whereas no change was observed in AhR-transfected or control hippocampus. These results suggest that constitutively activated AhR signaling disrupts neuronal migration during hippocampal development. Further studies are needed to investigate whether such developmental disruption in the hippocampus leads to the abnormal cognition and behavior of rodent offspring upon maternal exposure to AhR xenobiotic ligands.

  7. Binding constant of cell adhesion receptors and substrate-immobilized ligands depends on the distribution of ligands

    NASA Astrophysics Data System (ADS)

    Li, Long; Hu, Jinglei; Xu, Guangkui; Song, Fan

    2018-01-01

    Cell-cell adhesion and the adhesion of cells to tissues and extracellular matrix, which are pivotal for immune response, tissue development, and cell locomotion, depend sensitively on the binding constant of receptor and ligand molecules anchored on the apposing surfaces. An important question remains of whether the immobilization of ligands affects the affinity of binding with cell adhesion receptors. We have investigated the adhesion of multicomponent membranes to a flat substrate coated with immobile ligands using Monte Carlo simulations of a statistical mesoscopic model with biologically relevant parameters. We find that the binding of the adhesion receptors to ligands immobilized on the substrate is strongly affected by the ligand distribution. In the case of ligand clusters, the receptor-ligand binding constant can be significantly enhanced due to the less translational entropy loss of lipid-raft domains in the model cell membranes upon the formation of additional complexes. For ligands randomly or uniformly immobilized on the substrate, the binding constant is rather decreased since the receptors localized in lipid-raft domains have to pay an energetic penalty in order to bind ligands. Our findings help to understand why cell-substrate adhesion experiments for measuring the impact of lipid rafts on the receptor-ligand interactions led to contradictory results.

  8. Aryl hydrocarbon receptor and intestinal immunity.

    PubMed

    Lamas, Bruno; Natividad, Jane M; Sokol, Harry

    2018-04-07

    Aryl hydrocarbon receptor (AhR) is a member of the basic helix-loop-helix-(bHLH) superfamily of transcription factors, which are associated with cellular responses to environmental stimuli, such as xenobiotics and oxygen levels. Unlike other members of bHLH, AhR is the only bHLH transcription factor that is known to be ligand activated. Early AhR studies focused on understanding the role of AhR in mediating the toxicity and carcinogenesis properties of the prototypic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In recent years, however, it has become apparent that, in addition to its toxicological involvement, AhR is highly receptive to a wide array of endogenous and exogenous ligands, and that its activation leads to a myriad of key host physiological functions. In this study, we review the current understanding of the functions of AhR in the mucosal immune system with a focus on its role in intestinal barrier function and intestinal immune cells, as well as in intestinal homeostasis.

  9. AhR mediates an anti-inflammatory feedback mechanism in human Langerhans cells involving FcεRI and IDO.

    PubMed

    Koch, S; Stroisch, T J; Vorac, J; Herrmann, N; Leib, N; Schnautz, S; Kirins, H; Förster, I; Weighardt, H; Bieber, T

    2017-11-01

    Aryl hydrocarbon receptor (AhR), an important regulator of immune responses, is activated by UVB irradiation in the skin. Langerhans cells (LC) in the epidermis of patients with atopic dermatitis (AD) carry the high-affinity receptor for IgE, FcεRI, and are crucially involved in the pathogenesis of AD by inducing inflammatory responses and regulating tolerogenic processes. We investigated AhR and AhR repressor (AhRR) expression and functional consequences of AhR activation in human ex vivo skin cells and in in vitro-generated LC. Epidermal cells from healthy skin were analyzed for their expression of AhR and AhRR. LC generated from CD34 + hematopoietic stem cells (CD34LC) were treated with the UV photoproduct and AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). Cell surface receptors, transcription factors, and the tolerogenic tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) were analyzed using flow cytometry and quantitative PCR. Epidermal LC and CD34LC express AhR and AhRR. AhR was also found in keratinocytes, which lack AhRR. AhR activation of LC by FICZ caused downregulation of FcεRI in CD34LC without affecting their maturation. AhR-mediated regulation of FcεRI did not involve any known transcription factors related to this receptor. Furthermore, we could show upregulation of IDO mediated by AhR engagement. Our study shows that AhR activation by FICZ reduces FcεRI and upregulates IDO expression in LC. This AhR-mediated anti-inflammatory feedback mechanism may dampen the allergen-induced inflammation in AD. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

  10. Potential involvement of placental AhR in unexplained recurrent spontaneous abortion.

    PubMed

    Wu, Y; Chen, X; Chang, X; Huang, Y J; Bao, S; He, Q; Li, Y; Zheng, J; Duan, T; Wang, K

    2016-01-01

    Recurrent spontaneous abortion (RSA) is a common complication of pregnancy. Recent studies have demonstrated that the aryl hydrocarbon receptor (AhR) might play important roles in establishing and maintaining early pregnancy. In this study, we found that placental AhR protein levels were significantly lower and placental CYP1A1 mRNA levels were higher in unexplained RSA (URSA) patients than in control subjects. The results of immunohistochemical analyzes showed that placental AhR was expressed in syncytiotrophoblast cells and that the level of AhR was markedly lower in these cells in URSA subjects than in control subjects. β-Naphthoflavone (β-NF, an AhR ligand) at 5μM significantly inhibited proliferation and migration in HTR-8/SVneo cells and was associated with the activation of AhR. Moreover, overexpressing AhR in JAR cells significantly increased CYP1A1 mRNA levels and inhibited cell migration. These results indicate that AhR is highly activated in URSA placentas and that the activation of AhR in the placenta might impair trophoblast cell proliferation and migration, possibly leading to the occurrence of URSA. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. EGF receptor ligands: recent advances.

    PubMed

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR.

  12. EGF receptor ligands: recent advances

    PubMed Central

    Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

    2016-01-01

    Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR. PMID:27635238

  13. Computational analysis of the receptor binding specificity of novel influenza A/H7N9 viruses.

    PubMed

    Zhou, Xinrui; Zheng, Jie; Ivan, Fransiskus Xaverius; Yin, Rui; Ranganathan, Shoba; Chow, Vincent T K; Kwoh, Chee-Keong

    2018-05-09

    Influenza viruses are undergoing continuous and rapid evolution. The fatal influenza A/H7N9 has drawn attention since the first wave of infections in March 2013, and raised more grave concerns with its increased potential to spread among humans. Experimental studies have revealed several host and virulence markers, indicating differential host binding preferences which can help estimate the potential of causing a pandemic. Here we systematically investigate the sequence pattern and structural characteristics of novel influenza A/H7N9 using computational approaches. The sequence analysis highlighted mutations in protein functional domains of influenza viruses. Molecular docking and molecular dynamics simulation revealed that the hemagglutinin (HA) of A/Taiwan/1/2017(H7N9) strain enhanced the binding with both avian and human receptor analogs, compared with the previous A/Shanghai/02/2013(H7N9) strain. The Molecular Mechanics - Poisson Boltzmann Surface Area (MM-PBSA) calculation revealed the change of residue-ligand interaction energy and detected the residues with conspicuous binding preference. The results are novel and specific to the emerging influenza A/Taiwan/1/2017(H7N9) strain compared with A/Shanghai/02/2013(H7N9). Its enhanced ability to bind human receptor analogs, which are abundant in the human upper respiratory tract, may be responsible for the recent outbreak. Residues showing binding preference were detected, which could facilitate monitoring the circulating influenza viruses.

  14. Ligand binding was acquired during evolution of nuclear receptors

    PubMed Central

    Escriva, Hector; Safi, Rachid; Hänni, Catherine; Langlois, Marie-Claire; Saumitou-Laprade, Pierre; Stehelin, Dominique; Capron, André; Pierce, Raymond; Laudet, Vincent

    1997-01-01

    The nuclear receptor (NR) superfamily comprises, in addition to ligand-activated transcription factors, members for which no ligand has been identified to date. We demonstrate that orphan receptors are randomly distributed in the evolutionary tree and that there is no relationship between the position of a given liganded receptor in the tree and the chemical nature of its ligand. NRs are specific to metazoans, as revealed by a screen of NR-related sequences in early- and non-metazoan organisms. The analysis of the NR gene duplication pattern during the evolution of metazoans shows that the present NR diversity arose from two waves of gene duplications. Strikingly, our results suggest that the ancestral NR was an orphan receptor that acquired ligand-binding ability during subsequent evolution. PMID:9192646

  15. Aryl Hydrocarbon Receptor Ligands in Cigarette Smoke Induce Production of Interleukin-22 to Promote Pancreatic Fibrosis in Models of Chronic Pancreatitis.

    PubMed

    Xue, Jing; Zhao, Qinglan; Sharma, Vishal; Nguyen, Linh P; Lee, Yvonne N; Pham, Kim L; Edderkaoui, Mouad; Pandol, Stephen J; Park, Walter; Habtezion, Aida

    2016-12-01

    Cigarette smoke has been identified as an independent risk factor for chronic pancreatitis (CP). Little is known about the mechanisms by which smoking promotes development of CP. We assessed the effects of aryl hydrocarbon receptor (AhR) ligands found in cigarette smoke on immune cell activation in humans and pancreatic fibrosis in animal models of CP. We obtained serum samples from patients with CP treated at Stanford University hospital and healthy individuals (controls) and isolated CD4 + T cells. Levels of interleukin-22 (IL22) were measured by enzyme-linked immunosorbent assay and smoking histories were collected. T cells from healthy nonsmokers and smokers were stimulated and incubated with AhR agonists (2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo[a]pyrene) or antagonists and analyzed by flow cytometry. Mice were given intraperitoneal injections of caerulein or saline, with or without lipopolysaccharide, to induce CP. Some mice were given intraperitoneal injections of AhR agonists at the start of caerulein injection, with or without an antibody against IL22 (anti-IL22) starting 2 weeks after the first caerulein injection, or recombinant mouse IL22 or vehicle (control) intraperitoneally 4 weeks after the first caerulein injection. Mice were exposed to normal air or cigarette smoke for 6 h/d for 7 weeks and expression of AhR gene targets was measured. Pancreata were collected from all mice and analyzed by histology and quantitative reverse transcription polymerase chain reaction. Pancreatic stellate cells and T cells were isolated and studied using immunoblot, immunofluorescence, flow cytometry, and enzyme-linked immunosorbent analyses. Mice given AhR agonists developed more severe pancreatic fibrosis (based on decreased pancreas size, histology, and increased expression of fibrosis-associated genes) than mice not given agonists after caerulein injection. In mice given saline instead of caerulein, AhR ligands did not induce fibrosis. Pancreatic T cells

  16. Ligand- and receptor-based docking with LiBELa

    NASA Astrophysics Data System (ADS)

    dos Santos Muniz, Heloisa; Nascimento, Alessandro S.

    2015-08-01

    Methodologies on molecular docking are constantly improving. The problem consists on finding an optimal interplay between the computational cost and a satisfactory physical description of ligand-receptor interaction. In pursuit of an advance in current methods we developed a mixed docking approach combining ligand- and receptor-based strategies in a docking engine, where tridimensional descriptors for shape and charge distribution of a reference ligand guide the initial placement of the docking molecule and an interaction energy-based global minimization follows. This hybrid docking was evaluated with soft-core and force field potentials taking into account ligand pose and scoring. Our approach was found to be competitive to a purely receptor-based dock resulting in improved logAUC values when evaluated with DUD and DUD-E. Furthermore, the smoothed potential as evaluated here, was not advantageous when ligand binding poses were compared to experimentally determined conformations. In conclusion we show that a combination of ligand- and receptor-based strategy docking with a force field energy model results in good reproduction of binding poses and enrichment of active molecules against decoys. This strategy is implemented in our tool, LiBELa, available to the scientific community.

  17. AhR ligands, malassezin, and indolo[3,2-b]carbazole are selectively produced by Malassezia furfur strains isolated from seborrheic dermatitis.

    PubMed

    Gaitanis, George; Magiatis, Prokopios; Stathopoulou, Konstantina; Bassukas, Ioannis D; Alexopoulos, Evangelos C; Velegraki, Aristea; Skaltsounis, Alexios-Leandros

    2008-07-01

    Malassezia yeasts are connected with seborrheic dermatitis (SD) whereas M. furfur pathogenicity is associated with the production of bioactive indoles. In this study, the production of indoles by M. furfur isolates from healthy and diseased skin was compared, the respective HPLC patterns were analyzed, and substances that are preferentially synthesized by strains isolated from SD lesions were isolated and characterized. Malassezin, pityriacitrin, indole-3-carbaldehyde, and indolo[3,2-b]carbazole (ICZ) were isolated by HPLC from extracts of M. furfur grown in L-tryptophan agar, and identified by nuclear magnetic resonance and mass spectroscopy. Of these, ICZ, a potent ligand of the aryl hydrocarbon receptor (AhR), is described for the first time to our knowledge as a M. furfur metabolite. HPLC-photodiode array detection analysis of strain extracts from 7 healthy subjects and 10 SD patients showed that M. furfur isolates from only SD patients consistently produce malassezin and ICZ. This discriminatory production of AhR agonists provides initial evidence for a previously unreported mechanism triggering development of SD and indicates that the variable pathogenicity patterns recorded for M. furfur-associated SD conditions may be attributed to selective production (P<0.001) of measurable bioactive indoles.

  18. Lineage-specific co-evolution of the Egf receptor/ligand signaling system.

    PubMed

    Laisney, Juliette A G C; Braasch, Ingo; Walter, Ronald B; Meierjohann, Svenja; Schartl, Manfred

    2010-01-27

    The epidermal growth factor receptor (Egfr) with its numerous ligands has fundamental roles in development, cell differentiation and physiology. Dysfunction of the receptor-ligand system contributes to many human malignancies. Consistent with such various tasks, the Egfr gene family has expanded during vertebrate evolution as a consequence of several rounds of whole genome duplication. Of particular interest is the effect of the fish-specific whole genome duplication (FSGD) on the ligand-receptor system, as it has supplied this largest group of vertebrates with additional opportunities for sub- and/or neofunctionalization in this signaling system. We identified the predicted components of the Egf receptor-ligand signaling system in teleost fishes (medaka, platyfish, stickleback, pufferfishes and zebrafish). We found two duplicated egfr genes, egfra and egfrb, in all available teleost genomes. Surprisingly only one copy for each of the seven Egfr ligands could be identified in most fishes, with zebrafish hbegf being the only exception. Special focus was put on medaka, for which we more closely investigated all Egf receptors and Egfr ligands. The different expression patterns of egfra, egfrb and their ligands in medaka tissues and embryo stages suggest differences in role and function. Preferential co-expression of different subsets of Egfr ligands corroborates the possible subfunctionalization and specialization of the two receptors in adult tissues. Bioinformatic analyses of the ligand-receptor interface between Egfr and its ligands show a very weak evolutionary conservation within this region. Using in vitro analyses of medaka Egfra, we could show that this receptor is only activated by medaka ligands, but not by human EGF. Altogether, our data suggest a lineage-specific Egfr/Egfr ligand co-evolution. Our data indicate that medaka Egfr signaling occurs via its two copies, Egfra and Egfrb, each of them being preferentially coexpressed with different subsets of Egfr

  19. Lineage-specific co-evolution of the Egf receptor/ligand signaling system

    PubMed Central

    2010-01-01

    Background The epidermal growth factor receptor (Egfr) with its numerous ligands has fundamental roles in development, cell differentiation and physiology. Dysfunction of the receptor-ligand system contributes to many human malignancies. Consistent with such various tasks, the Egfr gene family has expanded during vertebrate evolution as a consequence of several rounds of whole genome duplication. Of particular interest is the effect of the fish-specific whole genome duplication (FSGD) on the ligand-receptor system, as it has supplied this largest group of vertebrates with additional opportunities for sub- and/or neofunctionalization in this signaling system. Results We identified the predicted components of the Egf receptor-ligand signaling system in teleost fishes (medaka, platyfish, stickleback, pufferfishes and zebrafish). We found two duplicated egfr genes, egfra and egfrb, in all available teleost genomes. Surprisingly only one copy for each of the seven Egfr ligands could be identified in most fishes, with zebrafish hbegf being the only exception. Special focus was put on medaka, for which we more closely investigated all Egf receptors and Egfr ligands. The different expression patterns of egfra, egfrb and their ligands in medaka tissues and embryo stages suggest differences in role and function. Preferential co-expression of different subsets of Egfr ligands corroborates the possible subfunctionalization and specialization of the two receptors in adult tissues. Bioinformatic analyses of the ligand-receptor interface between Egfr and its ligands show a very weak evolutionary conservation within this region. Using in vitro analyses of medaka Egfra, we could show that this receptor is only activated by medaka ligands, but not by human EGF. Altogether, our data suggest a lineage-specific Egfr/Egfr ligand co-evolution. Conclusions Our data indicate that medaka Egfr signaling occurs via its two copies, Egfra and Egfrb, each of them being preferentially coexpressed

  20. Receptor-based 3D QSAR analysis of estrogen receptor ligands - merging the accuracy of receptor-based alignments with the computational efficiency of ligand-based methods

    NASA Astrophysics Data System (ADS)

    Sippl, Wolfgang

    2000-08-01

    One of the major challenges in computational approaches to drug design is the accurate prediction of binding affinity of biomolecules. In the present study several prediction methods for a published set of estrogen receptor ligands are investigated and compared. The binding modes of 30 ligands were determined using the docking program AutoDock and were compared with available X-ray structures of estrogen receptor-ligand complexes. On the basis of the docking results an interaction energy-based model, which uses the information of the whole ligand-receptor complex, was generated. Several parameters were modified in order to analyze their influence onto the correlation between binding affinities and calculated ligand-receptor interaction energies. The highest correlation coefficient ( r 2 = 0.617, q 2 LOO = 0.570) was obtained considering protein flexibility during the interaction energy evaluation. The second prediction method uses a combination of receptor-based and 3D quantitative structure-activity relationships (3D QSAR) methods. The ligand alignment obtained from the docking simulations was taken as basis for a comparative field analysis applying the GRID/GOLPE program. Using the interaction field derived with a water probe and applying the smart region definition (SRD) variable selection, a significant and robust model was obtained ( r 2 = 0.991, q 2 LOO = 0.921). The predictive ability of the established model was further evaluated by using a test set of six additional compounds. The comparison with the generated interaction energy-based model and with a traditional CoMFA model obtained using a ligand-based alignment ( r 2 = 0.951, q 2 LOO = 0.796) indicates that the combination of receptor-based and 3D QSAR methods is able to improve the quality of the underlying model.

  1. Towards Targeting the Aryl Hydrocarbon Receptor in Cystic Fibrosis

    PubMed Central

    Paolicelli, Giuseppe; De Luca, Antonella; Renga, Giorgia; Borghi, Monica; Pariano, Marilena; Stincardini, Claudia; Scaringi, Lucia; Ricci, Maurizio; Romani, Luigina

    2018-01-01

    Tryptophan (trp) metabolism is an important regulatory component of gut mucosal homeostasis and the microbiome. Metabolic pathways targeting the trp can lead to a myriad of metabolites, of both host and microbial origins, some of which act as endogenous low-affinity ligands for the aryl hydrocarbon receptor (AhR), a cytosolic, ligand-operated transcription factor that is involved in many biological processes, including development, cellular differentiation and proliferation, xenobiotic metabolism, and the immune response. Low-level activation of AhR by endogenous ligands is beneficial in the maintenance of immune health and intestinal homeostasis. We have defined a functional node whereby certain bacteria species contribute to host/microbial symbiosis and mucosal homeostasis. A microbial trp metabolic pathway leading to the production of indole-3-aldehyde (3-IAld) by lactobacilli provided epithelial protection while inducing antifungal resistance via the AhR/IL-22 axis. In this review, we highlight the role of AhR in inflammatory lung diseases and discuss the possible therapeutic use of AhR ligands in cystic fibrosis. PMID:29670460

  2. Modeling Multivalent Ligand-Receptor Interactions with Steric Constraints on Configurations of Cell-Surface Receptor Aggregates

    PubMed Central

    Monine, Michael I.; Posner, Richard G.; Savage, Paul B.; Faeder, James R.; Hlavacek, William S.

    2010-01-01

    Abstract We use flow cytometry to characterize equilibrium binding of a fluorophore-labeled trivalent model antigen to bivalent IgE-FcεRI complexes on RBL cells. We find that flow cytometric measurements are consistent with an equilibrium model for ligand-receptor binding in which binding sites are assumed to be equivalent and ligand-induced receptor aggregates are assumed to be acyclic. However, this model predicts extensive receptor aggregation at antigen concentrations that yield strong cellular secretory responses, which is inconsistent with the expectation that large receptor aggregates should inhibit such responses. To investigate possible explanations for this discrepancy, we evaluate four rule-based models for interaction of a trivalent ligand with a bivalent cell-surface receptor that relax simplifying assumptions of the equilibrium model. These models are simulated using a rule-based kinetic Monte Carlo approach to investigate the kinetics of ligand-induced receptor aggregation and to study how the kinetics and equilibria of ligand-receptor interaction are affected by steric constraints on receptor aggregate configurations and by the formation of cyclic receptor aggregates. The results suggest that formation of linear chains of cyclic receptor dimers may be important for generating secretory signals. Steric effects that limit receptor aggregation and transient formation of small receptor aggregates may also be important. PMID:20085718

  3. The inhibition of lung cancer cell migration by AhR-regulated autophagy

    PubMed Central

    Tsai, Chi-Hao; Li, Ching-Hao; Cheng, Yu-Wen; Lee, Chen-Chen; Liao, Po-Lin; Lin, Cheng-Hui; Huang, Shih-Hsuan; Kang, Jaw-Jou

    2017-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in multiple organs and tissues. Whereas AhR mediates the metabolism of xenobiotic and endogenous compounds, its novel function in cancer epithelial-mesenchymal transition (EMT) remains controversial. Autophagy also participates in tumour progression through its functions in cell homeostasis and facilitates adaptation to EMT progression. In the present study, we found that AhR-regulated autophagy positively modulates EMT in non-small cell lung cancer cells. The motility of A549, H1299, and CL1-5 cells were correlated with different AhR expression levels. Invasive potential and cell morphology also changed when AhR protein expression was altered. Moreover, AhR levels exerted a contrasting effect on autophagy potential. Autophagy was higher in CL1-5 and H1299 cells with lower AhR levels than in A549 cells. Both AhR overexpression and autophagy inhibition decreased CL1-5 metastasis in vivo. Furthermore, AhR promoted BNIP3 ubiquitination for proteasomal degradation. AhR silencing in A549 cells also reduced BNIP3 ubiquitination. Taken together, these results provide a novel insight into the cross-linking between AhR and autophagy, we addressed the mechanistic BNIP3 modulation by endogenous AhR, which affect cancer cell EMT progression. PMID:28195146

  4. The inhibition of lung cancer cell migration by AhR-regulated autophagy.

    PubMed

    Tsai, Chi-Hao; Li, Ching-Hao; Cheng, Yu-Wen; Lee, Chen-Chen; Liao, Po-Lin; Lin, Cheng-Hui; Huang, Shih-Hsuan; Kang, Jaw-Jou

    2017-02-14

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in multiple organs and tissues. Whereas AhR mediates the metabolism of xenobiotic and endogenous compounds, its novel function in cancer epithelial-mesenchymal transition (EMT) remains controversial. Autophagy also participates in tumour progression through its functions in cell homeostasis and facilitates adaptation to EMT progression. In the present study, we found that AhR-regulated autophagy positively modulates EMT in non-small cell lung cancer cells. The motility of A549, H1299, and CL1-5 cells were correlated with different AhR expression levels. Invasive potential and cell morphology also changed when AhR protein expression was altered. Moreover, AhR levels exerted a contrasting effect on autophagy potential. Autophagy was higher in CL1-5 and H1299 cells with lower AhR levels than in A549 cells. Both AhR overexpression and autophagy inhibition decreased CL1-5 metastasis in vivo. Furthermore, AhR promoted BNIP3 ubiquitination for proteasomal degradation. AhR silencing in A549 cells also reduced BNIP3 ubiquitination. Taken together, these results provide a novel insight into the cross-linking between AhR and autophagy, we addressed the mechanistic BNIP3 modulation by endogenous AhR, which affect cancer cell EMT progression.

  5. Aryl hydrocarbon receptor (AhR) a possible target for the treatment of skin disease.

    PubMed

    Napolitano, Maddalena; Patruno, Cataldo

    2018-07-01

    Aryl hydrocarbon receptor (AhR) is a transcription factor expressed in all skin cells type. It responds to exogenous and endogenous chemicals by inducing/repressing the expression of several genes with toxic or protective effects in a wide range of species and tissues. In healthy skin, AhR signalling contributes to keratinocytes differentiation, skin barrier function, skin pigmentation, and mediates oxidative stress. In the last years, some studies have shown that AhR seems to be involved in the pathogenesis of some skin diseases, even if the currently available data are contradictory. Indeed, while the blocking the AhR signalling activity could prevent or treat skin cancer, the AhR activation seems to be advantageous for the treatment of inflammatory skin diseases. Therefore, for its multifaceted role in skin diseases, AhR seems to be an attractive therapeutic target. Indeed, recently some molecules have been identified for the prevention of skin cancer and the treatment of inflammatory skin diseases. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. AhR activation increases IL-2 production by alloreactive CD4+ T cells initiating the differentiation of mucosal-homing Tim3+ Lag3+ Tr1 cells.

    PubMed

    Ehrlich, Allison K; Pennington, Jamie M; Tilton, Susan; Wang, Xisheng; Marshall, Nikki B; Rohlman, Diana; Funatake, Castle; Punj, Sumit; O'Donnell, Edmond; Yu, Zhen; Kolluri, Siva K; Kerkvliet, Nancy I

    2017-11-01

    Activation of the aryl hydrocarbon receptor (AhR) by immunosuppressive ligands promotes the development of regulatory T (Treg) cells. Although AhR-induced Foxp3 + Treg cells have been well studied, much less is known about the development and fate of AhR-induced Type 1 Treg (AhR-Tr1) cells. In the current study, we identified the unique transcriptional and functional changes in murine CD4 + T cells that accompany the differentiation of AhR-Tr1 cells during the CD4 + T-cell-dependent phase of an allospecific cytotoxic T lymphocyte (allo-CTL) response. AhR activation increased the expression of genes involved in T-cell activation, immune regulation and chemotaxis, as well as a global downregulation of genes involved in cell cycling.  Increased IL-2 production was responsible for the early AhR-Tr1 activation phenotype previously characterized as CD25 + CTLA4 + GITR + on day 2. The AhR-Tr1 phenotype was further defined by the coexpression of the immunoregulatory receptors Lag3 and Tim3 and non-overlapping expression of CCR4 and CCR9. Consistent with the increased expression of CCR9, real-time imaging showed enhanced migration of AhR-Tr1 cells to the lamina propria of the small intestine and colon. The discovery of mucosal imprinting of AhR-Tr1 cells provides an additional mechanism by which therapeutic AhR ligands can control immunopathology. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Molecular modeling of ligand-receptor interactions in the OR5 olfactory receptor.

    PubMed

    Singer, M S; Shepherd, G M

    1994-06-02

    Olfactory receptors belong to the superfamily of seven transmembrane domain, G protein-coupled receptors. In order to begin analysis of mechanisms of receptor activation, a computer model of the OR5 olfactory receptor has been constructed and compared with other members of this superfamily. We have tested docking of the odor molecule lyral, which is known to activate the OR5 receptor. The results point to specific ligand-binding residues on helices III through VII that form a binding pocket in the receptor. Some of these residues occupy sequence positions identical to ligand-binding residues conserved among other superfamily members. The results provide new insights into possible molecular mechanisms of odor recognition and suggest hypotheses to guide future experimental studies using site-directed mutagenesis.

  8. Stabilization of peroxisome proliferator-activated receptor alpha by the ligand.

    PubMed

    Hirotani, M; Tsukamoto, T; Bourdeaux, J; Sadano, H; Osumi, T

    2001-10-19

    Peroxisome proliferator-activated receptor (PPAR) constitutes a subfamily among a large group of ligand-activated transcription factors, the nuclear receptor superfamily. We studied the effects of ligand on the intracellular behaviors of PPARalpha. Although nuclear localization of PPARalpha was not affected by a selective ligand, Wy14643, we observed that exogenously expressed PPARalpha was rapidly degraded in HeLa cells, and the ligand significantly stabilized the protein. The stability of PPARalpha was also improved by coexpression of the heterodimer partner retinoid X receptor (RXR) alpha, and further stabilization was not observed with the ligand. These results indicate that PPARalpha is stabilized through heterodimerization with RXR, and the excess protein unpaired with RXR is rapidly turned over, if not bound by an appropriate ligand. These observations on PPARalpha are in sharp contrast to the ligand-stimulated degradation reported on PPARgamma. The ligand-dependent stabilization would have physiological significance when the synthesis of PPARalpha is elevated exceeding the available level of RXR. Copyright 2001 Academic Press.

  9. TCDD and omeprazole prime platelets through the aryl hydrocarbon receptor (AhR) non-genomic pathway.

    PubMed

    Pombo, Mónica; Lamé, Michael W; Walker, Naomi J; Huynh, Danh H; Tablin, Fern

    2015-05-19

    The role of the aryl hydrocarbon receptor (AhR) in hemostasis has recently gained increased attention. Here, we demonstrate, by qRT-PCR and western blot, that human platelets express both AhR mRNA and AhR protein. AhR protein levels increase in a dose dependent manner when incubated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or omeprazole. Treatment of platelets with puromycin blocks increased AhR protein synthesis in the presence of AhR activators. Additionally, treatment of platelets with either activator results in phosphorylation of p38MAPK and cPLA2, two key signaling molecules in platelet activation pathways. Using the AhR competitive inhibitors alpha naphthoflavone and CH-223191, we show that phosphorylation of p38MAPK is AhR dependent. Further, inhibition of p38MAPK blocks downstream cPLA2 phosphorylation induced by TCDD or omeprazole. Treatment with AhR activators results in platelet priming, as demonstrated by increased platelet aggregation, which is inhibited by AhR antagonists. Our data support a model of the platelet AhR non-genomic pathway in which treatment with AhR activators results in increased expression of the AhR, phosphorylation of p38MAPK and cPLA2, leading to platelet priming in response to agonist. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. New Insights into Ligand-Receptor Pairing and Coevolution of Relaxin Family Peptides and Their Receptors in Teleosts

    PubMed Central

    Good, Sara; Yegorov, Sergey; Martijn, Joran; Franck, Jens; Bogerd, Jan

    2012-01-01

    Relaxin-like peptides (RLN/INSL) play diverse roles in reproductive and neuroendocrine processes in placental mammals and are functionally associated with two distinct types of receptors (RXFP) for each respective function. The diversification of RLN/INSL and RXFP gene families in vertebrates was predominantly driven by whole genome duplications (2R and 3R). Teleosts preferentially retained duplicates of genes putatively involved in neuroendocrine regulation, harboring a total of 10-11 receptors and 6 ligand genes, while most mammals have equal numbers of ligands and receptors. To date, the ligand-receptor relationships of teleost Rln/Insl peptides and their receptors have largely remained unexplored. Here, we use selection analyses based on sequence data from 5 teleosts and qPCR expression data from zebrafish to explore possible ligand-receptor pairings in teleosts. We find support for the hypothesis that, with the exception of RLN, which has undergone strong positive selection in mammalian lineages, the ligand and receptor genes shared between mammals and teleosts appear to have similar pairings. On the other hand, the teleost-specific receptors show evidence of subfunctionalization. Overall, this study underscores the complexity of RLN/INSL and RXFP ligand-receptor interactions in teleosts and establishes theoretical background for further experimental work in nonmammals. PMID:23008798

  11. Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

    PubMed

    Dagnino, Sonia; Bellet, Virginie; Grimaldi, Marina; Riu, Anne; Aït-Aïssa, Sélim; Cavaillès, Vincent; Fenet, Hélène; Balaguer, Patrick

    2014-02-01

    Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

  12. Ligand recognition by RAR and RXR receptors: binding and selectivity.

    PubMed

    Sussman, Fredy; de Lera, Angel R

    2005-10-06

    Fundamental biological functions, most notably embriogenesis, cell growth, cell differentiation, and cell apoptosis, are in part regulated by a complex genomic network that starts with the binding (and activation) of retinoids to their cognate receptors, members of the superfamily of nuclear receptors. We have studied ligand recognition of retinoic receptors (RXRalpha and RARgamma) using a molecular-mechanics-based docking method. The protocol used in this work is able to rank the affinity of pairs of ligands for a single retinoid receptor, the highest values corresponding to those that adapt better to the shape of the binding site and generate the optimal set of electrostatic and apolar interactions with the receptor. Moreover, our studies shed light onto some of the energetic contributions to retinoid receptor ligand selectivity. In this regard we show that there is a difference in polarity between the binding site regions that anchor the carboxylate in RAR and RXR, which translates itself into large differences in the energy of interaction of both receptors with the same ligand. We observe that the latter energy change is canceled off by the solvation energy penalty upon binding. This energy compensation is borne out as well by experiments that address the effect of site-directed mutagenesis on ligand binding to RARgamma. The hypothesis that the difference in binding site polarity might be exploited to build RXR-selective ligands is tested with some compounds having a thiazolidinedione anchoring group.

  13. Farnesoid X Receptor and Liver X Receptor Ligands Initiate Formation of Coated Platelets

    PubMed Central

    Unsworth, Amanda J.; Bye, Alexander P.; Tannetta, Dionne S.; Desborough, Michael J.R.; Kriek, Neline; Sage, Tanya; Allan, Harriet E.; Crescente, Marilena; Yaqoob, Parveen; Warner, Timothy D.; Jones, Chris I.

    2017-01-01

    Objectives— The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. Approach and Results— We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin αIIbβ3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. Conclusions— We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of αIIbβ3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo. PMID:28619996

  14. Modeling Conformational Transitions and Energetics of Ligand Binding with the Glutamate Receptor Ligand Binding Domain

    NASA Astrophysics Data System (ADS)

    Kurnikova, Maria

    2009-03-01

    Understanding of protein motion and energetics of conformational transitions is crucial to understanding protein function. The glutamate receptor ligand binding domain (GluR2 S1S2) is a two lobe protein, which binds ligand at the interface of two lobes and undergoes conformational transition. The cleft closure conformational transition of S1S2 has been implicated in gating of the ion channel formed by the transmembrane domain of the receptor. In this study we present a composite multi-faceted theoretical analysis of the detailed mechanism of this conformational transition based on rigid cluster decomposition of the protein structure [1] and identifying hydrogen bonds that are responsible for stabilizing the closed conformation [2]. Free energy of the protein reorganization upon ligand binding was calculated using combined Thermodynamic Integration (TI) and Umbrella Sampling (US) simulations [3]. Ligand -- protein interactions in the binding cleft were analyzed using Molecular Dynamics, continuum electrostatics and QM/MM models [4]. All model calculations compare well with corresponding experimental measurements. [4pt] [1] Protein Flexibility using Constraints from Molecular Dynamics Simulations T. Mamonova, B. Hespenheide, R. Straub, M. F. Thorpe, M. G. Kurnikova , Phys. Biol., 2, S137 (2005)[0pt] [2] Theoretical Study of the Glutamate Receptor Ligand Binding Domain Flexibility and Conformational Reorganization T. Mamonova, K. Speranskiy, and M. Kurnikova , Prot.: Struct., Func., Bioinf., 73,656 (2008)[0pt] [3] Energetics of the cleft closing transition and glutamate binding in the Glutamate Receptor ligand Binding Domain T. Mamonova, M. Yonkunas, and M. Kurnikova Biochemistry 47, 11077 (2008)[0pt] [4] On the Binding Determinants of the Glutamate Agonist with the Glutamate Receptor Ligand Binding Domain K. Speranskiy and M. Kurnikova Biochemistry 44, 11208 (2005)

  15. Receptor-ligand binding sites and virtual screening.

    PubMed

    Hattotuwagama, Channa K; Davies, Matthew N; Flower, Darren R

    2006-01-01

    Within the pharmaceutical industry, the ultimate source of continuing profitability is the unremitting process of drug discovery. To be profitable, drugs must be marketable: legally novel, safe and relatively free of side effects, efficacious, and ideally inexpensive to produce. While drug discovery was once typified by a haphazard and empirical process, it is now increasingly driven by both knowledge of the receptor-mediated basis of disease and how drug molecules interact with receptors and the wider physiome. Medicinal chemistry postulates that to understand a congeneric ligand series, or set thereof, is to understand the nature and requirements of a ligand binding site. Likewise, structural molecular biology posits that to understand a binding site is to understand the nature of ligands bound therein. Reality sits somewhere between these extremes, yet subsumes them both. Complementary to rules of ligand design, arising through decades of medicinal chemistry, structural biology and computational chemistry are able to elucidate the nature of binding site-ligand interactions, facilitating, at both pragmatic and conceptual levels, the drug discovery process.

  16. The tryptophan-derived endogenous arylhydrocarbon receptor ligand 6-formylindolo[3,2-b]carbazole (FICZ) is a nanomolar UVA-photosensitizer in epidermal keratinocytes

    PubMed Central

    Williams, Joshua D.; Cabello, Christopher M.; Qiao, Shuxi; Wondrak, Georg T.

    2014-01-01

    Endogenous UVA-chromophores may act as sensitizers of oxidative stress underlying cutaneous photoaging and photocarcinogenesis, but the molecular identity of non-DNA key chromophores displaying UVA-driven photodyamic activity in human skin remains largely undefined. Here we report that 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan photoproduct and endogenous high affinity aryl hydrocarbon receptor (AhR) agonist, acts as a nanomolar photosensitizer potentiating UVA-induced oxidative stress irrespective of AhR ligand activity. In human HaCaT and primary epidermal keratinocytes, photodynamic induction of apoptosis was elicited by the combined action of solar simulated UVA and FICZ, whereas exposure to the isolated action of UVA or FICZ did not impair viability. In a human epidermal tissue reconstruct, FICZ/UVA-cotreatment caused pronounced phototoxicity inducing keratinocyte cell death, and FICZ photodynamic activity was also substantiated in a murine skin exposure model. Array analysis revealed pronounced potentiation of cellular heat shock, ER stress, and oxidative stress response gene expression observed only upon FICZ/UVA-cotreatment. FICZ photosensitization caused intracellular oxidative stress, and comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (FPG)-sensitive oxidative DNA lesions suppressible by antioxidant cotreatment. Taken together, our data demonstrate that the endogenous AhR ligand FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin. PMID:25431849

  17. 5D-QSAR for spirocyclic sigma1 receptor ligands by Quasar receptor surface modeling.

    PubMed

    Oberdorf, Christoph; Schmidt, Thomas J; Wünsch, Bernhard

    2010-07-01

    Based on a contiguous and structurally as well as biologically diverse set of 87 sigma(1) ligands, a 5D-QSAR study was conducted in which a quasi-atomistic receptor surface modeling approach (program package Quasar) was applied. The superposition of the ligands was performed with the tool Pharmacophore Elucidation (MOE-package), which takes all conformations of the ligands into account. This procedure led to four pharmacophoric structural elements with aromatic, hydrophobic, cationic and H-bond acceptor properties. Using the aligned structures a 3D-model of the ligand binding site of the sigma(1) receptor was obtained, whose general features are in good agreement with previous assumptions on the receptor structure, but revealed some novel insights since it represents the receptor surface in more detail. Thus, e.g., our model indicates the presence of an H-bond acceptor moiety in the binding site as counterpart to the ligands' cationic ammonium center, rather than a negatively charged carboxylate group. The presented QSAR model is statistically valid and represents the biological data of all tested compounds, including a test set of 21 ligands not used in the modeling process, with very good to excellent accuracy [q(2) (training set, n=66; leave 1/3 out) = 0.84, p(2) (test set, n=21)=0.64]. Moreover, the binding affinities of 13 further spirocyclic sigma(1) ligands were predicted with reasonable accuracy (mean deviation in pK(i) approximately 0.8). Thus, in addition to novel insights into the requirements for binding of spirocyclic piperidines to the sigma(1) receptor, the presented model can be used successfully in the rational design of new sigma(1) ligands. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

  18. Augmentor α and β (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand-receptor interactions.

    PubMed

    Reshetnyak, Andrey V; Murray, Phillip B; Shi, Xiarong; Mo, Elizabeth S; Mohanty, Jyotidarsini; Tome, Francisco; Bai, Hanwen; Gunel, Murat; Lax, Irit; Schlessinger, Joseph

    2015-12-29

    Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. The orphan receptor anaplastic lymphoma kinase (ALK) is one of very few RTKs that remain without a firmly established protein ligand. Here we present a novel cytokine, FAM150B, which we propose naming augmentor-α (AUG-α), as a ligand for ALK. AUG-α binds ALK with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK or the related receptor leukocyte tyrosine kinase (LTK) demonstrate that AUG-α binds and robustly activates both ALK and LTK. We show that the previously established LTK ligand FAM150A (AUG-β) is specific for LTK and only weakly binds to ALK. Furthermore, expression of AUG-α stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer partly driven by ALK. These experiments reveal the hierarchy and specificity of two cytokines as ligands for ALK and LTK and set the stage for elucidating their roles in development and disease states.

  19. Ligand Entry and Exit Pathways in the β2-adrenergic Receptor

    PubMed Central

    Wang, Ting; Duan, Yong

    2009-01-01

    The recently determined crystal structure of the human β2-adrenergic (β2AR) G-protein coupled receptor provides an excellent structural basis for exploring β2AR -ligand binding and dissociation process. Based on this crystal structure, we simulated ligand exit from the β2AR receptor by applying the random acceleration molecular dynamics (RAMD) simulation method. The simulation results showed that the extracellular opening on the receptor surface was the most frequently observed egress point (referred to as pathway A) and a few other pathways through inter-helical clefts were also observed with significantly lower frequencies. In the egress trajectories along pathway A, the D192-K305 salt bridge between the extracellular loop 2 (ECL2) and the apex of the transmembrane helix 7 (TM7) was exclusively broken. The spatial occupancy maps of the ligand computed from the 100 RAMD simulation trajectories indicated that the receptor-ligand interactions that restrained the ligand in the binding pocket were the major resistance encountered by the ligand during exit and no second barrier was notable. We next performed RAMD simulations by using a putative ligand-free conformation of the receptor as input structure. This conformation was obtained in a standard MD simulation in the absence of the ligand and it differed from the ligand-bound conformation in a hydrophobic patch bridging ECL2 and TM7 due to the rotation of F193 of ECL2. Results from the RAMD simulations with this putative ligand-free conformation suggest that the cleft formed by the hydrophobic bridge, TM2, TM3 and TM7 on the extracellular surface likely serves as a more specific ligand-entry site and the ECL2-TM7 hydrophobic junction can be partially interrupted upon the entry of ligand that pushes F193 to rotate, resulting in a conformation as observed in the ligand-bound crystal structure. These results may help design β2AR-targeting drugs with improved efficacy as well as understand the receptor subtype

  20. Characterization of cannabinoid receptor ligands in tissues natively expressing cannabinoid CB2 receptors

    PubMed Central

    Marini, Pietro; Cascio, Maria-Grazia; King, Angela; Pertwee, Roger G; Ross, Ruth A

    2013-01-01

    Background and Purpose Although cannabinoid CB2 receptor ligands have been widely characterized in recombinant systems in vitro, little pharmacological characterization has been performed in tissues natively expressing CB2 receptors. The aim of this study was to compare the pharmacology of CB2 receptor ligands in tissue natively expressing CB2 receptors (human, rat and mouse spleen) and hCB2-transfected CHO cells. Experimental Approach We tested the ability of well-known cannabinoid CB2 receptor ligands to stimulate or inhibit [35S]GTPγS binding to mouse, rat and human spleen membranes and to hCB2-transfected CHO cell membranes. cAMP assays were also performed in hCB2-CHO cells. Key Results The data presented demonstrate that: (i) CP 55,940, WIN 55,212-2 and JWH 133 behave as CB2 receptor full agonists both in spleen and hCB2-CHO cells, in both [35S]GTPγS and cAMP assays; (ii) JWH 015 behaves as a low-efficacy agonist in spleen as well as in hCB2-CHO cells when tested in the [35S]GTPγS assay, while it displays full agonism when tested in the cAMP assay using hCB2-CHO cells; (iii) (R)-AM 1241 and GW 405833 behave as agonists in the [35S]GTPγS assay using spleen, instead it behaves as a low-efficacy inverse agonist in hCB2-CHO cells; and (iv) SR 144528, AM 630 and JTE 907 behave as CB2 receptor inverse agonists in all the tissues. Conclusion and Implications Our results demonstrate that CB2 receptor ligands can display differential pharmacology when assays are conducted in tissues that natively express CB2 receptors and imply that conclusions from recombinant CB2 receptors should be treated with caution. PMID:23711022

  1. A human intervention study with foods containing natural Ah-receptor agonists does not significantly show AhR-mediated effects as measured in blood cells and urine.

    PubMed

    de Waard, Pim W J; Peijnenburg, Ad A C M; Baykus, Hakan; Aarts, Jac M M J G; Hoogenboom, Ron L A P; van Schooten, Frederik J; de Kok, Theo M C M

    2008-10-22

    Binding and activation of the aryl hydrocarbon receptor (AhR) is thought to be an essential step in the toxicity of the environmental pollutants dioxins and dioxin-like PCBs. However, also a number of natural compounds, referred to as NAhRAs (natural Ah-receptor agonists), which are present in, for example, fruits and vegetables, can bind and activate this receptor. To study their potential effects in humans, we first investigated the effect of the prototypical AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on gene expression in ex vivo exposed freshly isolated human lymphocytes, and compared the resulting gene expression profile with those caused by the well-known NAhRA indolo[3,2-b]carbazole (ICZ), originating from cruciferous vegetables, and by a hexane extract of NAhRA-containing grapefruit juice (GJE). Only ICZ induced a gene expression profile similar to TCDD in the lymphocytes, and both significantly up-regulated CYP1B1 and TIPARP (TCDD-inducible poly (ADP-ribose) polymerase) mRNA. Next, we performed a human intervention study with NAhRA-containing cruciferous vegetables and grapefruit juice. The expression of the prototypical AhR-responsive genes CYP1A1, CYP1B1 and NQO1 in whole blood cells and in freshly isolated lymphocytes was not significantly affected. Also enzyme activities of CYP1A2, CYP2A6, N-acetyltransferase 2 (NAT2) and xanthine oxidase (XO), as judged by caffeine metabolites in urine, were unaffected, except for a small down-regulation of NAT2 activity by grapefruit juice. Examination of blood plasma with DR CALUX showed a 12% increased AhR agonist activity 3 and 24 h after consumption of cruciferous vegetables, but did not show a significant effect of grapefruit juice consumption. We conclude that intake of NAhRAs from food may result in minor AhR-related effects measurable in human blood and urine.

  2. Ligand and receptor dynamics contribute to the mechanism of graded PPARγ agonism

    PubMed Central

    Hughes, Travis S.; Chalmers, Michael J.; Novick, Scott; Kuruvilla, Dana S.; Chang, Mi Ra; Kamenecka, Theodore M.; Rance, Mark; Johnson, Bruce A.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

    2011-01-01

    SUMMARY Ligand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution NMR studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators. PMID:22244763

  3. Extracting ligands from receptors by reversed targeted molecular dynamics.

    PubMed

    Wolf, Romain M

    2015-11-01

    Short targeted MD trajectories are used to expel ligands from binding sites. The expulsion is governed by a linear increase of the target RMSD value, growing from zero to an arbitrary chosen final RMSD that forces the ligand to a selected distance outside of the receptor. The RMSD lag (i.e., the difference between the imposed and the actual RMSD) can be used to follow barriers encountered by the ligand during its way out of the receptor. The force constant used for the targeted MD can transform the RMSD lag into a strain energy. Integration of the (time-dependent) strain energy over time yields a value with the dimensions of "action" (i.e, energy multiplied by time) and can serve as a measure for the overall effort required to extract the ligand from its binding site. Possibilities to compare (numerically and graphically) the randomly detected exit pathways are discussed. As an example, the method is tested on the exit of bisphenol A from the human estrogen-related receptor [Formula: see text] and of GW0072 from the peroxysome proliferator activated receptor.

  4. The AhR agonist VAF347 augments retinoic acid-induced differentiation in leukemia cells

    PubMed Central

    Ibabao, Christopher N.; Bunaciu, Rodica P.; Schaefer, Deanna M.W.; Yen, Andrew

    2015-01-01

    In binary cell-fate decisions, driving one lineage and suppressing the other are conjoined. We have previously reported that aryl hydrocarbon receptor (AhR) promotes retinoic acid (RA)-induced granulocytic differentiation of lineage bipotent HL-60 myeloblastic leukemia cells. VAF347, an AhR agonist, impairs the development of CD14+CD11b+ monocytes from granulo-monocytic (GM) stage precursors. We thus hypothesized that VAF347 propels RA-induced granulocytic differentiation and impairs D3-induced monocytic differentiation of HL-60 cells. Our results show that VAF347 enhanced RA-induced cell cycle arrest, CD11b integrin expression and neutrophil respiratory burst. Granulocytic differentiation is known to be driven by MAPK signaling events regulated by Fgr and Lyn Src-family kinases, the CD38 cell membrane receptor, the Vav1 GEF, the c-Cbl adaptor, as well as AhR, all of which are embodied in a putative signalsome. We found that the VAF347 AhR ligand regulates the signalsome. VAF347 augments RA-induced expression of AhR, Lyn, Vav1, and c-Cbl as well as p47phox. Several interactions of partners in the signalsome appear to be enhanced: Fgr interaction with c-Cbl, CD38, and with pS259c-Raf and AhR interaction with c-Cbl and Lyn. Thus, we report that, while VAF347 impedes monocytic differentiation induced by 1,25-dihydroxyvitamin D3, VAF347 promotes RA-induced differentiation. This effect seems to involve but not to be limited to Lyn, Vav1, c-Cbl, AhR, and Fgr. PMID:25941627

  5. The AhR agonist VAF347 augments retinoic acid-induced differentiation in leukemia cells.

    PubMed

    Ibabao, Christopher N; Bunaciu, Rodica P; Schaefer, Deanna M W; Yen, Andrew

    2015-01-01

    In binary cell-fate decisions, driving one lineage and suppressing the other are conjoined. We have previously reported that aryl hydrocarbon receptor (AhR) promotes retinoic acid (RA)-induced granulocytic differentiation of lineage bipotent HL-60 myeloblastic leukemia cells. VAF347, an AhR agonist, impairs the development of CD14(+)CD11b(+) monocytes from granulo-monocytic (GM) stage precursors. We thus hypothesized that VAF347 propels RA-induced granulocytic differentiation and impairs D3-induced monocytic differentiation of HL-60 cells. Our results show that VAF347 enhanced RA-induced cell cycle arrest, CD11b integrin expression and neutrophil respiratory burst. Granulocytic differentiation is known to be driven by MAPK signaling events regulated by Fgr and Lyn Src-family kinases, the CD38 cell membrane receptor, the Vav1 GEF, the c-Cbl adaptor, as well as AhR, all of which are embodied in a putative signalsome. We found that the VAF347 AhR ligand regulates the signalsome. VAF347 augments RA-induced expression of AhR, Lyn, Vav1, and c-Cbl as well as p47(phox). Several interactions of partners in the signalsome appear to be enhanced: Fgr interaction with c-Cbl, CD38, and with pS259c-Raf and AhR interaction with c-Cbl and Lyn. Thus, we report that, while VAF347 impedes monocytic differentiation induced by 1,25-dihydroxyvitamin D3, VAF347 promotes RA-induced differentiation. This effect seems to involve but not to be limited to Lyn, Vav1, c-Cbl, AhR, and Fgr.

  6. Increased accuracy of ligand sensing by receptor diffusion on cell surface

    NASA Astrophysics Data System (ADS)

    Aquino, Gerardo; Endres, Robert G.

    2010-10-01

    The physical limit with which a cell senses external ligand concentration corresponds to the perfect absorber, where all ligand particles are absorbed and overcounting of same ligand particles does not occur. Here, we analyze how the lateral diffusion of receptors on the cell membrane affects the accuracy of sensing ligand concentration. Specifically, we connect our modeling to neurotransmission in neural synapses where the diffusion of glutamate receptors is already known to refresh synaptic connections. We find that receptor diffusion indeed increases the accuracy of sensing for both the glutamate α -Amino-3-hydroxy-5-Methyl-4-isoxazolePropionic Acid (AMPA) and N -Methyl-D-aspartic Acid (NMDA) receptor, although the NMDA receptor is overall much noisier. We propose that the difference in accuracy of sensing of the two receptors can be linked to their different roles in neurotransmission. Specifically, the high accuracy in sensing glutamate is essential for the AMPA receptor to start membrane depolarization, while the NMDA receptor is believed to work in a second stage as a coincidence detector, involved in long-term potentiation and memory.

  7. Glucagon-Like Peptide-1 Receptor Ligand Interactions: Structural Cross Talk between Ligands and the Extracellular Domain

    PubMed Central

    West, Graham M.; Willard, Francis S.; Sloop, Kyle W.; Showalter, Aaron D.; Pascal, Bruce D.; Griffin, Patrick R.

    2014-01-01

    Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R) peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R). In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands. PMID:25180755

  8. Thiophene-Core Estrogen Receptor Ligands Having Superagonist Activity

    PubMed Central

    Min, Jian; Wang, Pengcheng; Srinivasan, Sathish; Nwachukwu, Jerome C.; Guo, Pu; Huang, Minjian; Carlson, Kathryn E.; Katzenellenbogen, John A.; Nettles, Kendall W.; Zhou, Hai-Bing

    2013-01-01

    To probe the importance of the heterocyclic core of estrogen receptor (ER) ligands, we prepared a series of thiophene-core ligands by Suzuki cross-coupling of aryl boronic acids with bromo-thiophenes, and we assessed their receptor binding and cell biological activities. The disposition of the phenol substituents on the thiophene core, at alternate or adjacent sites, and the nature of substituents on these phenols all contribute to binding affinity and subtype selectivity. Most of the bis(hydroxyphenyl)-thiophenes were ERβ selective, whereas the tris(hydroxyphenyl)-thiophenes were ERα selective; analogous furan-core compounds generally have lower affinity and less selectivity. Some diarylthiophenes show distinct superagonist activity in reporter gene assays, giving maximal activities 2–3 times that of estradiol, and modeling suggests that these ligands have a different interaction with a hydrogen-bonding residue in helix-11. Ligand-core modification may be a new strategy for developing ER ligands whose selectivity is based on having transcriptional activity greater than that of estradiol. PMID:23586645

  9. The aryl hydrocarbon receptor modulates acute and late mast cell responses.

    PubMed

    Sibilano, Riccardo; Frossi, Barbara; Calvaruso, Marco; Danelli, Luca; Betto, Elena; Dall'Agnese, Alessandra; Tripodo, Claudio; Colombo, Mario P; Pucillo, Carlo E; Gri, Giorgia

    2012-07-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor whose activity is modulated by xenobiotics as well as physiological ligands. These compounds may modulate inflammatory responses and contribute to the rising prevalence of allergic diseases observed in industrialized countries. Mast cells (MCs), located within tissues at the boundary of the external environment, represent a potential target of AhR ligands. In this study, we report that murine and human MCs constitutively express AhR, and its activation by the high-affinity ligand 6-formylindolo[3,2-b]carbazole (FICZ) determines a boost in degranulation. On the contrary, repeated exposure to FICZ inhibits MC degranulation. Accordingly, histamine release, in an in vivo passive systemic anaphylactic model, is exacerbated by a single dose and is attenuated by repetitive stimulation of AhR. FICZ-exposed MCs produce reactive oxygen species and IL-6 in response to cAMP-dependent signals. Moreover, AhR-activated MCs produce IL-17, a critical player in chronic inflammation and autoimmunity, suggesting a novel pathway for MC activation in the pathogenesis of these diseases. Indeed, histological analysis of patients with chronic obstructive pulmonary disease revealed an enrichment in AhR/IL-6 and AhR/IL-17 double-positive MCs within bronchial lamina propria. Thus, tissue-resident MCs could translate external chemical challenges through AhR by modulating allergic responses and contributing to the generation of inflammation-related diseases.

  10. Ligand and structure-based methodologies for the prediction of the activity of G protein-coupled receptor ligands

    NASA Astrophysics Data System (ADS)

    Costanzi, Stefano; Tikhonova, Irina G.; Harden, T. Kendall; Jacobson, Kenneth A.

    2009-11-01

    Accurate in silico models for the quantitative prediction of the activity of G protein-coupled receptor (GPCR) ligands would greatly facilitate the process of drug discovery and development. Several methodologies have been developed based on the properties of the ligands, the direct study of the receptor-ligand interactions, or a combination of both approaches. Ligand-based three-dimensional quantitative structure-activity relationships (3D-QSAR) techniques, not requiring knowledge of the receptor structure, have been historically the first to be applied to the prediction of the activity of GPCR ligands. They are generally endowed with robustness and good ranking ability; however they are highly dependent on training sets. Structure-based techniques generally do not provide the level of accuracy necessary to yield meaningful rankings when applied to GPCR homology models. However, they are essentially independent from training sets and have a sufficient level of accuracy to allow an effective discrimination between binders and nonbinders, thus qualifying as viable lead discovery tools. The combination of ligand and structure-based methodologies in the form of receptor-based 3D-QSAR and ligand and structure-based consensus models results in robust and accurate quantitative predictions. The contribution of the structure-based component to these combined approaches is expected to become more substantial and effective in the future, as more sophisticated scoring functions are developed and more detailed structural information on GPCRs is gathered.

  11. Allergic responses and aryl hydrocarbon receptor novel pathway of mast cell activation.

    PubMed

    Sibilano, Riccardo; Pucillo, Carlo E; Gri, Giorgia

    2015-01-01

    The activation of the transcription factor aryl hydrocarbon receptor (AhR) is modulated by a wide variety of xenobiotics and ligands deriving from products of metabolism. The study of the contribution of AhR to allergic diseases has gained much interest in recent years. Here we discuss the role that environmental factors and metabolic products, particularly acting on AhR-expressing mast cells (MCs), could have in the development of local allergic/atopic response. Thus, this review will cover: a brief overview of the AhR mechanism of action in the immune system; a description of different AhR ligands and their effects to IgE-mediated MC activation in the allergic response, with particular attention to the role of IL-17; a discussion about the potential involvement of AhR in immune tolerance; and a conclusion on human diseases in which direct AhR activation of MC might have a major impact. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Hexachlorobenzene induces cell proliferation, and aryl hydrocarbon receptor expression (AhR) in rat liver preneoplastic foci, and in the human hepatoma cell line HepG2. AhR is a mediator of ERK1/2 signaling, and cell cycle regulation in HCB-treated HepG2 cells.

    PubMed

    de Tomaso Portaz, Ana Clara; Caimi, Giselle Romero; Sánchez, Marcela; Chiappini, Florencia; Randi, Andrea S; Kleiman de Pisarev, Diana L; Alvarez, Laura

    2015-10-02

    Hexachlorobenzene (HCB) is a widespread environmental pollutant, and a liver tumor promoter in rodents. Depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, terminal differentiation, or apoptosis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in drug and xenobiotic metabolism. AhR can also modulate a variety of cellular and physiological processes that can affect cell proliferation and cell fate determination. The mechanisms by which AhR ligands, both exogenous and endogenous, affect these processes involve multiple interactions between AhR and other signaling pathways. In the present study, we examined the effect of HCB on cell proliferation and AhR expression, using an initiation-promotion hepatocarcinogenesis protocol in rat liver and in the human-derived hepatoma cell line, HepG2. Female Wistar rats were initiated with a single dose of 100 mg/kg of diethylnitrosamine (DEN) at the start of the experiment. Two weeks later, daily dosing of 100 mg/kg HCB was maintained for 10 weeks. Partial hepatectomy was performed 3 weeks after initiation. The number and area of glutathione S-transferase-P (GST-P)-positive foci, in the rat liver were used as biomarkers of liver precancerous lesions. Immunohistochemical staining showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells, along with enhanced AhR protein expression in hepatocytes within GST-P-positive foci of (DEN HCB) group, when compared to DEN. In a similar manner, Western blot analysis demonstrated that HCB induced PCNA and AhR protein expression in HepG2 cells. Flow cytometry assay indicated that the cells were accumulated at S and G2/M phases of the cell cycle. HCB increased cyclin D1 protein levels and ERK1/2 phosphorylation in a dose-dependent manner. Treatment of cells with a selective MEK1 inhibitor, prevented HCB-stimulatory effect on PCNA and cyclinD1, indicating that these effects

  13. Novel roles for AhR and ARNT in the regulation of alcohol dehydrogenases in human hepatic cells.

    PubMed

    Attignon, Eléonore A; Leblanc, Alix F; Le-Grand, Béatrice; Duval, Caroline; Aggerbeck, Martine; Rouach, Hélène; Blanc, Etienne B

    2017-01-01

    The mechanisms by which pollutants participate in the development of diverse pathologies are not completely understood. The pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activates the AhR (aryl hydrocarbon receptor) signaling pathway. We previously showed that TCDD (25 nM, 30 h) decreased the expression of several alcohol metabolism enzymes (cytochrome P450 2E1, alcohol dehydrogenases ADH1, 4 and 6) in differentiated human hepatic cells (HepaRG). Here, we show that, as rapidly as 8 h after treatment (25 nM TCDD) ADH expression decreased 40 % (p < 0.05). ADH1 and 4 protein levels decreased 40 and 27 %, respectively (p < 0.05), after 72 h (25 nM TCDD). The protein half-lives were not modified by TCDD which suggests transcriptional regulation of expression. The AhR antagonist CH-223191 or AhR siRNA reduced the inhibitory effect of 25 nM TCDD on ADH1A, 4 and 6 expression 50-100 % (p < 0.05). The genomic pathway (via the AhR/ARNT complex) and not the non-genomic pathway involving c-SRC mediated these effects. Other AhR ligands (3-methylcholanthrene and PCB 126) decreased ADH1B, 4 and 6 mRNAs by more than 78 and 55 %, respectively (p < 0.01). TCDD also regulated the expression of ADH4 in the HepG2 human hepatic cell line, in primary human hepatocytes and in C57BL/6J mouse liver. In conclusion, activation of the AhR/ARNT signaling pathway by AhR ligands represents a novel mechanism for regulating the expression of ADHs. These effects may be implicated in the toxicity of AhR ligands as well as in the alteration of ethanol or retinol metabolism and may be associated further with higher risk of liver diseases or/and alcohol abuse disorders.

  14. Pharmacokinetics and Pharmacodynamics of Nonsteroidal Androgen Receptor Ligands

    PubMed Central

    Gao, Wenqing; Kim, Juhyun; Dalton, James T.

    2007-01-01

    Testosterone and structurally related anabolic steroids have been used to treat hypogonadism, muscle wasting, osteoporosis, male contraception, cancer cachexia, anemia, and hormone replacement therapy in aging men or age-related frailty; while antiandrogens may be useful for treatment of conditions like acne, alopecia (male-pattern baldness), hirsutism, benign prostatic hyperplasia (BPH) and prostate cancer. However, the undesirable physicochemical and pharmacokinetic properties of steroidal androgen receptor (AR) ligands limited their clinical use. Nonsteroidal AR ligands with improved pharmacological and pharmacokinetic properties have been developed to overcome these problems. This review focuses on the pharmacokinetics, metabolism, and pharmacology of clinically used and emerging nonsteroidal AR ligands, including antagonists, agonists, and selective androgen receptor modulators. PMID:16841196

  15. An aryl hydrocarbon receptor ligand acts on dendritic cells and T cells to suppress the Th17 response in allergic rhinitis patients.

    PubMed

    Wei, Ping; Hu, Guo-Hua; Kang, Hou-Yong; Yao, Hong-Bing; Kou, Wei; Liu, Hong; Zhang, Cheng; Hong, Su-Ling

    2014-05-01

    A predominant Th17 population is a marker of allergic rhinitis (AR). The aryl hydrocarbon receptor (AhR) exhibits strong immunomodulation potential via regulation of the differentiation of T lymphocytes and dendritic cells (DCs) after activation by its ligand, such as 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). The aim of this study was to analyze the effect of AhR on Th17 differentiation by investigating the action of ITE on DCs and CD4(+) T cells from patients with AR. In all, 26 AR patients and 12 healthy controls were included in this study. The expression of interleukin (IL)-1β, IL-6, IL-10, and IL-17 in the culture supernatant and the presence of Th17 cells in CD4(+) T cells and DC-CD4(+) T-cell co-culture system were measured before and after treatment with ITE. We show that ITE significantly induced cell secretion of IL-10 and inhibited IL-1β and IL-6 production in DCs, and promoted IL-10 production and suppressed IL-17 expression in CD4(+) T cells in vitro. It also suppressed the expansion of Th17 cells in vitro. Our work demonstrates that ITE acts on DCs and CD4(+) T cells to inhibit the Th17 response that suppresses AR; the AhR-DC-Th17 axis may be an important pathway in the treatment of AR. ITE, a nontoxic AhR ligand, attenuated the Th17 response; thus, it appears to be a promising therapeutic candidate for suppressing the inflammatory responses associated with AR.

  16. Ligand-independent Dimer Formation of Epidermal Growth Factor Receptor (EGFR) Is a Step Separable from Ligand-induced EGFR Signaling

    PubMed Central

    Yu, Xiaochun; Sharma, Kailash D.; Takahashi, Tsuyoshi; Iwamoto, Ryo; Mekada, Eisuke

    2002-01-01

    Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between 835Ala and 918Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events. PMID:12134089

  17. Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities.

    PubMed

    Lew, Erin D; Oh, Jennifer; Burrola, Patrick G; Lax, Irit; Zagórska, Anna; Través, Paqui G; Schlessinger, Joseph; Lemke, Greg

    2014-09-29

    The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl. Optimal TAM signaling requires coincident TAM ligand engagement of both its receptor and the phospholipid phosphatidylserine (PtdSer): Gas6 lacking its PtdSer-binding 'Gla domain' is significantly weakened as a Tyro3/Mer agonist and is inert as an Axl agonist, even though it binds to Axl with wild-type affinity. In two settings of TAM-dependent homeostatic phagocytosis, Mer plays a predominant role while Axl is dispensable, and activation of Mer by Protein S is sufficient to drive phagocytosis.

  18. Ligand-independent pathway that controls stability of interferon alpha receptor

    PubMed Central

    Liu, Jianghuai; Plotnikov, Alexander; Banerjee, Anamika; Kumar, K.G. Suresh; Ragimbeau, Josiane; Marijanovic, Zrinka; Baker, Darren P.; Pellegrini, Sandra; Fuchs, Serge Y.

    2008-01-01

    SUMMARY Ligand-specific negative regulation of cytokine-induced signaling relies on down regulation of the cytokine receptors. Down regulation of the IFNAR1 sub-unit of the Type I interferon (IFN) receptor proceeds via lysosomal receptor proteolysis, which is triggered by ubiquitination that depends on IFNAR1 serine phosphorylation. While IFN-inducible phosphorylation, ubiquitination and degradation requires the catalytic activity of the Tyk2 Janus kinase, here we found the ligand- and Tyk2-independent pathway that promotes IFNAR1 phosphorylation, ubiquitination, and degradation when IFNAR1 is expressed at high levels. A major cellular kinase activity that is responsible for IFNAR1 phosphorylation in vitro does not depend on either ligand or Tyk2 activity. Inhibition of ligand-independent IFNAR1 degradation suppresses cell proliferation. We discuss the signaling events that might lead to ubiquitination and degradation of IFNAR1 via ligand-dependent and independent pathways and their potential physiologic significance. PMID:18166147

  19. Ligand regulation of a constitutively dimeric EGF receptor

    NASA Astrophysics Data System (ADS)

    Freed, Daniel M.; Alvarado, Diego; Lemmon, Mark A.

    2015-06-01

    Ligand-induced receptor dimerization has traditionally been viewed as the key event in transmembrane signalling by epidermal growth factor receptors (EGFRs). Here we show that the Caenorhabditis elegans EGFR orthologue LET-23 is constitutively dimeric, yet responds to its ligand LIN-3 without changing oligomerization state. SAXS and mutational analyses further reveal that the preformed dimer of the LET-23 extracellular region is mediated by its domain II dimerization arm and resembles other EGFR extracellular dimers seen in structural studies. Binding of LIN-3 induces only minor structural rearrangements in the LET-23 dimer to promote signalling. Our results therefore argue that EGFR can be regulated by allosteric changes within an existing receptor dimer--resembling signalling by insulin receptor family members, which share similar extracellular domain compositions but form covalent dimers.

  20. Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

    PubMed Central

    Coopman, K.; Wallis, R.; Robb, G.; Brown, A. J. H.; Wilkinson, G. F.; Timms, D.

    2011-01-01

    The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues. PMID:21868452

  1. The aryl hydrocarbon receptor AhR links atopic dermatitis and air pollution via induction of the neurotrophic factor artemin.

    PubMed

    Hidaka, Takanori; Ogawa, Eisaku; Kobayashi, Eri H; Suzuki, Takafumi; Funayama, Ryo; Nagashima, Takeshi; Fujimura, Taku; Aiba, Setsuya; Nakayama, Keiko; Okuyama, Ryuhei; Yamamoto, Masayuki

    2017-01-01

    Atopic dermatitis is increasing worldwide in correlation with air pollution. Various organic components of pollutants activate the transcription factor AhR (aryl hydrocarbon receptor). Through the use of AhR-CA mice, whose keratinocytes express constitutively active AhR and that develop atopic-dermatitis-like phenotypes, we identified Artn as a keratinocyte-specific AhR target gene whose product (the neurotrophic factor artemin) was responsible for epidermal hyper-innervation that led to hypersensitivity to pruritus. The activation of AhR via air pollutants induced expression of artemin, alloknesis, epidermal hyper-innervation and inflammation. AhR activation and ARTN expression were positively correlated in the epidermis of patients with atopic dermatitis. Thus, AhR in keratinocytes senses environmental stimuli and elicits an atopic-dermatitis pathology. We propose a mechanism of air-pollution-induced atopic dermatitis via activation of AhR.

  2. Tumor necrosis factor-α inhibits effects of aryl hydrocarbon receptor ligands on cell death in human lymphocytes.

    PubMed

    Ghatrehsamani, Mahdi; Soleimani, Masoud; Esfahani, Behjat A Moayedi; Shirzad, Hedayatollah; Hakemi, Mazdak G; Mossahebimohammadi, Majid; Eskandari, Nahid; Adib, Minoo

    2015-01-01

    Activation of aryl hydrocarbon receptor (AhR) leads to diverse outcome in various kinds of cells. AhR activation may induce apoptosis or prevent of apoptosis and cell death. Recent studies suggest that apoptosis effects of AhR can be modulated by inflammatory cytokine like tumor necrosis factor alpha (TNF-α). In this study, we try to investigate the possible interaction of TNF-α with the 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a ligand of AhR, on peripheral lymphocytes. Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by discontinuous density gradient centrifugation on ficoll. Isolated PBMCs were divided into four groups: Control group, TNF-α administered group, TCDD administered group, co-administered group with TCDD and TNF-α. Cells were maintained for a week in lymphocyte culture condition. Then, TNF-α was added to group 2 and 4. Finally, apoptosis and necrosis were analyzed in all samples using flowcytometry. In group 4, the mean percent of necrosis and apoptosis in TCDD treatment groups was significantly larger than other groups; (P < 0.05). Furthermore, there was no significant difference between the mean percent of cell death in TNF-α administered group and TCDD administered group (P > 0.05). However, the mean percent of cell death in co-administered group with TCDD and TNF-α was significantly lower than other groups; (P < 0.05). TNF-α could significantly inhibit effects of TCDD on lymphocytes apoptosis. Combination effects of TNF-α and TCDD on lymphocyte increase cell survival.

  3. Enhanced thyroid hormone breakdown in hepatocytes by mutual induction of the constitutive androstane receptor (CAR, NR1I3) and arylhydrocarbon receptor by benzo[a]pyrene and phenobarbital.

    PubMed

    Schraplau, Anne; Schewe, Bettina; Neuschäfer-Rube, Frank; Ringel, Sebastian; Neuber, Corinna; Kleuser, Burkhard; Püschel, Gerhard P

    2015-02-03

    Xenobiotics may interfere with the hypothalamic-pituitary-thyroid endocrine axis by inducing enzymes that inactivate thyroid hormones and thereby reduce the metabolic rate. This induction results from an activation of xeno-sensing nuclear receptors. The current study shows that benzo[a]pyrene, a frequent contaminant of processed food and activator of the arylhydrocarbon receptor (AhR) activated the promoter and induced the transcription of the nuclear receptor constitutive androstane receptor (CAR, NR1I3) in rat hepatocytes. Likewise, phenobarbital induced the AhR transcription. This mutual induction of the nuclear receptors enhanced the phenobarbital-dependent induction of the prototypic CAR target gene Cyp2b1 as well as the AhR-dependent induction of UDP-glucuronosyltransferases. In both cases, the induction by the combination of both xenobiotics was more than the sum of the induction by either substance alone. By inducing the AhR, phenobarbital enhanced the benzo[a]pyrene-dependent reduction of thyroid hormone half-life and the benzo[a]pyrene-dependent increase in the rate of thyroid hormone glucuronide formation in hepatocyte cultures. CAR ligands might thus augment the endocrine disrupting potential of AhR activators by an induction of the AhR. Copyright © 2014. Published by Elsevier Ireland Ltd.

  4. Ligand-Assisted Protein Structure (LAPS): An Experimental Paradigm for Characterizing Cannabinoid-Receptor Ligand-Binding Domains.

    PubMed

    Janero, David R; Korde, Anisha; Makriyannis, Alexandros

    2017-01-01

    Detailed characterization of the ligand-binding motifs and structure-function correlates of the principal GPCRs of the endocannabinoid-signaling system, the cannabinoid 1 (CB1R) and cannabinoid 2 (CB2R) receptors, is essential to inform the rational design of drugs that modulate CB1R- and CB2R-dependent biosignaling for therapeutic gain. We discuss herein an experimental paradigm termed "ligand-assisted protein structure" (LAPS) that affords a means of characterizing, at the amino acid level, CB1R and CB2R structural features key to ligand engagement and receptor-dependent information transmission. For this purpose, LAPS integrates three key disciplines and methodologies: (a) medicinal chemistry: design and synthesis of high-affinity, pharmacologically active probes as reporters capable of reacting irreversibly with particular amino acids at (or in the immediate vicinity of) the ligand-binding domain of the functionally active receptor; (b) molecular and cellular biology: introduction of discrete, conservative point mutations into the target GPCR and determination of their effect on probe binding and pharmacological activity; (c) analytical chemistry: identification of the site(s) of probe-GPCR interaction through focused, bottom-up, amino acid-level proteomic identification of the probe-receptor complex using liquid chromatography tandem mass spectrometry. Subsequent in silico methods including ligand docking and computational modeling provide supplementary data on the probe-receptor interaction as defined by LAPS. Examples of LAPS as applied to human CB2R orthosteric binding site characterization for a biarylpyrazole antagonist/inverse agonist and a classical cannabinoid agonist belonging to distinct chemical classes of cannabinergic compounds are given as paradigms for further application of this methodology to other therapeutic protein targets. LAPS is well positioned to complement other experimental and in silico methods in contemporary structural biology such

  5. AhR and Arnt differentially regulate NF-κB signaling and chemokine responses in human bronchial epithelial cells

    PubMed Central

    2014-01-01

    Background The aryl hydrocarbon receptor (AhR) has gradually emerged as a regulator of inflammation in the lung and other tissues. AhR may interact with the p65-subunit of the nuclear factor (NF)-κB transcription factors, but reported outcomes of AhR/NF-κB-interactions are conflicting. Some studies suggest that AhR possess pro-inflammatory activities while others suggest that AhR may be anti-inflammatory. The present study explored the impact of AhR and its binding partner AhR nuclear translocator (Arnt) on p65-activation and two differentially regulated chemokines, CXCL8 (IL-8) and CCL5 (RANTES), in human bronchial epithelial cells (BEAS-2B). Results Cells were exposed to CXCL8- and CCL5-inducing chemicals, 1-nitropyrene (1-NP) and 1-aminopyrene (1-AP) respectively, or the synthetic double-stranded RNA analogue, polyinosinic-polycytidylic acid (Poly I:C) which induced both chemokines. Only CXCL8, and not CCL5, appeared to be p65-dependent. Yet, constitutively active unligated AhR suppressed both CXCL8 and CCL5, as shown by siRNA knock-down and the AhR antagonist α-naphthoflavone. Moreover, AhR suppressed activation of p65 by TNF-α and Poly I:C as assessed by luciferase-assay and p65-phosphorylation at serine 536, without affecting basal p65-activity. In contrast, Arnt suppressed only CXCL8, but did not prevent the p65-activation directly. However, Arnt suppressed expression of the NF-κB-subunit RelB which is under transcriptional regulation by p65. Furthermore, AhR-ligands alone at high concentrations induced a moderate CXCL8-response, without affecting CCL5, but suppressed both CXCL8 and CCL5-responses by Poly I:C. Conclusion AhR and Arnt may differentially and independently regulate chemokine-responses induced by both inhaled pollutants and pulmonary infections. Constitutively active, unligated AhR suppressed the activation of p65, while Arnt may possibly interfere with the action of activated p65. Moreover, ligand-activated AhR suppressed CXCL8 and CCL5

  6. Ligand-Induced Conformational Change in the α7 Nicotinic Receptor Ligand Binding Domain

    PubMed Central

    Henchman, Richard H.; Wang, Hai-Long; Sine, Steven M.; Taylor, Palmer; McCammon, J. Andrew

    2005-01-01

    Molecular dynamics simulations of a homology model of the ligand binding domain of the α7 nicotinic receptor are conducted with a range of bound ligands to induce different conformational states. Four simulations of 15 ns each are run with no ligand, antagonist d-tubocurarine (dTC), agonist acetylcholine (ACh), and agonist ACh with potentiator Ca2+, to give insight into the conformations of the active and inactive states of the receptor and suggest the mechanism for conformational change. The main structural factor distinguishing the active and inactive states is that a more open, symmetric arrangement of the five subunits arises for the two agonist simulations, whereas a more closed and asymmetric arrangement results for the apo and dTC cases. Most of the difference arises in the lower portion of the ligand binding domain near its connection to the adjacent transmembrane domain. The transfer of the more open state to the transmembrane domain could then promote ion flow through the channel. Variation in how subunits pack together with no ligand bound appears to give rise to asymmetry in the apo case. The presence of dTC expands the receptor but induces rotations in alternate directions in adjacent subunits that lead to an asymmetric arrangement as in the apo case. Ca2+ appears to promote a slightly greater expansion in the subunits than ACh alone by stabilizing the C-loop and ACh positions. Although the simulations are unlikely to be long enough to view the full conformational changes between open and closed states, a collection of different motions at a range of length scales are observed that are likely to participate in the conformational change. PMID:15665135

  7. Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken.

    PubMed

    Cooley, James R; Yatskievych, Tatiana A; Antin, Parker B

    2014-03-01

    Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis. Copyright © 2013 Wiley Periodicals, Inc.

  8. Relationships between serum-induced AhR bioactivity or mitochondrial inhibition and circulating polychlorinated biphenyls (PCBs).

    PubMed

    Park, Wook Ha; Kang, Sora; Lee, Hong Kyu; Salihovic, Samira; Bavel, Bert van; Lind, P Monica; Pak, Youngmi Kim; Lind, Lars

    2017-08-24

    Metabolic syndrome and mitochondrial dysfunction have been linked to elevated serum levels of persistent organic pollutants (POPs). However, it is not clear which specific POPs contribute to aryl hydrocarbon receptor (AhR)-dependent bioactivity or inhibit mitochondrial function in human subjects. Here, we measured the cumulative bioactivity of AhR ligand mixture (AhR bioactivity) and the effects on mitochondrial function (ATP concentration) in recombinant Hepa1c1c7 cells incubated with raw serum samples obtained from 911 elderly subjects in the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) cohort. Plasma concentrations of 30 POPs and plastic chemicals have previously been determined in the same PIVUS subjects. Linear regression analysis demonstrated that total toxic equivalence (TEQ) values and polychlorinated biphenyls (PCBs) were significantly correlated with AhR bioactivity (positively) and ATP concentration (negatively). Serum AhR bioactivities were positively associated with some PCBs, regardless of their dioxin-like properties, but only dioxin-like PCBs stimulated AhR bioactivity. By contrast, PCBs mediated a reduction in ATP content independently of their dioxin-like properties. This study suggests that AhR bioactivity and ATP concentrations in serum-treated cells may be valuable surrogate biomarkers of POP exposure and could be useful for the estimation of the effects of POPs on human health.

  9. Structural Insights into σ₁ Receptor Interactions with Opioid Ligands by Molecular Dynamics Simulations.

    PubMed

    Kurciński, Mateusz; Jarończyk, Małgorzata; Lipiński, Piotr F J; Dobrowolski, Jan Cz; Sadlej, Joanna

    2018-02-18

    Despite considerable advances over the past years in understanding the mechanisms of action and the role of the σ₁ receptor, several questions regarding this receptor remain unanswered. This receptor has been identified as a useful target for the treatment of a diverse range of diseases, from various central nervous system disorders to cancer. The recently solved issue of the crystal structure of the σ₁ receptor has made elucidating the structure-activity relationship feasible. The interaction of seven representative opioid ligands with the crystal structure of the σ₁ receptor (PDB ID: 5HK1) was simulated for the first time using molecular dynamics (MD). Analysis of the MD trajectories has provided the receptor-ligand interaction fingerprints, combining information on the crucial receptor residues and frequency of the residue-ligand contacts. The contact frequencies and the contact maps suggest that for all studied ligands, the hydrophilic (hydrogen bonding) interactions with Glu172 are an important factor for the ligands' affinities toward the σ₁ receptor. However, the hydrophobic interactions with Tyr120, Val162, Leu105, and Ile124 also significantly contribute to the ligand-receptor interplay and, in particular, differentiate the action of the agonistic morphine from the antagonistic haloperidol.

  10. Distribution of Endo180 receptor and ligand in developing articular cartilage.

    PubMed

    Howard, M J; Chambers, M G; Mason, R M; Isacke, C M

    2004-01-01

    To investigate the expression of a novel member of the mannose receptor family, Endo180 (also known as uPARAP), and the distribution of Endo180 ligand(s) in the articular cartilage and growth plate of normal CBA mice and STR/ort mice, a well characterized model of spontaneous osteoarthritis. A polyclonal anti-Endo180 antibody was used to determine receptor expression. The Endo180 extracellular domain fused to a human immunoglobulin Fc tail was used to detect ligand. Endo180 receptor was strongly expressed in chondrocytes both in vitro and throughout the articular cartilage of young CBA and STR/ort mice. Expression decreased in older animals. In STR/ort mice with osteoarthritic lesions, no upregulation of Endo180 was detected. In the developing growth plate, Endo180 was expressed strongly by the proliferating chondrocytes. In contrast, Endo180 ligand was detected most strongly in hypertrophic zone of the growth plate and only at low levels in articular cartilage. In cultured chondrocytes, Endo180 was localized on the cell surface and in intracellular vesicles. Constitutively recycling endocytic receptors function to internalize ligand from the extracellular milieu and the ability of Endo180 to bind both glycosylated ligands and collagens suggests a role in extracellular matrix remodeling. Expression of Endo180 in articular cartilage chondrocytes of young, but not old, mice and the reciprocal expression of Endo180 and its ligands in the growth plate suggest that this receptor is involved in cartilage development but not in cartilage homeostasis. In addition, our data indicates that Endo180 does not appear to play a role in the development or progression of murine osteoarthritis.

  11. Molecular characterization of a family of ligands for eph-related tyrosine kinase receptors.

    PubMed Central

    Beckmann, M P; Cerretti, D P; Baum, P; Vanden Bos, T; James, L; Farrah, T; Kozlosky, C; Hollingsworth, T; Shilling, H; Maraskovsky, E

    1994-01-01

    A family of tyrosine kinase receptors related to the product of the eph gene has been described recently. One of these receptors, elk, has been shown to be expressed only in brain and testes. Using a direct expression cloning technique, a ligand for the elk receptor has been isolated by screening a human placenta cDNA library with a fusion protein containing the extracellular domain of the receptor. This isolated cDNA encodes a transmembrane protein. While the sequence of the ligand cDNA is unique, it is related to a previously described sequence known as B61. Northern blot analysis of human tissue mRNA showed that the elk ligand's mRNA is 3.5 kb long and is found in placenta, heart, lung, liver, skeletal muscle, kidney and pancreas. Southern blot analysis showed that the gene is highly conserved in a wide variety of species. Both elk ligand and B61 mRNAs are inducible by tumour necrosis factor in human umbilical vein endothelial cells. In addition, both proteins show promiscuity in binding to the elk and the related hek receptors. Since these two ligand sequences are similar, and since elk and hek are members of a larger family of eph-related receptor molecules, we refer to these ligands as LERKs (ligands for eph-related kinases). Images PMID:8070404

  12. T-cell expression of AhR inhibits the maintenance of pTreg cells in the gastrointestinal tract in acute GVHD.

    PubMed

    Dant, Trisha A; Lin, Kaifeng L; Bruce, Danny W; Montgomery, Stephanie A; Kolupaev, Oleg V; Bommiasamy, Hemamalini; Bixby, Lisa M; Woosley, John T; McKinnon, Karen P; Gonzalez, Frank J; Blazar, Bruce R; Vincent, Benjamin G; Coghill, James M; Serody, Jonathan S

    2017-07-20

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that affects the function and development of immune cells. Here, we show that recipient mice receiving AhR -/- T cells have improved survival and decreased acute graft-versus-host disease (aGVHD) in 2 different murine allogeneic bone marrow transplant (BMT) models. We also show that CD4 + T cells lacking AhR demonstrate reduced accumulation in secondary lymphoid tissue because of low levels of proliferation 4 days after BMT. Additionally, we found a significant increase in the quantity of peripherally induced regulatory donor T (pT reg ) cells in the colon of recipients transplanted with AhR -/- T cells 14 days after transplant. Blockade of AhR using a clinically available AhR antagonist greatly enhanced the in vitro generation of inducible T reg (iT reg ) cells from naïve CD4 + human T cells. We have identified AhR as a novel target on donor T cells that is critical to the pathogenesis of aGVHD.

  13. An Analytical Model for Determining Two-Dimensional Receptor-Ligand Kinetics

    PubMed Central

    Cheung, Luthur Siu-Lun; Konstantopoulos, Konstantinos

    2011-01-01

    Cell-cell adhesive interactions play a pivotal role in major pathophysiological vascular processes, such as inflammation, infection, thrombosis, and cancer metastasis, and are regulated by hemodynamic forces generated by blood flow. Cell adhesion is mediated by the binding of receptors to ligands, which are both anchored on two-dimensional (2-D) membranes of apposing cells. Biophysical assays have been developed to determine the unstressed (no-force) 2-D affinity but fail to disclose its dependence on force. Here we develop an analytical model to estimate the 2-D kinetics of diverse receptor-ligand pairs as a function of force, including antibody-antigen, vascular selectin-ligand, and bacterial adhesin-ligand interactions. The model can account for multiple bond interactions necessary to mediate adhesion and resist detachment amid high hemodynamic forces. Using this model, we provide a generalized biophysical interpretation of the counterintuitive force-induced stabilization of cell rolling observed by a select subset of receptor-ligand pairs with specific intrinsic kinetic properties. This study enables us to understand how single-molecule and multibond biophysics modulate the macroscopic cell behavior in diverse pathophysiological processes. PMID:21575567

  14. Probing receptor-ligand interactions by sedimentation equilibrium

    NASA Astrophysics Data System (ADS)

    Philo, John S.

    1997-05-01

    While sedimentation equilibrium is most commonly used to characterize the molecular weight and state of association of single proteins, this technique is also a very powerful tool for probing the interactions between two or more different proteins, and can characterize both the binding stoichiometry and the equilibrium constants. To resolve the complex binding interactions that can occur in such systems, it is crucial to globally fit data from many experiments to a common binding model, including samples made with different mixing ratios and a wide range of total concentration. It is often also essential to constrain the parameters during fitting so that the fits correctly reproduce the molar ratio of proteins used in making each sample. We have applied this methodology to probe mechanisms of receptor activation for a number of hematopoietic receptors and their cognate ligands, using receptor extracellular domains expressed as soluble proteins. Such data can potentially help in the design of improved or new protein therapeutics, as well as in efforts to create small- molecular mimetics of protein hormones through structure- based drug design. Sedimentation equilibrium has shown that stem cell factor, erythropoietin, and granulocyte-colony stimulating factor can each dimerize their respective receptors in solution, but the mechanism of ligand-induced receptor dimerization for these three systems are strikingly different.

  15. Xenobiotics and loss of cell adhesion drive distinct transcriptional outcomes by aryl hydrocarbon receptor signaling.

    PubMed

    Hao, Nan; Lee, Kian Leong; Furness, Sebastian G B; Bosdotter, Cecilia; Poellinger, Lorenz; Whitelaw, Murray L

    2012-12-01

    The aryl hydrocarbon receptor (AhR) is a signal-regulated transcription factor, which is canonically activated by the direct binding of xenobiotics. In addition, switching cells from adherent to suspension culture also activates the AhR, representing a nonxenobiotic, physiological activation of AhR signaling. Here, we show that the AhR is recruited to target gene enhancers in both ligand [isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439)]-treated and suspension cells, suggesting a common mechanism of target gene induction between these two routes of AhR activation. However, gene expression profiles critically differ between xenobiotic- and suspension-activated AhR signaling. Por and Cldnd1 were regulated predominantly by ligand treatments, whereas, in contrast, ApoER2 and Ganc were regulated predominantly by the suspension condition. Classic xenobiotic-metabolizing AhR targets such as Cyp1a1, Cyp1b1, and Nqo1 were regulated by both ligand and suspension conditions. Temporal expression patterns of AhR target genes were also found to vary, with examples of transient activation, transient repression, or sustained alterations in expression. Furthermore, sequence analysis coupled with chromatin immunoprecipitation assays and reporter gene analysis identified a functional xenobiotic response element (XRE) in the intron 1 of the mouse Tiparp gene, which was also bound by hypoxia-inducible factor-1α during hypoxia and features a concatemer of four XRE cores (GCGTG). Our data suggest that this XRE concatemer site concurrently regulates the expression of both the Tiparp gene and its cis antisense noncoding RNA after ligand- or suspension-induced AhR activation. This work provides novel insights into how AhR signaling drives different transcriptional programs via the ligand versus suspension modes of activation.

  16. Structure-based discovery of selective serotonin 5-HT(1B) receptor ligands.

    PubMed

    Rodríguez, David; Brea, José; Loza, María Isabel; Carlsson, Jens

    2014-08-05

    The development of safe and effective drugs relies on the discovery of selective ligands. Serotonin (5-hydroxytryptamine [5-HT]) G protein-coupled receptors are therapeutic targets for CNS disorders but are also associated with adverse drug effects. The determination of crystal structures for the 5-HT1B and 5-HT2B receptors provided an opportunity to identify subtype selective ligands using structure-based methods. From docking screens of 1.3 million compounds, 22 molecules were predicted to be selective for the 5-HT1B receptor over the 5-HT2B subtype, a requirement for safe serotonergic drugs. Nine compounds were experimentally verified as 5-HT1B-selective ligands, with up to 300-fold higher affinities for this subtype. Three of the ligands were agonists of the G protein pathway. Analysis of state-of-the-art homology models of the two 5-HT receptors revealed that the crystal structures were critical for predicting selective ligands. Our results demonstrate that structure-based screening can guide the discovery of ligands with specific selectivity profiles. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Exploring Molecular Mechanisms of Ligand Recognition by Opioid Receptors with Metadynamics†

    PubMed Central

    Provasi, Davide; Bortolato, Andrea; Filizola, Marta

    2009-01-01

    Opioid receptors are G protein-coupled receptors (GPCRs) of utmost significance in the development of potent analgesic drugs for the treatment of severe pain. An accurate evaluation at the molecular level of the ligand binding pathways into these receptors may play a key role in the design of new molecules with more desirable properties and reduced side effects. The recent characterization of high-resolution X-ray crystal structures of non-rhodopsin GPCRs for diffusible hormones and neurotransmitters presents an unprecedented opportunity to build improved homology models of opioid receptors, and to study in more detail their molecular mechanisms of ligand recognition. In this study, possible entry pathways of the non-selective antagonist naloxone (NLX) from the water environment into the well-accepted alkaloid binding pocket of a delta opioid receptor (DOR) molecular model based on the β2-adrenergic receptor crystal structure are explored using microsecond-scale well-tempered metadynamics simulations. Using as collective variables distances that account for the position of NLX and of the receptor extracellular loop 2 in relation to the DOR binding pocket, we were able to distinguish between the different states visited by the ligand (i.e., docked, undocked, and metastable bound intermediates), and to predict a free energy of binding close to experimental values after correcting for possible drawbacks of the sampling approach. The strategy employed herein holds promise for its application to the docking of diverse ligands to the opioid receptors as well as to other GPCRs. PMID:19785461

  18. Exploring molecular mechanisms of ligand recognition by opioid receptors with metadynamics.

    PubMed

    Provasi, Davide; Bortolato, Andrea; Filizola, Marta

    2009-10-27

    Opioid receptors are G protein-coupled receptors (GPCRs) of utmost significance in the development of potent analgesic drugs for the treatment of severe pain. An accurate evaluation at the molecular level of the ligand binding pathways into these receptors may play a key role in the design of new molecules with more desirable properties and reduced side effects. The recent characterization of high-resolution X-ray crystal structures of non-rhodopsin GPCRs for diffusible hormones and neurotransmitters presents an unprecedented opportunity to build improved homology models of opioid receptors, and to study in more detail their molecular mechanisms of ligand recognition. In this study, possible pathways for entry of the nonselective antagonist naloxone (NLX) from the water environment into the well-accepted alkaloid binding pocket of a delta opioid receptor (DOR) molecular model based on the beta2-adrenergic receptor crystal structure are explored using microsecond-scale well-tempered metadynamics simulations. Using as collective variables distances that account for the position of NLX and of the receptor extracellular loop 2 in relation to the DOR binding pocket, we were able to distinguish between the different states visited by the ligand (i.e., docked, undocked, and metastable bound intermediates) and to predict a free energy of binding close to experimental values after correcting for possible drawbacks of the sampling approach. The strategy employed herein holds promise for its application to the docking of diverse ligands to the opioid receptors as well as to other GPCRs.

  19. Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation

    PubMed Central

    Corrada, Dario; Soshilov, Anatoly A.; Denison, Michael S.

    2016-01-01

    The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the

  20. Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation.

    PubMed

    Corrada, Dario; Soshilov, Anatoly A; Denison, Michael S; Bonati, Laura

    2016-06-01

    The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the

  1. Dioxin Sensitivity-Related Two Critical Amino Acids of Arylhydrocarbon Receptor May Not Correlate with the Taxonomy or Phylogeny in Avian Species

    PubMed Central

    FUJISAWA, Nozomi; KAWAI, Yusuke K.; NAKAYAMA, Shouta M. M.; IKENAKA, Yoshinori; YAMAMOTO, Hideaki; ISHIZUKA, Mayumi

    2013-01-01

    ABSTRACT There are two arylhydrocarbon receptor (AhR) isoforms in birds, AhR1 and AhR2. The varying sensitivity of AhR is reported to be related to two critical amino acids at positions 325 and 381 in the AhR1 ligand-binding domain. In this study, seven avian species whose in vivo dioxin sensitivity was known, and 13 species with no data regarding their in vivo dioxin sensitivity were examined. The two critical amino acids in the ligand-binding domain were investigated in avian species, and the results were compared with the taxonomy or phylogenetic trees for the bird AhR proteins. We found that the two critical amino acids did not correlate with the taxonomy or phylogeny of these proteins, suggesting that dioxin sensitivity was independent of taxonomy. PMID:23912877

  2. Ligand-Receptor Interaction Modulates the Energy Landscape of Enzyme-Instructed Self-Assembly of Small Molecules.

    PubMed

    Haburcak, Richard; Shi, Junfeng; Du, Xuewen; Yuan, Dan; Xu, Bing

    2016-11-30

    The concurrence of enzymatic reaction and ligand-receptor interactions is common for proteins, but rare for small molecules and has yet to be explored. Here we show that ligand-receptor interaction modulates the morphology of molecular assemblies formed by enzyme-instructed assembly of small molecules. While the absence of ligand-receptor interaction allows enzymatic dephosphorylation of a precursor to generate the hydrogelator that self-assembles to form long nanofibers, the presence of the ligand-receptor interaction biases the pathway to form precipitous aggregates containing short nanofibers. While the hydrogelators self-assemble to form nanofibers or nanoribbons that are unable to bind with the ligand (i.e., vancomycin), the addition of surfactant breaks up the assemblies to restore the ligand-receptor interaction. In addition, an excess amount of the ligands can disrupt the nanofibers and result in the precipitates. As the first example of the use of ligand-receptor interaction to modulate the kinetics of enzymatic self-assembly, this work not only provides a solution to evaluate the interaction between aggregates and target molecules but also offers new insight for understanding the emergent behavior of sophisticated molecular systems having multiple and parallel processes.

  3. CoMSIA and Docking Study of Rhenium Based Estrogen Receptor Ligand Analogs

    PubMed Central

    Wolohan, Peter; Reichert, David E.

    2007-01-01

    OPLS all atom force field parameters were developed in order to model a diverse set of novel rhenium based estrogen receptor ligands whose relative binding affinities (RBA) to the estrogen receptor alpha isoform (ERα) with respect to 17β-Estradiol were available. The binding properties of these novel rhenium based organometallic complexes were studied with a combination of Comparative Molecular Similarity Indices Analysis (CoMSIA) and docking. A total of 29 estrogen receptor ligands consisting of 11 rhenium complexes and 18 organic ligands were docked inside the ligand-binding domain (LBD) of ERα utilizing the program Gold. The top ranked pose was used to construct CoMSIA models from a training set of 22 of the estrogen receptor ligands which were selected at random. In addition scoring functions from the docking runs and the polar volume (PV) were also studied to investigate their ability to predict RBA ERα. A partial least-squares analysis consisting of the CoMSIA steric, electrostatic and hydrophobic indices together with the polar volume proved sufficiently predictive having a correlation coefficient, r2, of 0.94 and a cross-validated correlation coefficient, q2, utilizing the leave one out method of 0.68. Analysis of the scoring functions from Gold showed particularly poor correlation to RBA ERα which did not improve when the rhenium complexes were extracted to leave the organic ligands. The combined CoMSIA and polar volume model ranked correctly the ligands in order of increasing RBA ERα, illustrating the utility of this method as a prescreening tool in the development of novel rhenium based estrogen receptor ligands. PMID:17280694

  4. Ligand-activated epidermal growth factor receptor (EGFR) signaling governs endocytic trafficking of unliganded receptor monomers by non-canonical phosphorylation.

    PubMed

    Tanaka, Tomohiro; Zhou, Yue; Ozawa, Tatsuhiko; Okizono, Ryuya; Banba, Ayako; Yamamura, Tomohiro; Oga, Eiji; Muraguchi, Atsushi; Sakurai, Hiroaki

    2018-02-16

    The canonical description of transmembrane receptor function is initial binding of ligand, followed by initiation of intracellular signaling and then internalization en route to degradation or recycling to the cell surface. It is known that low concentrations of extracellular ligand lead to a higher proportion of receptor that is recycled and that non-canonical mechanisms of receptor activation, including phosphorylation by the kinase p38, can induce internalization and recycling. However, no connections have been made between these pathways; i.e. it has yet to be established what happens to unbound receptors following stimulation with ligand. Here we demonstrate that a minimal level of activation of epidermal growth factor receptor (EGFR) tyrosine kinase by low levels of ligand is sufficient to fully activate downstream mitogen-activated protein kinase (MAPK) pathways, with most of the remaining unbound EGFR molecules being efficiently phosphorylated at intracellular serine/threonine residues by activated mitogen-activated protein kinase. This non-canonical, p38-mediated phosphorylation of the C-tail of EGFR, near Ser-1015, induces the clathrin-mediated endocytosis of the unliganded EGFR monomers, which occurs slightly later than the canonical endocytosis of ligand-bound EGFR dimers via tyrosine autophosphorylation. EGFR endocytosed via the non-canonical pathway is largely recycled back to the plasma membrane as functional receptors, whereas p38-independent populations are mainly sorted for lysosomal degradation. Moreover, ligand concentrations balance these endocytic trafficking pathways. These results demonstrate that ligand-activated EGFR signaling controls unliganded receptors through feedback phosphorylation, identifying a dual-mode regulation of the endocytic trafficking dynamics of EGFR. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Multivalent ligand-receptor-mediated interaction of small filled vesicles with a cellular membrane

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2017-07-01

    The ligand-receptor-mediated contacts of small sub-100-nm-sized lipid vesicles (or nanoparticles) with the cellular membrane are of interest in the contexts of cell-to-cell communication, endocytosis of membrane-coated virions, and drug (RNA) delivery. In all these cases, the interior of vesicles is filled by biologically relevant content. Despite the diversity of such systems, the corresponding ligand-receptor interaction possesses universal features. One of them is that the vesicle-membrane contacts can be accompanied by the redistribution of ligands and receptors between the contact and contact-free regions. In particular, the concentrations of ligands and receptors may become appreciably higher in the contact regions and their composition may there be different compared to that in the suspended state in the solution. A statistical model presented herein describes the corresponding distribution of various ligands and receptors and allows one to calculate the related change of the free energy with variation of the vesicle-engulfment extent. The results obtained are used to clarify the necessary conditions for the vesicle-assisted pathway of drug delivery.

  6. Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine.

    PubMed Central

    White, R; Sjöberg, M; Kalkhoven, E; Parker, M G

    1997-01-01

    The oestrogen receptor is a member of the nuclear receptor family of transcription factors which, on binding the steroid hormone 17beta-oestradiol, interacts with co-activator proteins and stimulates gene expression. Replacement of a single tyrosine in the hormone-binding domain generated activated forms of the receptor which stimulated transcription in the absence of hormone. This increased activation is related to a decrease in hydrophobicity and a reduction in size of the side chain of the amino acid with which the tyrosine is replaced. Ligand-independent, in common with ligand-dependent transcriptional activation, requires an amphipathic alpha-helix at the C-terminus of the ligand-binding domain which is essential for the interaction of the receptor with a number of potential co-activator proteins. In contrast to the wild-type protein, constitutively active receptors were able to bind both the receptor-interacting protein RIP-140 and the steroid receptor co-activator SRC-1 in a ligand-independent manner, although in the case of SRC-1 this was only evident when the receptors were prebound to DNA. We propose, therefore, that this tyrosine is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone. Modification of this residue may generate a conformational change in the ligand-binding domain of the receptor to form an interacting surface which allows the recruitment of co-activators independent of hormone binding. This suggests that this tyrosine may be a target for a different signalling pathway which forms an alternative mechanism of activating oestrogen receptor-mediated transcription. PMID:9135157

  7. Revealing a steroid receptor ligand as a unique PPAR[gamma] agonist

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

    Lin, Shengchen; Han, Ying; Shi, Yuzhe

    2012-06-28

    Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPAR{gamma} agonist, thereby uncovering a novel signaling route for this steroid drug. Similar to rosiglitazone, RU-486 modulates the expression of key PPAR{gamma} target genes and promotes adipocyte differentiation, but with a lower adipogenic activity. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPAR{gamma} ligand-binding pocket with distinctive properties and epitopes, providing the molecular mechanisms for the discrimination ofmore » RU-486 from thiazolidinediones (TZDs) drugs. Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPAR{gamma} ligands in the treatment of insulin resistance.« less

  8. Functional Expression of Aryl Hydrocarbon Receptor on Mast Cells Populating Human Endometriotic Tissues

    PubMed Central

    Orsaria, Maria; Marzinotto, Stefania; Londero, Ambrogio P; Bulfoni, Michela; Candotti, Veronica; Zanello, Andrea; Ballico, Maurizio; Mimmi, Maria C; Calcagno, Angelo; Marchesoni, Diego; Di Loreto, Carla; Beltrami, Antonio P; Cesselli, Daniela; Gri, Giorgia

    2016-01-01

    Endometriosis is an inflammatory disease characterized by the presence of ectopic endometrial tissue outside the uterus. A diffuse infiltration of mast cells (MCs) is observed throughout endometriotic lesions, but little is known about how these cells contribute to the network of molecules that modulate the growth of ectopic endometrial implants and promote endometriosis-associated inflammation. The Aryl Hydrocarbon Receptor (AhR), a transcription factor known to respond to environmental toxins and endogenous compounds, is present in MCs. In response to AhR activation, MCs produce IL-17 and reactive oxygen species, highlighting the potential impact of AhR ligands on inflammation via MCs. Here, we investigated the possibility that endometrial MCs promote an inflammatory microenvironment by sensing AhR ligands, thus sustaining endometriosis development. Using human endometriotic tissue (ET) samples, we performed the following experiments: i) examined the cytokine expression profile; ii) counted AhR-expressing MCs; iii) verified the phenotype of AhR-expressing MCs to establish whether MCs have a tolerogenic (IL-10-positive) or inflammatory (IL-17-positive) phenotype; iv) measured the presence of AhR ligands (tryptophan-derived kynurenine) and tryptophan-metabolizing enzymes (indoleamine 2,3-dioxygenase 1 (IDO1)); v) treated ET organ cultures with an AhR antagonist in vitro to measure changes in the cytokine milieu; and vi) measured the growth of endometrial stromal cells cultured with AhR-activated MC-conditioned medium. We found that ET tissue was conducive to cytokine production, orchestrating chronic inflammation and a population of AhR-expressing MCs that are both IL-17 and IL-10-positive. ET was rich in IDO1 and the AhR-ligand kynurenine compared with control tissue, possibly promoting MC activation through AhR. ET was susceptible to treatment with an AhR antagonist, and endometrial stromal cell growth was improved in the presence of soluble factors released by

  9. Functional expression of aryl hydrocarbon receptor on mast cells populating human endometriotic tissues.

    PubMed

    Mariuzzi, Laura; Domenis, Rossana; Orsaria, Maria; Marzinotto, Stefania; Londero, Ambrogio P; Bulfoni, Michela; Candotti, Veronica; Zanello, Andrea; Ballico, Maurizio; Mimmi, Maria C; Calcagno, Angelo; Marchesoni, Diego; Di Loreto, Carla; Beltrami, Antonio P; Cesselli, Daniela; Gri, Giorgia

    2016-09-01

    Endometriosis is an inflammatory disease characterized by the presence of ectopic endometrial tissue outside the uterus. A diffuse infiltration of mast cells (MCs) is observed throughout endometriotic lesions, but little is known about how these cells contribute to the network of molecules that modulate the growth of ectopic endometrial implants and promote endometriosis-associated inflammation. The aryl hydrocarbon receptor (AhR), a transcription factor known to respond to environmental toxins and endogenous compounds, is present in MCs. In response to AhR activation, MCs produce IL-17 and reactive oxygen species, highlighting the potential impact of AhR ligands on inflammation via MCs. Here, we investigated the possibility that endometrial MCs promote an inflammatory microenvironment by sensing AhR ligands, thus sustaining endometriosis development. Using human endometriotic tissue (ET) samples, we performed the following experiments: (i) examined the cytokine expression profile; (ii) counted AhR-expressing MCs; (iii) verified the phenotype of AhR-expressing MCs to establish whether MCs have a tolerogenic (IL-10-positive) or inflammatory (IL-17-positive) phenotype; (iv) measured the presence of AhR ligands (tryptophan-derived kynurenine) and tryptophan-metabolizing enzymes (indoleamine 2,3-dioxygenase 1 (IDO1)); (v) treated ET organ cultures with an AhR antagonist in vitro to measure changes in the cytokine milieu; and (vi) measured the growth of endometrial stromal cells cultured with AhR-activated MC-conditioned medium. We found that ET tissue was conducive to cytokine production, orchestrating chronic inflammation and a population of AhR-expressing MCs that are both IL-17 and IL-10-positive. ET was rich in IDO1 and the AhR-ligand kynurenine compared with control tissue, possibly promoting MC activation through AhR. ET was susceptible to treatment with an AhR antagonist, and endometrial stromal cell growth was improved in the presence of soluble factors

  10. Aromatic interactions impact ligand binding and function at serotonin 5-HT2C G protein-coupled receptors: receptor homology modelling, ligand docking, and molecular dynamics results validated by experimental studies

    NASA Astrophysics Data System (ADS)

    Córdova-Sintjago, Tania; Villa, Nancy; Fang, Lijuan; Booth, Raymond G.

    2014-02-01

    The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2 G protein-coupled receptor (GPCR) family consists of types 2A, 2B, and 2C that share ∼75% transmembrane (TM) sequence identity. Agonists for 5-HT2C receptors are under development for psychoses; whereas, at 5-HT2A receptors, antipsychotic effects are associated with antagonists - in fact, 5-HT2A agonists can cause hallucinations and 5-HT2B agonists cause cardiotoxicity. It is known that 5-HT2A TM6 residues W6.48, F6.51, and F6.52 impact ligand binding and function; however, ligand interactions with these residues at the 5-HT2C receptor have not been reported. To predict and validate molecular determinants for 5-HT2C-specific activation, results from receptor homology modelling, ligand docking, and molecular dynamics simulation studies were compared with experimental results for ligand binding and function at wild type and W6.48A, F6.51A, and F6.52A point-mutated 5-HT2C receptors.

  11. Cognitive impairments of alcoholic cirrhotic patients: correlation with endogenous benzodiazepine receptor ligands and increased affinity of platelet receptors.

    PubMed Central

    Kapczinski, F; Curran, H V; Przemioslo, R; Williams, R; Fluck, E; Fernandes, C; File, S E

    1996-01-01

    OBJECTIVES--To determine whether differences in cognitive function between alcoholic and non-alcoholic cirrhotic patients relate to differences in endogenous ligands for the benzodiazepine receptor and/or benzodiazepine binding. METHODS--Seventeen grade-I hepatic encephalopathic patients (nine alcoholic, eight non-alcoholic) were compared with 10 matched controls on plasma concentrations of endogenous ligands for the neuronal benzodiazepine receptor, benzodiazepine binding in platelets, and performance on tests of cognitive function. RESULTS--Both groups of patients were impaired on verbal recall and on reaction time tasks compared with controls; alcoholic patients were also impaired on Reitan's trails test and digit cancellation. Four of the 17 patients had detectable concentrations of endogenous benzodiazepine ligands and they were more impaired than other patients on trails and cancellation tests. The groups did not differ in the density of benzodiazepine platelet receptors, but receptor affinity was higher in alcoholic patients than in controls; furthermore, receptor affinity correlated with the time to complete the cancellation task and with reaction time. CONCLUSION--Alcoholic cirrhotic patients may have enhanced concentrations of ligands for neuronal and peripheral benzodiazepine receptors and these may contribute to cognitive impairments in these patients. PMID:8648337

  12. Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

    PubMed

    Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

    1999-02-01

    We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

  13. Non-conventional Frizzled ligands and Wnt receptors.

    PubMed

    Hendrickx, Marijke; Leyns, Luc

    2008-05-01

    The Wnt family of secreted signaling factors plays numerous roles in embryonic development and in stem cell biology. In the adult, Wnt signaling is involved in tissue homeostasis and mutations that lead to the overexpression of Wnt can be linked to cancer. Wnt signaling is transduced intracellularly by the Frizzled (Fzd) family of receptors. In the canonical pathway, accumulation of beta-catenin and the subsequent formation of a complex with T cell factors (TCF) or lymphoid enhancing factors (Lef) lead to target gene activation. The identification of Ryk as an alternative Wnt receptor and the discovery of the novel Fzd ligands Norrie disease protein (NDP) and R-Spondin, changed the traditional view of Wnts binding to Fzd receptors. Mouse R-Spondin cooperates with Wnt signaling and Low density lipoprotein (LDL) receptor related protein (LRP) to activate beta-catenin dependent gene expression and is involved in processes such as limb and placental development in the mouse. NDP is the product of the Norrie disease gene and controls vascular development in the retina, inner ear and in the female reproductive system during pregnancy. In this review a functional overview of the interactions of the different Wnt and non-Wnt ligands with the Fzd receptors is given as well as a survey of Wnts binding to Ryk and we discuss the biological significance of these interactions.

  14. Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

    NASA Technical Reports Server (NTRS)

    Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

    1995-01-01

    A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

  15. Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down.

    PubMed

    Forrester, Alison R; Elias, Martina S; Woodward, Emma L; Graham, Mark; Williams, Faith M; Reynolds, Nick J

    2014-01-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood. To elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers. Using primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E. In NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD>β-NF>ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype. Ligand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier

  16. Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down

    PubMed Central

    Forrester, Alison R.; Elias, Martina S.; Woodward, Emma L.; Graham, Mark; Williams, Faith M.; Reynolds, Nick J.

    2014-01-01

    Background 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent activator of the aryl hydrocarbon receptor (AhR) and causes chloracne in humans. The pathogenesis and role of AhR in chloracne remains incompletely understood. Objective To elucidate the mechanisms contributing to the development of the chloracne-like phenotype in a human epidermal equivalent model and identify potential biomarkers. Methods Using primary normal human epidermal keratinocytes (NHEK), we studied AhR activation by XRE-luciferase, AhR degradation and CYP1A1 induction. We treated epidermal equivalents with high affinity TCDD or two non-chloracnegens: β-naphthoflavone (β-NF) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Using Western blotting and immunochemistry for filaggrin (FLG), involucrin (INV) and transglutaminase-1 (TGM-1), we compared the effects of the ligands on keratinocyte differentiation and development of the chloracne-like phenotype by H&E. Results In NHEKs, activation of an XRE-luciferase and CYP1A1 protein induction correlated with ligand binding affinity: TCDD > β-NF > ITE. AhR degradation was induced by all ligands. In epidermal equivalents, TCDD induced a chloracne-like phenotype, whereas β-NF or ITE did not. All three ligands induced involucrin and TGM-1 protein expression in epidermal equivalents whereas FLG protein expression decreased following treatment with TCDD and β-NF. Inhibition of AhR by α-NF blocked TCDD-induced AhR activation in NHEKs and blocked phenotypic changes in epidermal equivalents; however, AhR knock down did not reproduce the phenotype. Conclusion Ligand-induced CYP1A1 and AhR degradation did not correlate with their chloracnegenic potential, indicating that neither CYP1A1 nor AhR are suitable biomarkers. Mechanistic studies showed that the TCDD-induced chloracne-like phenotype depends on AhR activation whereas AhR knock down did not appear sufficient to induce the phenotype. PMID:24161567

  17. Purinergic P2X receptors: structural models and analysis of ligand-target interaction.

    PubMed

    Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Marucci, Gabriella; Thomas, Ajiroghene; Volpini, Rosaria

    2015-01-07

    The purinergic P2X receptors are ligand-gated cation channels activated by the endogenous ligand ATP. They assemble as homo- or heterotrimers from seven cloned subtypes (P2X1-7) and all trimer subunits present a common topology consisting in intracellular N- and C- termini, two transmembrane domains and a large extracellular domain. These membrane proteins are present in virtually all mammalian tissues and regulate a large variety of responses in physio- and pathological conditions. The development of ligands that selectively activate or block specific P2X receptor subtypes hence represents a promising strategy to obtain novel pharmacological tools for the treatment of pain, cancer, inflammation, and neurological, cardiovascular, and endocrine diseases. The publication of the crystal structures of zebrafish P2X4 receptor in inactive and ATP-bound active forms provided structural data for the analysis of the receptor structure, the interpretation of mutagenesis data, and the depiction of ligand binding and receptor activation mechanism. In addition, the availability of ATP-competitive ligands presenting selectivity for P2X receptor subtypes supports the design of new potent and selective ligands with possibly improved pharmacokinetic profiles, with the final aim to obtain new drugs. This study describes molecular modelling studies performed to develop structural models of the human and rat P2X receptors in inactive and active states. These models allowed to analyse the role of some non-conserved residues at ATP binding site and to study the receptor interaction with some non-specific or subtype selective agonists and antagonists. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  18. Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system.

    PubMed

    Rovira, X; Vivó, M; Serra, J; Roche, D; Strange, P G; Giraldo, J

    2009-01-01

    Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

  19. Sigma Receptor (σR) Ligands with Antiproliferative and Anticancer Activity.

    PubMed

    Georgiadis, Markos-Orestis; Karoutzou, Olga; Foscolos, Angeliki-Sofia; Papanastasiou, Ioannis

    2017-08-25

    Sigma receptor (σR) ligands have proven to be useful as cancer diagnostics and anticancer therapeutics and their ligands have been developed as molecular probes in oncology. Moreover, various σR ligands generate cancer cell death in vitro and in vivo. These σR ligands have exhibited promising results against numerous human and rodent cancers and are investigated under preclinical and clinical study trials, indicating a new category of drugs in cancer therapy.

  20. Ligands raise the constraint that limits constitutive activation in G protein-coupled opioid receptors.

    PubMed

    Vezzi, Vanessa; Onaran, H Ongun; Molinari, Paola; Guerrini, Remo; Balboni, Gianfranco; Calò, Girolamo; Costa, Tommaso

    2013-08-16

    Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and μ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

  1. Effects of anthocyanins on the AhR-CYP1A1 signaling pathway in human hepatocytes and human cancer cell lines

    PubMed Central

    Kamenickova, Alzbeta; Anzenbacherova, Eva; Pavek, Petr; Soshilov, Anatoly A.; Denison, Michael S.; Zapletalova, Michaela; Anzenbacher, Pavel; Dvorak, Zdenek

    2013-01-01

    Anthocyanins are plant pigments occurring in flowers and berry fruits. Since a phenomenon of food-drug interactions is increasingly emerging, we examined the effects of 21 major anthocyanins and the extracts from 3 food supplements containing anthocyanins on the aryl hydrocarbon receptor (AhR) – cytochrome P450 CYP1A1 signaling pathway in human hepatocytes and human hepatic HepG2 and intestinal LS174T cancer cells. Pelargonidin-3-O-rutinoside (PEL-2) and cyanidin-3,5-O-diglucoside (CYA-3) dose-dependently activated AhR, as revealed by gene reporter assay. PEL-2 and CYA-3 induced CYP1A1 mRNA but not protein in HepG2 and LS174T cells. Neither compound induced CYP1A1 mRNA and protein in four different primary human hepatocytes cultures. The effects of PEL-2 and CYA-3 on AhR occurred by ligand-dependent and ligand-independent mechanisms, respectively, as demonstrated by ligand binding assay. In a direct enzyme inhibition assay, none of the antocyanins tested inhibited the CYP1A1 marker activity to less than 50% even at 100 µM concentration. PEL-2 and CYA-3 at 100 µM inhibited CYP1A1 to 79% and 65%, respectively. In conclusion, with exception of PEL-2 and CYA-3, there were no effects of 19 major anthocyanins and 3 food supplements containing anthocyanins on AhR-CYP1A1 signaling, implying zero potential of these compounds for food-drug interactions with respect to AhR-CYP1A1 pathway. PMID:23735880

  2. Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

    PubMed Central

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe; van Deurs, Bo; Grøvdal, Lene Melsæther

    2013-01-01

    The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown. PMID:23472148

  3. Detection of receptor ligands by monitoring selective stabilization of a Renilla luciferase-tagged, constitutively active mutant, G-protein-coupled receptor

    PubMed Central

    Ramsay, Douglas; Bevan, Nicola; Rees, Stephen; Milligan, Graeme

    2001-01-01

    The wild-type β2-adrenoceptor and a constitutively active mutant of this receptor were C-terminally tagged with luciferase from the sea pansy Renilla reniformis. C-terminal addition of Renilla luciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant β2-adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. Treatment of cells expressing constitutively active mutant β2-adrenoceptor-Renilla luciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity. The pEC50 for ligand-induced luciferase upregulation and ligand affinity to bind the receptor were highly correlated. Similar upregulation could be observed following sustained treatment with agonist ligands. These effects were only observed at a constitutively active mutant of the β2-adrenoceptor. Co-expression of the wild-type β2-adrenoceptor C-terminally tagged with the luciferase from Photinus pyralis did not result in ligand-induced upregulation of the levels of activity of this luciferase. Co-expression of the constitutively active mutant β2-adrenoceptor-Renilla luciferase and an equivalent mutant of the α1b-adrenoceptor C-terminally tagged with green fluorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. This approach offers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor. As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein-coupled receptors. PMID:11350868

  4. NcoA2-Dependent Inhibition of HIF-1α Activation Is Regulated via AhR.

    PubMed

    Tsai, Chi-Hao; Li, Ching-Hao; Liao, Po-Lin; Cheng, Yu-Wen; Lin, Cheng-Hui; Huang, Shih-Hsuan; Kang, Jaw-Jou

    2015-12-01

    High endogenous levels of aryl hydrocarbon receptor (AhR) contribute to hypoxia signaling pathway inhibition following exposure to the potent AhR ligand benzo[a]pyrene (B[a]P) and could alter cellular homeostasis and disease condition. Increasing evidence indicates that AhR might compete with AhR nuclear translocator (ARNT) for complex formation with hypoxia-inducible factor-1α (HIF-1α) for transactivation, which could alter several physiological variables. Nuclear receptor coactivator 2 (NcoA2) is a transcription coactivator that regulates transcription factor activation and inhibition of basic helix-loop-helix Per (Period)-ARNT-SIM (single-minded) (bHLH-PAS) family proteins, such as HIF-1α, ARNT, and AhR, through protein-protein interactions. In this study, we demonstrated that both hypoxia and hypoxia-mimic conditions decreased NcoA2 protein expression in HEK293T cells. Hypoxia response element (HRE) and xenobiotic-responsive element (XRE) transactivation also were downregulated with NcoA2 knockdown under hypoxic conditions. In addition, B[a]P significantly decreased NcoA2 protein expression be accompanied with AhR degradation. We next evaluated whether the absence of AhR could affect NcoA2 protein function under hypoxia-mimetic conditions. NcoA2 and HIF-1α nuclear localization decreased in both B[a]P-pretreated and AhR-knockdown HepG2 cells under hypoxia-mimic conditions. Interestingly, NcoA2 overexpression downregulated HRE transactivation by competing with HIF-1α and AhR to form protein complexes with ARNT. Both NcoA2 knockdown and overexpression inhibited endothelial cell tube formation in vitro. We also demonstrated using the in vivo plug assay that NcoA2-regulated vascularization decreased in mice. Taken together, these results revealed a biphasic role of NcoA2 between AhR and hypoxic conditions, thus providing a novel mechanism underlying the cross talk between AhR and hypoxia that affects disease development and progression. © The Author 2015

  5. Discriminative stimulus effects of the imidazoline I2 receptor ligands BU224 and phenyzoline in rats.

    PubMed

    Qiu, Yanyan; Zhang, Yanan; Li, Jun-Xu

    2015-02-15

    Although imidazoline I2 receptor ligands have been used as discriminative stimuli, the role of efficacy of I2 receptor ligands as a critical determinant in drug discrimination has not been explored. This study characterized the discriminative stimulus effects of selective imidazoline I2 receptor ligands BU224 (a low-efficacy I2 receptor ligand) and phenyzoline (a higher efficacy I2 receptor ligand) in rats. Two groups of male Sprague-Dawley rats were trained to discriminate 5.6mg/kg BU224 or 32mg/kg phenyzoline (i.p.) from their vehicle in a two-lever food-reinforced drug discrimination procedure, respectively. All rats acquired the discriminations after an average of 18 (BU224) and 56 (phenyzoline) training sessions, respectively. BU224 and phenyzoline completely substituted for one another symmetrically. Several I2 receptor ligands (tracizoline, CR4056, RS45041, and idazoxan) all occasioned>80% drug-associated lever responding in both discriminations. The I2 receptor ligand 2-BFI and a monoamine oxidase inhibitor harmane occasioned>80% drug-associated lever responding in rats discriminating BU224. Other drugs that occasioned partial or less substitution to BU224 cue included clonidine, methamphetamine, ketamine, morphine, methadone and agmatine. Clonidine, methamphetamine and morphine also only produced partial substitution to phenyzoline cue. Naltrexone, dopamine D2 receptor antagonist haloperidol and serotonin (5-HT)2A receptor antagonist MDL100907 failed to alter the discriminative stimulus effects of BU224 or phenyzoline. Combined, these results are the first to demonstrate that BU224 and phenyzoline can serve as discriminative stimuli and that the low-efficacy I2 receptor ligand BU224 shares similar discriminative stimulus effects with higher-efficacy I2 receptor ligands such as phenyzoline and 2-BFI. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Accelerated molecular dynamics simulations of ligand binding to a muscarinic G-protein-coupled receptor.

    PubMed

    Kappel, Kalli; Miao, Yinglong; McCammon, J Andrew

    2015-11-01

    Elucidating the detailed process of ligand binding to a receptor is pharmaceutically important for identifying druggable binding sites. With the ability to provide atomistic detail, computational methods are well poised to study these processes. Here, accelerated molecular dynamics (aMD) is proposed to simulate processes of ligand binding to a G-protein-coupled receptor (GPCR), in this case the M3 muscarinic receptor, which is a target for treating many human diseases, including cancer, diabetes and obesity. Long-timescale aMD simulations were performed to observe the binding of three chemically diverse ligand molecules: antagonist tiotropium (TTP), partial agonist arecoline (ARc) and full agonist acetylcholine (ACh). In comparison with earlier microsecond-timescale conventional MD simulations, aMD greatly accelerated the binding of ACh to the receptor orthosteric ligand-binding site and the binding of TTP to an extracellular vestibule. Further aMD simulations also captured binding of ARc to the receptor orthosteric site. Additionally, all three ligands were observed to bind in the extracellular vestibule during their binding pathways, suggesting that it is a metastable binding site. This study demonstrates the applicability of aMD to protein-ligand binding, especially the drug recognition of GPCRs.

  7. Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering.

    PubMed

    Cai, Haogang; Muller, James; Depoil, David; Mayya, Viveka; Sheetz, Michael P; Dustin, Michael L; Wind, Shalom J

    2018-04-30

    Elucidating the rules for receptor triggering in cell-cell and cell-matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This comprises monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer. The TCR ligand could be co-planar with the supported lipid bilayer (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.

  8. Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

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

    Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai

    A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GRmore » LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.« less

  9. Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

    PubMed Central

    Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

    1999-01-01

    We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. PMID:9927419

  10. Suppression of aberrant choroidal neovascularization through activation of the aryl hydrocarbon receptor.

    PubMed

    Choudhary, Mayur; Safe, Stephen; Malek, Goldis

    2018-05-01

    The aryl hydrocarbon receptor (AhR) is a ligand activated transcription factor, initially discovered for its role in regulating xenobiotic metabolism. There is extensive evidence supporting a multi-faceted role for AhR, modulating physiological pathways important in cell health and disease. Recently we demonstrated that the AhR plays a role in the pathogenesis of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. We found that loss of AhR exacerbates choroidal neovascular (CNV) lesion formation in a murine model. Herein we tested the therapeutic impact of AhR activation on CNV lesion formation and factors associated with aberrant neovascularization. We screened a panel of synthetic drugs and endogenous AhR ligands, assessed their ability to activate AhR in choroidal endothelial cells, and inhibit angiogenesis in vitro. Drugs with an anti-angiogenic profile were then administered to a murine model of CNV. Two compounds, leflunomide and flutamide, significantly inhibited CNV formation concurrent with positive modifying effects on angiogenesis, inflammation, extracellular matrix remodeling, and fibrosis. These results validate the role of the AhR pathway in regulating CNV pathogenesis, identify mechanisms of AhR-based therapies in the eye, and argue in favor of developing AhR as a drug target for the treatment of neovascular AMD. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Enhanced sampling of glutamate receptor ligand-binding domains.

    PubMed

    Lau, Albert Y

    2018-04-14

    The majority of excitatory synaptic transmission in the central nervous system is mediated by ionotropic glutamate receptors (iGluRs). These membrane-bound protein assemblies consist of modular domains that can be genetically isolated and expressed, which has resulted in a plethora of crystal structures of individual domains in different conformations bound to different ligands. These structures have presented opportunities for molecular dynamics (MD) simulation studies. To examine the free energies that govern molecular behavior, simulation strategies and algorithms have been developed, collectively called enhanced sampling methods This review focuses on the use of enhanced sampling MD simulations of isolated iGluR ligand-binding domains to characterize thermodynamic properties important to receptor function. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Multiparameter flow cytometry of a pH sensitive ligand bound to receptors and inside cells

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

    Fay, S.P.; Habbersett, R.; Posner, R.G.

    1993-01-01

    Because fluoresceinated ligands of the neutrophil formyl peptide receptor can be protonated either upon binding to the receptor on the cell surface or in acidified intracellular compartments, the authors synthesized a ligand conjugated to the pH sensitive fluorescent probe SNAFL (CHO-Met-Leu-Phe-Phe-Lys-SNAFL). In the three laser flow cytometer at LANL, protonated dye is excited at 488 nm and emits at 530 nm; unprotonated dye is excited at 568 nm and emits at 650 nm. Detection at the isobestic and isoemissive points at 528 and 600 nm is used to keep track of variations in ligand concentration from sample to sample. Themore » SNAFL-ligand bound to HL-60 cells (which overexpress the formyl peptide receptor) was compared to the free ligand in solution over a pH range from 6.5 to 9.0. The results suggest that the ligand bound to cell surface receptors was protonated in the binding pocket, possibly by virtue of its proximity to His 90, based on sequence data. When the cells were raised from 4[degrees] to 37[degrees], they also observed a time-dependent acidification of the ligand, indicative of ligand-receptor processing beginning 3-4 minutes after internalization.« less

  13. Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells

    PubMed Central

    2013-01-01

    Background The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. Results To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Conclusions Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases. PMID:23731667

  14. B61 is a ligand for the ECK receptor protein-tyrosine kinase.

    PubMed

    Bartley, T D; Hunt, R W; Welcher, A A; Boyle, W J; Parker, V P; Lindberg, R A; Lu, H S; Colombero, A M; Elliott, R L; Guthrie, B A

    1994-04-07

    A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

  15. Elimination of a ligand gating site generates a supersensitive olfactory receptor.

    PubMed

    Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I

    2016-06-21

    Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors.

  16. Elimination of a ligand gating site generates a supersensitive olfactory receptor

    PubMed Central

    Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I.

    2016-01-01

    Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors. PMID:27323929

  17. Structural Analysis on the Pathologic Mutant Glucocorticoid Receptor Ligand-Binding Domains.

    PubMed

    Hurt, Darrell E; Suzuki, Shigeru; Mayama, Takafumi; Charmandari, Evangelia; Kino, Tomoshige

    2016-02-01

    Glucocorticoid receptor (GR) gene mutations may cause familial or sporadic generalized glucocorticoid resistance syndrome. Most of the missense forms distribute in the ligand-binding domain and impair its ligand-binding activity and formation of the activation function (AF)-2 that binds LXXLL motif-containing coactivators. We performed molecular dynamics simulations to ligand-binding domain of pathologic GR mutants to reveal their structural defects. Several calculated parameters including interaction energy for dexamethasone or the LXXLL peptide indicate that destruction of ligand-binding pocket (LBP) is a primary character. Their LBP defects are driven primarily by loss/reduction of the electrostatic interaction formed by R611 and T739 of the receptor to dexamethasone and a subsequent conformational mismatch, which deacylcortivazol resolves with its large phenylpyrazole moiety and efficiently stimulates transcriptional activity of the mutant receptors with LBP defect. Reduced affinity of the LXXLL peptide to AF-2 is caused mainly by disruption of the electrostatic bonds to the noncore leucine residues of this peptide that determine the peptide's specificity to GR, as well as by reduced noncovalent interaction against core leucines and subsequent exposure of the AF-2 surface to solvent. The results reveal molecular defects of pathologic mutant receptors and provide important insights to the actions of wild-type GR.

  18. Solution structure of the chick TGFbeta type II receptor ligand-binding domain.

    PubMed

    Marlow, Michael S; Brown, Christopher B; Barnett, Joey V; Krezel, Andrzej M

    2003-02-28

    The transforming growth factor beta (TGFbeta) signaling pathway influences cell proliferation, immune responses, and extracellular matrix reorganization throughout the vertebrate life cycle. The signaling cascade is initiated by ligand-binding to its cognate type II receptor. Here, we present the structure of the chick type II TGFbeta receptor determined by solution NMR methods. Distance and angular constraints were derived from 15N and 13C edited NMR experiments. Torsion angle dynamics was used throughout the structure calculations and refinement. The 20 final structures were energy minimized using the generalized Born solvent model. For these 20 structures, the average backbone root-mean-square distance from the average structure is below 0.6A. The overall fold of this 109-residue domain is conserved within the superfamily of these receptors. Chick receptors fully recognize and respond to human TGFbeta ligands despite only 60% identity at the sequence level. Comparison with the human TGFbeta receptor determined by X-ray crystallography reveals different conformations in several regions. Sequence divergence and crystal packing interactions under low pH conditions are likely causes. This solution structure identifies regions were structural changes, however subtle, may occur upon ligand-binding. We also identified two very well conserved molecular surfaces. One was found to bind ligand in the crystallized human TGFbeta3:TGFbeta type II receptor complex. The other, newly identified area can be the interaction site with type I and/or type III receptors of the TGFbeta signaling complex.

  19. Phosphorylation and Intramolecular Stabilization of the Ligand Binding Domain in the Nuclear Receptor Steroidogenic Factor 1

    PubMed Central

    Desclozeaux, Marion; Krylova, Irina N.; Horn, Florence; Fletterick, Robert J.; Ingraham, Holly A.

    2002-01-01

    Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor with no known ligand. We showed previously that phosphorylation at serine 203 located N′-terminal to the ligand binding domain (LBD) enhanced cofactor recruitment, analogous to the ligand-mediated recruitment in ligand-dependent receptors. In this study, results of biochemical analyses and an LBD helix assembly assay suggest that the SF-1 LBD adopts an active conformation, with helices 1 and 12 packed against the predicted alpha-helical bundle, in the apparent absence of ligand. Fine mapping of the previously defined proximal activation function in SF-1 showed that the activation function mapped fully to helix 1 of the LBD. Limited proteolyses demonstrate that phosphorylation of S203 in the hinge region mimics the stabilizing effects of ligand on the LBD. Moreover, similar effects were observed in an SF-1/thyroid hormone LBD chimera receptor, illustrating that the S203 phosphorylation effects are transferable to a heterologous ligand-dependent receptor. Our collective data suggest that the hinge together with helix 1 is an individualized specific motif, which is tightly associated with its cognate LBD. For SF-1, we find that this intramolecular association and hence receptor activity are further enhanced by mitogen-activated protein kinase phosphorylation, thus mimicking many of the ligand-induced changes observed for ligand-dependent receptors. PMID:12242296

  20. Mix-and-match: ligand-receptor pairs in stomatal development and beyond.

    PubMed

    Torii, Keiko U

    2012-12-01

    Stomata are small valves on the plant epidermis balancing gas exchange and water loss. Stomata are formed according to positional cues. In Arabidopsis, two EPIDERMAL PATTERNING FACTOR (EPF) peptides, EPF1 and EPF2, are secreted from stomatal precursors enforcing proper stomatal patterning. Here, I review recent studies revealing the ligand-receptor pairs and revising the previously predicted relations between receptors specifying stomatal patterning: ERECTA-family and TOO MANY MOUTHS (TMM). Furthermore, EPF-LIKE9 (EPFL9/Stomagen) promotes stomatal differentiation from internal tissues. Two EPFL peptides specify inflorescence architecture, a process beyond stomatal development, as ligands for ERECTA. Thus, broadly expressed receptor kinases may regulate multiple developmental processes through perceiving different peptide ligands, each with a specialized expression pattern. TMM in the epidermis may fine-tune multiple EPF/EPFL signals to prevent signal interference. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. P2X receptor ligands and pain.

    PubMed

    Shieh, Char-Chang; Jarvis, Michael F; Lee, Chih-Hung; Perner, Richard J

    2006-08-01

    P2X receptors belong to a superfamily of ligand-gated ion channels that conduct the influx of Ca(2+), Na(+) and K(+) cations following activation by extracellular nucleotides such as ATP. Molecular cloning studies have identified seven subunits, namely P2X(1-7), that share approximately 40 - 50% identity in amino acid sequences within the subfamily. Using gene-silencing, pharmacological and electrophysiological approaches, recent studies have revealed roles for P2X(2), P2X(3), P2X(4) and P2X(7) receptors in nociceptive signalling. Homomeric P2X(3) and heteromeric P2X(2/3) receptors are highly localised in the peripheral sensory afferent neurons that conduct nociceptive sensory information to the spinal chord and brain. The discovery of A-317491, a selective and potent non-nucleotide P2X(3) antagonist, provided a pharmacological tool to determine the site and mode of action of P2X(3)-containing receptors in different pain behaviours, including neuropathic, inflammatory and visceral pain. Other P2X receptors (P2X(4) and P2X(7)) that are predominantly expressed in microglia, macrophages and cells of immune origin can trigger the release of cytokines, such as IL-1-beta and TNF-alpha. Genetic disruption of P2X(4) and P2X(7) signalling has been demonstrated to reduce inflammatory and neuropathic pain, suggesting that these two receptors might serve as integrators of neuroinflammation and pain. This article provides an overview of recent scientific literature and patents focusing on P2X(3), P2X(4) and P2X(7) receptors, and the identification of small molecule ligands for the potential treatment of neuropathic and inflammatory pain.

  2. The Retinoid X Receptors and Their Ligands

    PubMed Central

    Dawson, Marcia I.; Xia, Zebin

    2014-01-01

    This chapter presents an overview of the current status of studies on the structural and molecular biology of the retinoid X receptor subtypes α, β, and γ (RXRs, NR2B1–3), their nuclear and cytoplasmic functions, post-transcriptional processing, and recently reported ligands. Points of interest are the different changes in the ligand-binding pocket induced by variously shaped agonists, the communication of the ligand–bound pocket with the coactivator binding surface and the heterodimerization interface, and recently identified ligands that are natural products, those that function as environmental toxins or drugs that had been originally designed to interact with other targets, as well as those that were deliberately designed as RXR-selective transcriptional agonists, synergists, or antagonists. Of these synthetic ligands, the general trend in design appears to be away from fully aromatic rigid structures to those containing partial elements of the flexible tetraene side chain of 9-cis-retinoic acid. PMID:22020178

  3. Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

    NASA Astrophysics Data System (ADS)

    Richter, David; Moraga, Ignacio; Winkelmann, Hauke; Birkholz, Oliver; Wilmes, Stephan; Schulte, Markos; Kraich, Michael; Kenneweg, Hella; Beutel, Oliver; Selenschik, Philipp; Paterok, Dirk; Gavutis, Martynas; Schmidt, Thomas; Garcia, K. Christopher; Müller, Thomas D.; Piehler, Jacob

    2017-07-01

    The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand-receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

  4. Molecular modeling study of the differential ligand-receptor interaction at the μ, δ and κ opioid receptors

    NASA Astrophysics Data System (ADS)

    Filizola, Marta; Carteni-Farina, Maria; Perez, Juan J.

    1999-07-01

    3D models of the opioid receptors μ, δ and κ were constructed using BUNDLE, an in-house program to build de novo models of G-protein coupled receptors at the atomic level. Once the three opioid receptors were constructed and before any energy refinement, models were assessed for their compatibility with the results available from point-site mutations carried out on these receptors. In a subsequent step, three selective antagonists to each of three receptors (naltrindole, naltrexone and nor-binaltorphamine) were docked onto each of the three receptors and subsequently energy minimized. The nine resulting complexes were checked for their ability to explain known results of structure-activity studies. Once the models were validated, analysis of the distances between different residues of the receptors and the ligands were computed. This analysis permitted us to identify key residues tentatively involved in direct interaction with the ligand.

  5. The role of aryl hydrocarbon receptor (AhR) in the pathology of pleomorphic adenoma in parotid gland.

    PubMed

    Drozdzik, Agnieszka; Kowalczyk, Robert; Lipski, Mariusz; Łapczuk, Joanna; Urasinska, Elzbieta; Kurzawski, Mateusz

    2016-01-01

    Pleomorphic adenoma (benign mixed tumor) is one of the most common salivary gland tumors. However, molecular mechanisms implicated in its development are not entirely defined. Therefore, the study aimed at definition of aryl hydrocarbon receptor (AhR) involvement in pleomorphic adenoma pathology, as the AhR controlled gene system was documented to play a role in development of various human tumors. The study was carried out in pleomorphic adenoma and control parotid gland tissues where gene expression of AHR, AhR nuclear translocator (ARNT), AhR repressor (AHRR), as well as AhR controlled genes: CYP1A1 and CYP1B1, at mRNA and protein (immunohistochemistry) levels were studied. Functional evaluation of AhR system was evaluated in HSY cells (human parotid gland adenocarcinoma cells) using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as AhR specific inducer. Pleomorphic adenoma specimens showed cytoplasmic and nuclear AhR expression in epithelial cells as well as in mesenchymal cells. In parotid gland AhR was expressed in cytoplasm of duct cells. Quantitative expression at mRNA level showed significantly higher expression of AHR, ARNT and CYP1B1, and comparable levels of CYP1A1 in pleomorphic adenoma tissue in comparison to healthy parotid gland. The HSY cell study revealed significantly higher expression level of AHRR in HSY as compared with MCF-7 cells (human breast adenocarcinoma cell line used as reference). Upon TCDD stimulation a drop in AHRR level in HSY cells and an increase in MCF-7 cells were observed. The HSY and MCF-7 cell proliferation rate (measured by WST-1 test) was not affected by TCDD. Summarizing both in vitro and in vivo observations it can be stated that AhR system may play a role in the pathology of pleomorphic adenoma. Copyright © 2015. Published by Elsevier Ltd.

  6. ALX receptor ligands define a biochemical endotype for severe asthma

    PubMed Central

    Ricklefs, Isabell; Barkas, Ioanna; Duvall, Melody G.; Grossman, Nicole L.; Israel, Elliot; Bleecker, Eugene R.; Castro, Mario; Erzurum, Serpil C.; Fahy, John V.; Gaston, Benjamin M.; Denlinger, Loren C.; Mauger, David T.; Wenzel, Sally E.; Comhair, Suzy A.; Coverstone, Andrea M.; Fajt, Merritt L.; Hastie, Annette T.; Johansson, Mats W.; Peters, Michael C.; Phillips, Brenda R.; Levy, Bruce D.

    2017-01-01

    BACKGROUND. In health, inflammation resolution is an active process governed by specialized proresolving mediators and receptors. ALX/FPR2 receptors (ALX) are targeted by both proresolving and proinflammatory ligands for opposing signaling events, suggesting pivotal roles for ALX in the fate of inflammatory responses. Here, we determined if ALX expression and ligands were linked to severe asthma (SA). METHODS. ALX expression and levels of proresolving ligands (lipoxin A4 [LXA4], 15-epi-LXA4, and annexin A1 [ANXA1]), and a proinflammatory ligand (serum amyloid A [SAA]) were measured in bronchoscopy samples collected in Severe Asthma Research Program-3 (SA [n = 69], non-SA [NSA, n = 51] or healthy donors [HDs, n = 47]). RESULTS. Bronchoalveolar lavage (BAL) fluid LXA4 and 15-epi-LXA4 were decreased and SAA was increased in SA relative to NSA. BAL macrophage ALX expression was increased in SA. Subjects with LXA4loSAAhi levels had increased BAL neutrophils, more asthma symptoms, lower lung function, increased relative risk for asthma exacerbation, sinusitis, and gastroesophageal reflux disease, and were assigned more frequently to SA clinical clusters. SAA and aliquots of LXA4loSAAhi BAL fluid induced IL-8 production by lung epithelial cells expressing ALX receptors, which was inhibited by coincubation with 15-epi-LXA4. CONCLUSIONS. Together, these findings have established an association between select ALX receptor ligands and asthma severity that define a potentially new biochemical endotype for asthma and support a pivotal functional role for ALX signaling in the fate of lung inflammation. TRIAL REGISTRATION. Severe Asthma Research Program-3 (SARP-3; ClinicalTrials.gov NCT01606826) FUNDING Sources. National Heart, Lung and Blood Institute, the NIH, and the German Society of Pediatric Pneumology. PMID:28724795

  7. Expression of human peroxisome proliferator-activated receptors ligand binding domain-maltose binding protein fusion protein in Escherichia coli: a convenient and reliable method for preparing receptor for screening ligands.

    PubMed

    Li, Changqing; Tian, Mi; Yuan, Ye; Zhou, Qinxin

    2008-12-01

    Human peroxisome proliferator-activated receptors (hPPARs) are ligand-activated transcription factors and are the target for the treatment of many diseases. Screening of their ligands is mainly based on assays of ligand binding to the ligand binding domain (LBD) of hPPARs.However, such assays are difficult because of the preparation of hPPARs LBD. In order to yield functional hPPARs LBD for screening ligands, hPPARs LBD was fused with maltose-binding protein(MBP) using the pMAL-p2x expression system through the gene engineering technique. The radioligand binding assay showed that MBP did not affect ligand binding with hPPARs LBD in the fusion proteins, which means that MBP-hPPARs LBD can be used instead of hPPARs LBD in ligand screening work. The results show that the new strategy using MBP as a fusion tag for preparing hPPARs LBD for screening ligands is a convenient and reliable method. It may be used to easily obtain the other nuclear receptors.

  8. Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms.

    PubMed

    Ries, Jonas; Yu, Shuizi Rachel; Burkhardt, Markus; Brand, Michael; Schwille, Petra

    2009-09-01

    Analysis of receptor-ligand interactions in vivo is key to biology but poses a considerable challenge to quantitative microscopy. Here we combine static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy to solve this task at cellular resolution in complex biological environments. We quantified the mobility of fibroblast growth factor receptors Fgfr1 and Fgfr4 in cell membranes of living zebrafish embryos and determined their in vivo binding affinities to their ligand Fgf8.

  9. Neuropeptide S receptor ligands: a patent review (2005-2016).

    PubMed

    Ruzza, Chiara; Calò, Girolamo; Di Maro, Salvatore; Pacifico, Salvatore; Trapella, Claudio; Salvadori, Severo; Preti, Delia; Guerrini, Remo

    2017-03-01

    Neuropeptide S (NPS) is a 20-residue peptide and endogenous ligand of the NPS receptor (NPSR). This receptor was a formerly orphan GPCR whose activation increases calcium and cyclic adenosine monophosphate levels. The NPS/NPSR system is expressed in several brain regions where it controls important biological functions including locomotor activity, arousal and sleep, anxiety, food intake, memory, pain, and drug addiction. Areas covered: This review furnishes an updated overview of the patent literature covering NPSR ligands since 2005, when the first example of an NPSR antagonist was disclosed. Expert opinion: Several potent NPSR antagonists are available as valuable pharmacological tools despite showing suboptimal pharmacokinetic properties in vivo. The optimization of these ligands is needed to speed up their potential clinical advancement as pharmaceuticals to treat drug addiction. In order to support the design of novel NPSR antagonists, we performed a ligand-based conformational analysis recognizing some structural requirements for NPSR antagonism. The identification of small-molecule NPSR agonists now represents an unmet challenge to be addressed. These molecules will allow investigation of the beneficial effects of selective NPSR activation in a large panel of psychiatric disorders and to foresee their therapeutic potential as anxiolytics, nootropics, and analgesics.

  10. Effects of artificial sweeteners on the AhR- and GR-dependent CYP1A1 expression in primary human hepatocytes and human cancer cells.

    PubMed

    Kamenickova, Alzbeta; Pecova, Michaela; Bachleda, Petr; Dvorak, Zdenek

    2013-12-01

    Food constituents may cause a phenomenon of food-drug interactions. In the current study, we examined the effects of artificial sweeteners (aspartame, acesulfame, cyclamate, saccharin) on the aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR)-dependent expression of CYP1A1 in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cell lines. Sweeteners were tested in concentrations up to those occurring in non-alcoholic beverages. Basal and ligand-inducible AhR- and GR-dependent reporter gene activation in stably transfected HepG2 and HeLa cells, respectively, were not affected by either of the sweeteners tested after 24h of incubation. The expression of CYP1A1 mRNA and protein in primary cultures of human hepatocytes and in LS174T and HepG2 cells was not induced by any of the tested sweeteners. Overall, aspartame, acesulfame, saccharin and cyclamate had no effects on CYP1A1 expression and transcriptional activities of AhR and GR. These data imply the safety of artificial sweeteners in terms of interference with AhR, GR and CYP1A1. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Ligand Activation of TAM Family Receptors-Implications for Tumor Biology and Therapeutic Response.

    PubMed

    Davra, Viralkumar; Kimani, Stanley G; Calianese, David; Birge, Raymond B

    2016-11-29

    The TAM family of receptors (i.e., Tyro3, Axl, and Mertk), and their ligands Growth arrest specific factor 6 (Gas6) and Protein S (Pros1) contribute to several oncogenic processes, such as cell survival, invasion, migration, chemo-resistance, and metastasis, whereby expression often correlates with poor clinical outcomes. In recent years, there has been great interest in the study of TAM receptors in cancer, stemming both from their roles as oncogenic signaling receptors, as well as their roles in tumor immunology. As a result, several classes of TAM inhibitors that include small molecule tyrosine kinase inhibitors, monoclonal antibodies, decoy receptors, as well as novel strategies to target TAM ligands are being developed. This paper will review the biology of TAM receptors and their ligands with a focus on cancer, as well as evidence-based data for the continued pursuit of TAM/Gas6 inhibitors in clinical practice.

  12. In Vitro Antitumor Effects of AHR Ligands Aminoflavone (AFP 464) and Benzothiazole (5F 203) in Human Renal Carcinoma Cells.

    PubMed

    Luzzani, Gabriela A; Callero, Mariana A; Kuruppu, Anchala I; Trapani, Valentina; Flumian, Carolina; Todaro, Laura; Bradshaw, Tracey D; Loaiza Perez, Andrea I

    2017-12-01

    We investigated activity and mechanism of action of two AhR ligand antitumor agents, AFP 464 and 5F 203 on human renal cancer cells, specifically examining their effects on cell cycle progression, apoptosis, and migration. TK-10, SN12C, Caki-1, and ACHN human renal cancer cell lines were treated with AFP 464 and 5F 203. We evaluated cytotoxicity by MTS assays, cell cycle arrest, and apoptosis by flow cytometry and corroborated a mechanism of action involving AhR signal transduction activation. Changes in migration properties by wound healing assays were investigated: 5F 203-sensitive cells show decreased migration after treatment, therefore, we measured c-Met phosphorylation by Western blot in these cells. A 5F 203 induced a decrease in cell viability which was more marked than AFP 464. This cytotoxicity was reduced after treatment with the AhR inhibitor α-NF for both compounds indicating AhR signaling activation plays a role in the mechanism of action. A 5F 203 is sequestered by TK-10 cells and induces CYP1A1 expression; 5F 203 potently inhibited migration of TK-10, Caki-1, and SN12C cells, and inhibited c-Met receptor phosphorylation in TK-10 cells. AhR ligand antitumor agents AFP 464 and 5F 203 represent potential new candidates for the treatment of renal cancer. A 5F 203 only inhibited migration of sensitive cells and c-Met receptor phosphorylation in TK-10 cells. c-Met receptor signal transduction is important in migration and metastasis. Therefore, we consider that 5F 203 offers potential for the treatment of metastatic renal carcinoma. J. Cell. Biochem. 118: 4526-4535, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. Ligand-Induced Dynamics of Neurotrophin Receptors Investigated by Single-Molecule Imaging Approaches

    PubMed Central

    Marchetti, Laura; Luin, Stefano; Bonsignore, Fulvio; de Nadai, Teresa; Beltram, Fabio; Cattaneo, Antonino

    2015-01-01

    Neurotrophins are secreted proteins that regulate neuronal development and survival, as well as maintenance and plasticity of the adult nervous system. The biological activity of neurotrophins stems from their binding to two membrane receptor types, the tropomyosin receptor kinase and the p75 neurotrophin receptors (NRs). The intracellular signalling cascades thereby activated have been extensively investigated. Nevertheless, a comprehensive description of the ligand-induced nanoscale details of NRs dynamics and interactions spanning from the initial lateral movements triggered at the plasma membrane to the internalization and transport processes is still missing. Recent advances in high spatio-temporal resolution imaging techniques have yielded new insight on the dynamics of NRs upon ligand binding. Here we discuss requirements, potential and practical implementation of these novel approaches for the study of neurotrophin trafficking and signalling, in the framework of current knowledge available also for other ligand-receptor systems. We shall especially highlight the correlation between the receptor dynamics activated by different neurotrophins and the respective signalling outcome, as recently revealed by single-molecule tracking of NRs in living neuronal cells. PMID:25603178

  14. Vitamin D receptor ligands for osteoporosis.

    PubMed

    Cheskis, Boris J; Freedman, Leonard P; Nagpal, Sunil

    2006-10-01

    1alpha,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the biologically active metabolite of vitamin D, mediates its actions via the vitamin D receptor (VDR), a member of the superfamily of steroid/thyroid hormone/retinoid receptors. 1,25-(OH)2D3 is required for calcium and phosphorus homeostasis, and for normal skeletal development as well as maintenance of skeletal architecture. Two VDR ligands, calcitriol (1,25-(OH)2D3) and its synthetic analog alfacalcidol (1alpha-hydroxyvitamin D3), have been approved for the treatment of osteoporosis. However, the use of calcitriol and alfacalcidol is limited by their major side effect, hypercalcemia, which is mediated mainly by VDR activity in the small intestine. In order to identify VDR ligands with less hypercalcemia liability, a number of pharmaceutical companies are pursuing efforts to develop synthetic vitamin D analogs. This review discusses the mechanism of action of vitamin D, and summarizes the currently approved anti-osteoporotic VDR agonists and compounds that are under development. The future directions of vitamin D research for the discovery of novel VDR agonists for osteoporosis are also discussed.

  15. Molecular Dynamics Methodologies for Probing Cannabinoid Ligand/Receptor Interaction

    PubMed Central

    Lynch, Diane L.; Hurst, Dow P.; Shore, Derek M.; Pitman, Mike C.; Reggio, Patricia H.

    2018-01-01

    The cannabinoid type 1 and 2 G-protein-coupled receptors are currently important pharmacological targets with significant drug discovery potential. These receptors have been shown to display functional selectivity or biased agonism, a property currently thought to have substantial therapeutic potential. Although recent advances in crystallization techniques have provided a wealth of structural information about this important class of membrane-embedded proteins, these structures lack dynamical information. In order to fully understand the interplay of structure and function for this important class of proteins, complementary techniques that address the dynamical aspects of their function are required such as NMR as well as a variety of other spectroscopies. Complimentary to these experimental approaches is molecular dynamics, which has been effectively used to help unravel, at the atomic level, the dynamics of ligand binding and activation of these membrane-bound receptors. Here, we discuss and present several representative examples of the application of molecular dynamics simulations to the understanding of the signatures of ligand-binding and -biased signaling at the cannabinoid type 1 and 2 receptors. PMID:28750815

  16. Membrane-bound LERK2 ligand can signal through three different Eph-related receptor tyrosine kinases.

    PubMed Central

    Brambilla, R; Schnapp, A; Casagranda, F; Labrador, J P; Bergemann, A D; Flanagan, J G; Pasquale, E B; Klein, R

    1995-01-01

    The Eph-related family of receptor tyrosine kinases consists of at least 13 members, several of which display distinctive expression patterns in the developing and adult nervous system. Recently, a small family of ligands, structurally related to the B61 protein, was identified. Binding of these ligands to Eph-related receptors did not, however, elicit measurable biological signals in cultured cells. In order to study functional interactions between B61-related ligands and Eph-related receptors, we constructed chimeric receptors, containing an Eph-related ectodomain and the cytoplasmic domain of the TrkB neurotrophin receptor. Expression and activation of such chimeric receptors in NIH 3T3 cells induced transformation in focus formation assays. Membrane-bound LERK2 ligand is shown to signal through three different Eph-related receptors, namely Cek5, Cek10 and Elk. LERK2, however, fails to interact functionally with the Cek9 receptor. Quantitative analysis including binding assays indicates that Cek10 is the preferred LERK2 receptor. Preliminary mutagenesis of the LERK2 protein suggests a negative regulatory role for its cytoplasmic domain in LERK2 signaling. Images PMID:7621826

  17. Measuring two-dimensional receptor-ligand binding kinetics by micropipette.

    PubMed Central

    Chesla, S E; Selvaraj, P; Zhu, C

    1998-01-01

    We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2. PMID:9726957

  18. An introduction to the molecular basics of aryl hydrocarbon receptor biology.

    PubMed

    Abel, Josef; Haarmann-Stemmann, Thomas

    2010-11-01

    Depending on their chemical structure and properties, environmental chemicals and other xenobiotics that enter the cell can affect cellular function by either nonselective binding to cellular macromolecules or by interference with cellular receptors, which would initiate a more defined cell biological response. One of these intracellular chemosensor molecules is the aryl hydrocarbon receptor (AhR), a transcription factor of the bHLH/PAS family that is known to mediate the biochemical and toxic effects of dioxins, polyaromatic hydrocarbons and related compounds. Numerous investigations have revealed that the AhR is not only a master regulator of drug metabolism activated by anthropogenic chemicals, but is also triggered by natural and endogenous ligands and can influence cell biological endpoints such as growth and differentiation. Cutting-edge research has identified new intriguing functions of the AhR, such as during proteasomal degradation of steroid hormone receptors, the cellular UVB stress response and the differentiation of certain T-cell subsets. In this review we provide both a survey of the fundamental basics of AhR biology and an insight into new functional aspects of AhR signaling to further stimulate research on this intriguing transcription factor at the interface between toxicology, cell biology and immunology.

  19. A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts.

    PubMed

    Lenkei, Z; Beaudet, A; Chartrel, N; De Mota, N; Irinopoulou, T; Braun, B; Vaudry, H; Llorens-Cortes, C

    2000-11-01

    Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC(50) = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.

  20. Aryl hydrocarbon receptor-dependent regulation of miR-196a expression controls lung fibroblast apoptosis but not proliferation

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

    Hecht, Emelia; Zago, Michela; Sarill, Miles

    2014-11-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR{sup −/−}) and wild-type (AhR{supmore » +/+}) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR{sup −/−} cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR{sup −/−} compared to AhR{sup +/+} cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR{sup +/+} fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR{sup +/+} lung fibroblasts in response to serum, corresponding to a decrease in p27{sup KIP1} protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27{sup KIP1} in AhR{sup −/−} fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR. - Highlights: • The AhR controls proliferation and apoptosis in lung cells. • The AhR regulates

  1. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

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

    Nie,Y.; Hobbs, J.; Vigues, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain amore » large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.« less

  2. Decreased expression of the aryl hydrocarbon receptor in ocular Behcet's disease.

    PubMed

    Wang, Chaokui; Ye, Zi; Kijlstra, Aize; Zhou, Yan; Yang, Peizeng

    2014-01-01

    Recent studies show that the aryl hydrocarbon receptor (AhR) is involved in immune responses. AhR is activated following interaction with its ligands, such as 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). In this study, we investigated the role of AhR activation by its endogenous ligands in the pathogenesis of ocular Behcet's disease (BD). The expression of AhR was significantly decreased in active BD patients as compared to inactive BD patients and normal controls. Both FICZ and ITE inhibited Th1 and Th17 polarization and induced the expression of IL-22 by PBMCs and by CD4(+)T cells in active BD patients and normal controls. Stimulation of purified CD4(+)T cells with FICZ or ITE caused a decreased expression of RORC, IL-17, IL-23R, and CCR6 and an increased phosphorylation of STAT3 and STAT5. The present study suggests that a decreased AhR expression is associated with disease activity in BD patients. The activation of AhR by either FICZ or ITE was able to inhibit Th1 and Th17 cell polarization. Further studies are needed to investigate whether modulation of AhR might be used in the treatment of BD.

  3. Decreased Expression of the Aryl Hydrocarbon Receptor in Ocular Behcet's Disease

    PubMed Central

    Wang, Chaokui; Ye, Zi; Kijlstra, Aize; Zhou, Yan; Yang, Peizeng

    2014-01-01

    Recent studies show that the aryl hydrocarbon receptor (AhR) is involved in immune responses. AhR is activated following interaction with its ligands, such as 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). In this study, we investigated the role of AhR activation by its endogenous ligands in the pathogenesis of ocular Behcet's disease (BD). The expression of AhR was significantly decreased in active BD patients as compared to inactive BD patients and normal controls. Both FICZ and ITE inhibited Th1 and Th17 polarization and induced the expression of IL-22 by PBMCs and by CD4+T cells in active BD patients and normal controls. Stimulation of purified CD4+T cells with FICZ or ITE caused a decreased expression of RORC, IL-17, IL-23R, and CCR6 and an increased phosphorylation of STAT3 and STAT5. The present study suggests that a decreased AhR expression is associated with disease activity in BD patients. The activation of AhR by either FICZ or ITE was able to inhibit Th1 and Th17 cell polarization. Further studies are needed to investigate whether modulation of AhR might be used in the treatment of BD. PMID:25045206

  4. Rational Quantitative Structure-Activity Relationship (RQSAR) Screen for PXR and CAR Isoform-Specific Nuclear Receptor Ligands

    PubMed Central

    Dring, Ann M.; Anderson, Linnea E.; Qamar, Saima; Stoner, Matthew A.

    2010-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are closely related orphan nuclear receptor proteins that share several ligands and target overlapping sets of genes involved in homeostasis and all phases of drug metabolism. CAR and PXR are involved in the development of certain diseases, including diabetes, metabolic syndrome and obesity. Ligand screens for these receptors so far have typically focused on steroid hormone analogs with pharmacophore-based approaches, only to find relatively few new hits. Multiple CAR isoforms have been detected in human liver, with the most abundant being the constitutively active reference, CAR1, and the ligand-dependent isoform CAR3. It has been assumed that any compound that binds CAR1 should also activate CAR3, and so CAR3 can be used as a ligand-activated surrogate for CAR1 studies. The possibility of CAR3-specific ligands has not, so far, been addressed. To investigate the differences between CAR1, CAR3 and PXR, and to look for more CAR ligands that may be of use in quantitative structure-activity relationship (QSAR) studies, we performed a luciferase transactivation assay screen of 60 mostly non-steroid compounds. Known active compounds with different core chemistries were chosen as starting points and structural variants were rationally selected for screening. Distinct differences in agonist versus inverse agonist/antagonist effects were seen in 49 compounds that had some ligand effect on at least one receptor and 18 that had effects on all three receptors; eight were CAR1 ligands only, three were CAR3 only ligands and four affected PXR only. This work provides evidence for new CAR ligands, some of which have CAR3-specific effects, and provides observational data on CAR and PXR ligands with which to inform in silico strategies. Compounds that demonstrated unique activity on any one receptor are potentially valuable diagnostic tools for the investigation of in vivo molecular targets. PMID:20869355

  5. Essential oils of culinary herbs and spices display agonist and antagonist activities at human aryl hydrocarbon receptor AhR.

    PubMed

    Bartoňková, Iveta; Dvořák, Zdeněk

    2018-01-01

    Essential oils (EOs) of culinary herbs and spices are used to flavor, color and preserve foods and drinks. Dietary intake of EOs is significant, deserving an attention of toxicologists. We examined the effects of 31 EOs of culinary herbs and spices on the transcriptional activity of human aryl hydrocarbon receptor (AhR), which is a pivotal xenobiotic sensor, having also multiple roles in human physiology. Tested EOs were sorted out into AhR-inactive ones (14 EOs) and AhR-active ones, including full agonists (cumin, jasmine, vanilla, bay leaf), partial agonists (cloves, dill, thyme, nutmeg, oregano) and antagonists (tarragon, caraway, turmeric, lovage, fennel, spearmint, star anise, anise). Major constituents (>10%) of AhR-active EOs were studied in more detail. We identified AhR partial agonists (carvacrol, ligustilide, eugenol, eugenyl acetate, thymol, ar-turmerone) and antagonists (trans-anethole, butylidine phtalide, R/S-carvones, p-cymene), which account for AhR-mediated activities of EOs of fennel, anise, star anise, caraway, spearmint, tarragon, cloves, dill, turmeric, lovage, thyme and oregano. We also show that AhR-mediated effects of some individual constituents of EOs differ from those manifested in mixtures. In conclusion, EOs of culinary herbs and spices are agonists and antagonists of human AhR, implying a potential for food-drug interactions and interference with endocrine pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Structural basis of ligand recognition in 5-HT3 receptors

    PubMed Central

    Kesters, Divya; Thompson, Andrew J; Brams, Marijke; van Elk, René; Spurny, Radovan; Geitmann, Matthis; Villalgordo, Jose M; Guskov, Albert; Helena Danielson, U; Lummis, Sarah C R; Smit, August B; Ulens, Chris

    2013-01-01

    The 5-HT3 receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT3 receptor. In the serotonin-bound structure, we observe hydrophilic interactions with loop E-binding site residues, which might enable transitions to channel opening. In the granisetron-bound structure, we observe a critical cation–π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5-HT3 receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high-affinity ligand binding in the human 5-HT3 receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti-emetics in the 5-HT3 receptor. PMID:23196367

  7. Small potent ligands to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor.

    PubMed

    Axén, Andreas; Andersson, Hanna; Lindeberg, Gunnar; Rönnholm, Harriet; Kortesmaa, Jarkko; Demaegdt, Heidi; Vauquelin, Georges; Karlén, Anders; Hallberg, Mathias

    2007-07-01

    Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor. Displacement of the C-terminal of angiotensin IV with an o-substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (K(i) = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a gamma-turn in the C-terminal of its bioactive conformation. Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT(4) receptor.

  8. REACTIVITY PROFILE OF CONFORMATIONALLY-FLEXIBLE RETINOID RECEPTOR LIGANDS

    EPA Science Inventory

    Retinoids and associated derivatives represent a class of endogenousr hormones that bind to and activate different families of retinoic acid receptors (RARs, RXRs), and control many aspects of normal vertebrate development. Identification of potential RAR and RXRs ligands is of i...

  9. Dissection of the insulin-sensitizing effect of liver X receptor ligands.

    PubMed

    Commerford, S Renee; Vargas, Leo; Dorfman, Suzanne E; Mitro, Nico; Rocheford, Erik C; Mak, Puiying A; Li, Xue; Kennedy, Patrick; Mullarkey, Tara L; Saez, Enrique

    2007-12-01

    The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Treatment of insulin-resistant mice with synthetic LXR ligands enhances glucose tolerance, inducing changes in gene expression expected to decrease hepatic gluconeogenesis (via indirect suppression of gluconeogenic enzymes) and increase peripheral glucose disposal (via direct up-regulation of glut4 in fat). To evaluate the relative contribution of each of these effects on whole-body insulin sensitivity, we performed hyperinsulinemic-euglycemic clamps in high-fat-fed insulin-resistant rats treated with an LXR agonist or a peroxisome proliferator-activated receptor gamma ligand. Both groups showed significant improvement in insulin action. Interestingly, rats treated with LXR ligand had lower body weight and smaller fat cells than controls. Insulin-stimulated suppression of the rate of glucose appearance (Ra) was pronounced in LXR-treated rats, but treatment failed to enhance peripheral glucose uptake (R'g), despite increased expression of glut4 in epididymal fat. To ascertain whether LXR ligands suppress hepatic gluconeogenesis directly, mice lacking LXRalpha (the primary isotype in liver) were treated with LXR ligand, and gluconeogenic gene expression was assessed. LXR activation decreased expression of gluconeogenic genes in wild-type and LXRbeta null mice, but failed to do so in animals lacking LXRalpha. Our observations indicate that despite inducing suggestive gene expression changes in adipose tissue in this model of diet-induced insulin resistance, the antidiabetic effect of LXR ligands is primarily due to effects in the liver that appear to require LXRalpha. These findings have important implications for clinical development of LXR agonists as insulin sensitizers.

  10. Dynamical Binding Modes Determine Agonistic and Antagonistic Ligand Effects in the Prostate-Specific G-Protein Coupled Receptor (PSGR).

    PubMed

    Wolf, Steffen; Jovancevic, Nikolina; Gelis, Lian; Pietsch, Sebastian; Hatt, Hanns; Gerwert, Klaus

    2017-11-22

    We analysed the ligand-based activation mechanism of the prostate-specific G-protein coupled receptor (PSGR), which is an olfactory receptor that mediates cellular growth in prostate cancer cells. Furthermore, it is an olfactory receptor with a known chemically near identic antagonist/agonist pair, α- and β-ionone. Using a combined theoretical and experimental approach, we propose that this receptor is activated by a ligand-induced rearrangement of a protein-internal hydrogen bond network. Surprisingly, this rearrangement is not induced by interaction of the ligand with the network, but by dynamic van der Waals contacts of the ligand with the involved amino acid side chains, altering their conformations and intraprotein connectivity. Ligand recognition in this GPCR is therefore highly stereo selective, but seemingly lacks any ligand recognition via polar contacts. A putative olfactory receptor-based drug design scheme will have to take this unique mode of protein/ligand action into account.

  11. Synthesis of 3-alkyl naphthalenes as novel estrogen receptor ligands

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

    Fang, Jing; Akwabi-Ameyaw, Adwoa; Britton, Jonathan E.

    2009-06-24

    A series of estrogen receptor ligands based on a 3-alkyl naphthalene scaffold was synthesized using an intramolecular enolate-alkyne cycloaromatization as the key step. Several of these compounds bearing a C6-OH group were shown to be high affinity ligands. All compounds had similar ER{alpha} and ER{beta} binding affinity ranging from micromolar to low nanomolar.

  12. Expression of the ERBB Family of Ligands and Receptors in Gastric Cancer.

    PubMed

    Byeon, Sun-Ju; Lee, Hye Seung; Kim, Min-A; Lee, Byung Lan; Kim, Woo Ho

    2017-01-01

    Gastric cancer (GC) is the second most common cancer and the third leading cause of cancer-related death in Korea. Alterations in the ERBB (homology to the erythroblastoma viral gene product, v-erbB) receptor family and ERBB-related signaling pathways are frequently observed in GC. However, the roles of the ERBB receptors and their ligands in GC are not well established. We evaluated the expression levels of various ERBB receptor ligands (i.e., heparin-binding epidermal growth factor-like growth factor [HBEGF], transforming growth factor-α [TGFA], amphiregulin [AREG], epiregulin [EREG], epidermal growth factor [EGF], and betacellulin [BTC]) and 3 ERBB family receptors (i.e., epidermal growth factor receptor [EGFR], human EGFR2 [HER2], and ERBB3) in 313 cases of GC using immunohistochemistry, fluorescence in situ hybridization, and mRNA in situ hybridization. A high expression of EGFR, HER2, and ERBB3 was observed in 30, 32, and 27 cases, respectively. A high expression of HBEGF, TGFA, AREG, EREG, EGF, and BTC was observed in 91, 97, 151, 74, 26, and 37 cases, respectively. A high expression of TGFA was associated with better survival, while a high expression of BTC was associated with worse survival. These results were confirmed using Cox proportional hazards analysis. HBEGF, TGFA, AREG, tumor-node-metastasis classification, Lauren's classification, and ERBB3 were significant survival parameters in multivariate analysis. Among the ERBB family receptors and ligands examined, 3 ligands (i.e., TGFA, HBEGF, and AREG) and ERBB3 had a prognostic impact. © 2017 S. Karger AG, Basel.

  13. The AhR is involved in the regulation of LoVo cell proliferation through cell cycle-associated proteins.

    PubMed

    Yin, Jiuheng; Sheng, Baifa; Han, Bin; Pu, Aimin; Yang, Kunqiu; Li, Ping; Wang, Qimeng; Xiao, Weidong; Yang, Hua

    2016-05-01

    Some ingredients in foods can activate the aryl hydrocarbon receptor (AhR) and arrest cell proliferation. In this study, we hypothesized that 6-formylindolo [3, 2-b] carbazole (FICZ) arrests the cell cycle in LoVo cells (a colon cancer line) through the AhR. The AhR agonist FICZ and the AhR antagonist CH223191 were used to treat LoVo cells. Real-time PCR and Western blot analyses were performed to detect the expression of the AhR, CYP1A1, CDK4, cyclinD1, cyclin E, CDK2, P27, and pRb. The distribution and activation of the AhR were detected with immunofluorescence. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometric analysis were performed to measure cell viability, cell cycle stage, and apoptosis. Our results show that FICZ inhibited LoVo cell proliferation by inducing G1 cell cycle arrest but had no effect on epithelial apoptosis. Further analysis found that FICZ downregulated cyclinD1 and upregulated p27 expression to arrest Rb phosphorylation. The downregulation of cyclinD1 and upregulation of p27 were abolished by co-treatment with CH223191. We conclude that the AhR, when activated by FICZ (an endogenous AhR ligand), can arrest the cell cycle and block LoVo cell proliferation. © 2016 International Federation for Cell Biology.

  14. Assessment and Challenges of Ligand Docking into Comparative Models of G-Protein Coupled Receptors

    PubMed Central

    Frimurer, Thomas M.; Meiler, Jens

    2013-01-01

    The rapidly increasing number of high-resolution X-ray structures of G-protein coupled receptors (GPCRs) creates a unique opportunity to employ comparative modeling and docking to provide valuable insight into the function and ligand binding determinants of novel receptors, to assist in virtual screening and to design and optimize drug candidates. However, low sequence identity between receptors, conformational flexibility, and chemical diversity of ligands present an enormous challenge to molecular modeling approaches. It is our hypothesis that rapid Monte-Carlo sampling of protein backbone and side-chain conformational space with Rosetta can be leveraged to meet this challenge. This study performs unbiased comparative modeling and docking methodologies using 14 distinct high-resolution GPCRs and proposes knowledge-based filtering methods for improvement of sampling performance and identification of correct ligand-receptor interactions. On average, top ranked receptor models built on template structures over 50% sequence identity are within 2.9 Å of the experimental structure, with an average root mean square deviation (RMSD) of 2.2 Å for the transmembrane region and 5 Å for the second extracellular loop. Furthermore, these models are consistently correlated with low Rosetta energy score. To predict their binding modes, ligand conformers of the 14 ligands co-crystalized with the GPCRs were docked against the top ranked comparative models. In contrast to the comparative models themselves, however, it remains difficult to unambiguously identify correct binding modes by score alone. On average, sampling performance was improved by 103 fold over random using knowledge-based and energy-based filters. In assessing the applicability of experimental constraints, we found that sampling performance is increased by one order of magnitude for every 10 residues known to contact the ligand. Additionally, in the case of DOR, knowledge of a single specific ligand

  15. Molecular Basis of Ligand Dissociation from G Protein-Coupled Receptors and Predicting Residence Time.

    PubMed

    Guo, Dong; IJzerman, Adriaan P

    2018-01-01

    G protein-coupled receptors (GPCRs) are integral membrane proteins and represent the largest class of drug targets. During the past decades progress in structural biology has enabled the crystallographic elucidation of the architecture of these important macromolecules. It also provided atomic-level visualization of ligand-receptor interactions, dramatically boosting the impact of structure-based approaches in drug discovery. However, knowledge obtained through crystallography is limited to static structural information. Less information is available showing how a ligand associates with or dissociates from a given receptor, whose importance is in fact increasingly recognized by the drug research community. Owing to recent advances in computer power and algorithms, molecular dynamics stimulations have become feasible that help in analyzing the kinetics of the ligand binding process. Here, we review what is currently known about the dynamics of GPCRs in the context of ligand association and dissociation, as determined through both crystallography and computer simulations. We particularly focus on the molecular basis of ligand dissociation from GPCRs and provide case studies that predict ligand dissociation pathways and residence time.

  16. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    NASA Astrophysics Data System (ADS)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  17. Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

    PubMed Central

    1991-01-01

    Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand. PMID:1848864

  18. Interactions of ligands with active and inactive conformations of the dopamine D2 receptor.

    PubMed

    Malmberg, A; Mohell, N; Backlund Höök, B; Johansson, A M; Hacksell, U; Nordvall, G

    1998-04-10

    The affinities of 19 pharmacologically diverse dopamine D2 receptor ligands were determined for the active and inactive conformations of cloned human dopamine D2 receptors expressed in Ltk cells. The agonist [3H]quinpirole was used to selectively label the guanine nucleotide-binding protein-coupled, active receptor conformation. The antagonist [3H]raclopride, in the presence of the non-hydrolysable GTP-analogue Gpp(NH)p and sodium ions and in the absence of magnesium ions, was used to label the free inactive receptor conformation. The intrinsic activities of the ligands were determined in a forskolin-stimulated cyclic AMP assay using the same cells. An excellent correlation was shown between the affinity ratios (KR/KRG) of the ligands for the two receptor conformations and their intrinsic activity (r=0.96). The ligands included eight structurally related and enantiopure 2-aminotetralin derivatives; the enantiomers of 5-hydroxy-2-(dipropylamino)tetralin, 5-methoxy-2-(dipropylamino)tetralin, 5-fluoro-2-(dipropylamino)tetralin and 2-(dipropylamino)tetralin. The (S)-enantiomers behaved as full agonists in the cyclic AMP assay and displayed a large KR/KRG ratio. The (R)-enantiomers were classified as partial agonists and had lower ratios. The structure-affinity relationships of these compounds at the active and the inactive receptor conformations were analysed separately, and used in conjunction with a homology based receptor model of the dopamine D2 receptor. This led to proposed binding modes for agonists, antagonists and partial agonists in the 2-aminotetralin series. The concepts used in this study should be of value in the design of ligands with predetermined affinity and intrinsic activity.

  19. Theoretical investigation of interaction between the set of ligands and α7 nicotinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Glukhova, O. E.; Prytkova, T. R.; Shmygin, D. S.

    2016-03-01

    Nicotinic acetylcholine receptors (nAChRs) are neuron receptor proteins that provide a transmission of nerve impulse through the synapses. They are composed of a pentametric assembly of five homologous subunits (5 α7 subunits for α7nAChR, for example), oriented around the central pore. These receptors might be found in the chemical synapses of central and peripheral nervous system, and also in the neuromuscular synapses. Transmembrane domain of the one of such receptors constitutes ion channel. The conductive properties of ion channel strongly depend on the receptor conformation changes in the response of binding with some molecule, f.e. acetylcholine. Investigation of interaction between ligands and acetylcholine receptor is important for drug design. In this work we investigate theoretically the interaction between the set of different ligands (such as vanillin, thymoquinone, etc.) and the nicotinic acetylcholine receptor (primarily with subunit of the α7nAChR) by different methods and packages (AutodockVina, GROMACS, KVAZAR, HARLEM, VMD). We calculate interaction energy between different ligands in the subunit using molecular dynamics. On the base of obtained calculation results and using molecular docking we found an optimal location of different ligands in the subunit.

  20. Generating "fragment-based virtual library" using pocket similarity search of ligand-receptor complexes.

    PubMed

    Khashan, Raed S

    2015-01-01

    As the number of available ligand-receptor complexes is increasing, researchers are becoming more dedicated to mine these complexes to aid in the drug design and development process. We present free software which is developed as a tool for performing similarity search across ligand-receptor complexes for identifying binding pockets which are similar to that of a target receptor. The search is based on 3D-geometric and chemical similarity of the atoms forming the binding pocket. For each match identified, the ligand's fragment(s) corresponding to that binding pocket are extracted, thus forming a virtual library of fragments (FragVLib) that is useful for structure-based drug design. The program provides a very useful tool to explore available databases.

  1. Structure-based discovery and binding site analysis of histamine receptor ligands.

    PubMed

    Kiss, Róbert; Keserű, György M

    2016-12-01

    The application of structure-based drug discovery in histamine receptor projects was previously hampered by the lack of experimental structures. The publication of the first X-ray structure of the histamine H1 receptor has been followed by several successful virtual screens and binding site analysis studies of H1-antihistamines. This structure together with several other recently solved aminergic G-protein coupled receptors (GPCRs) enabled the development of more realistic homology models for H2, H3 and H4 receptors. Areas covered: In this paper, the authors review the development of histamine receptor models and their application in drug discovery. Expert opinion: In the authors' opinion, the application of atomistic histamine receptor models has played a significant role in understanding key ligand-receptor interactions as well as in the discovery of novel chemical starting points. The recently solved H1 receptor structure is a major milestone in structure-based drug discovery; however, our analysis also demonstrates that for building H3 and H4 receptor homology models, other GPCRs may be more suitable as templates. For these receptors, the authors envisage that the development of higher quality homology models will significantly contribute to the discovery and optimization of novel H3 and H4 ligands.

  2. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

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

    Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar

    2011-03-18

    Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependentmore » functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.« less

  3. Rigid-body Ligand Recognition Drives Cytotoxic T-lymphocyte Antigen 4 (CTLA-4) Receptor Triggering

    PubMed Central

    Yu, Chao; Sonnen, Andreas F.-P.; George, Roger; Dessailly, Benoit H.; Stagg, Loren J.; Evans, Edward J.; Orengo, Christine A.; Stuart, David I.; Ladbury, John E.; Ikemizu, Shinji; Gilbert, Robert J. C.; Davis, Simon J.

    2011-01-01

    The inhibitory T-cell surface-expressed receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which belongs to the class of cell surface proteins phosphorylated by extrinsic tyrosine kinases that also includes antigen receptors, binds the related ligands, B7-1 and B7-2, expressed on antigen-presenting cells. Conformational changes are commonly invoked to explain ligand-induced “triggering” of this class of receptors. Crystal structures of ligand-bound CTLA-4 have been reported, but not the apo form, precluding analysis of the structural changes accompanying ligand binding. The 1.8-Å resolution structure of an apo human CTLA-4 homodimer emphasizes the shared evolutionary history of the CTLA-4/CD28 subgroup of the immunoglobulin superfamily and the antigen receptors. The ligand-bound and unbound forms of both CTLA-4 and B7-1 are remarkably similar, in marked contrast to B7-2, whose binding to CTLA-4 has elements of induced fit. Isothermal titration calorimetry reveals that ligand binding by CTLA-4 is enthalpically driven and accompanied by unfavorable entropic changes. The similarity of the thermodynamic parameters determined for the interactions of CTLA-4 with B7-1 and B7-2 suggests that the binding is not highly specific, but the conformational changes observed for B7-2 binding suggest some level of selectivity. The new structure establishes that rigid-body ligand interactions are capable of triggering CTLA-4 phosphorylation by extrinsic kinase(s). PMID:21156796

  4. Molecular determinants of ligand binding modes in the histamine H(4) receptor: linking ligand-based three-dimensional quantitative structure-activity relationship (3D-QSAR) models to in silico guided receptor mutagenesis studies.

    PubMed

    Istyastono, Enade P; Nijmeijer, Saskia; Lim, Herman D; van de Stolpe, Andrea; Roumen, Luc; Kooistra, Albert J; Vischer, Henry F; de Esch, Iwan J P; Leurs, Rob; de Graaf, Chris

    2011-12-08

    The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

  5. Importance of the pharmacological profile of the bound ligand in enrichment on nuclear receptors: toward the use of experimentally validated decoy ligands.

    PubMed

    Lagarde, Nathalie; Zagury, Jean-François; Montes, Matthieu

    2014-10-27

    The evaluation of virtual ligand screening methods is of major importance to ensure their reliability. Taking into account the agonist/antagonist pharmacological profile should improve the quality of the benchmarking data sets since ligand binding can induce conformational changes in the nuclear receptor structure and such changes may vary according to the agonist/antagonist ligand profile. We indeed found that splitting the agonist and antagonist ligands into two separate data sets for a given nuclear receptor target significantly enhances the quality of the evaluation. The pharmacological profile of the ligand bound in the binding site of the target structure was also found to be an additional critical parameter. We also illustrate that active compound data sets for a given pharmacological activity can be used as a set of experimentally validated decoy ligands for another pharmacological activity to ensure a reliable and challenging evaluation of virtual screening methods.

  6. Quantification of Ligand Binding to G-Protein Coupled Receptors on Cell Membranes by Ellipsometry

    PubMed Central

    Kriechbaumer, Verena; Nabok, Alexei; Widdowson, Robert; Smith, David P.; Abell, Ben M.

    2012-01-01

    G-protein-coupled receptors (GPCRs) are prime drug targets and targeted by approximately 60% of current therapeutic drugs such as β-blockers, antipsychotics and analgesics. However, no biophysical methods are available to quantify their interactions with ligand binding in a native environment. Here, we use ellipsometry to quantify specific interactions of receptors within native cell membranes. As a model system, the GPCR-ligand CXCL12α and its receptor CXCR4 are used. Human-derived Ishikawa cells were deposited onto gold coated slides via Langmuir-Schaefer film deposition and interactions between the receptor CXCR4 on these cells and its ligand CXCL12α were detected via total internal reflection ellipsometry (TIRE). This interaction could be inhibited by application of the CXCR4-binding drug AMD3100. Advantages of this approach are that it allows measurement of interactions in a lipid environment without the need for labelling, protein purification or reconstitution of membrane proteins. This technique is potentially applicable to a wide variety of cell types and their membrane receptors, providing a novel method to determine ligand or drug interactions targeting GPCRs and other membrane proteins. PMID:23049983

  7. Ligand-based receptor tyrosine kinase partial agonists: New paradigm for cancer drug discovery?

    PubMed

    Riese, David J

    2011-02-01

    INTRODUCTION: Receptor tyrosine kinases (RTKs) are validated targets for oncology drug discovery and several RTK antagonists have been approved for the treatment of human malignancies. Nonetheless, the discovery and development of RTK antagonists has lagged behind the discovery and development of agents that target G-protein coupled receptors. In part, this is because it has been difficult to discover analogs of naturally-occurring RTK agonists that function as antagonists. AREAS COVERED: Here we describe ligands of ErbB receptors that function as partial agonists for these receptors, thereby enabling these ligands to antagonize the activity of full agonists for these receptors. We provide insights into the mechanisms by which these ligands function as antagonists. We discuss how information concerning these mechanisms can be translated into screens for novel small molecule- and antibody-based antagonists of ErbB receptors and how such antagonists hold great potential as targeted cancer chemotherapeutics. EXPERT OPINION: While there have been a number of important key findings into this field, the identification of the structural basis of ligand functional specificity is still of the greatest importance. While it is true that, with some notable exceptions, peptide hormones and growth factors have not proven to be good platforms for oncology drug discovery; addressing the fundamental issues of antagonistic partial agonists for receptor tyrosine kinases has the potential to steer oncology drug discovery in new directions. Mechanism based approaches are now emerging to enable the discovery of RTK partial agonists that may antagonize both agonist-dependent and -independent RTK signaling and may hold tremendous promise as targeted cancer chemotherapeutics.

  8. Major advances in the development of histamine H4 receptor ligands.

    PubMed

    Smits, Rogier A; Leurs, Rob; de Esch, Iwan J P

    2009-08-01

    The search for new and potent histamine H4 receptor ligands is leading to a steadily increasing number of scientific publications and patent applications. Several interesting and structurally diverse compounds have been found, but fierce IP competition for a preferred 2-aminopyrimidine scaffold is becoming apparent. Recent investigations into the role of the histamine H(4)R in (patho)physiology and the use of H4R ligands in in vivo disease models reveal enormous potential in the field of inflammation and allergy, among others. The development of ligands that display activity at two or more histamine receptor (HR) subtypes is another clinical opportunity that is currently being explored. Taken together, the histamine H4R field is gearing up for clinical studies and has the potential to deliver another generation of blockbuster drugs.

  9. Targeting ligand-operated chaperone sigma-1 receptors in the treatment of neuropsychiatric disorders

    PubMed Central

    Teruo, Hayashi; Shang-Yi, Tsai; Tomohisa, Mori; Michiko, Fujimoto; Tsung-Ping, Su

    2011-01-01

    Introduction Current conventional therapeutic drugs for the treatment of psychiatric or neurodegenerative disorders have certain limitations of use. Psychotherapeutic drugs such as typical and atypical antipsychotics, tricyclic antidepressants, and selective monoamine reuptake inhibitors, aim to normalize the hyper- or hypo-neurotransmission of monoaminergic systems. Despite their great contribution to the outcomes of psychiatric patients, these agents often exert severe side effects and require chronic treatments to promote amelioration of symptoms. Furthermore, drugs available for the treatment of neurodegenerative disorders are severely limited. Areas covered This review discusses recent evidence that has shed light on sigma-1 receptor ligands, which may serve as a new class of antidepressants or neuroprotective agents. Sigma-1 receptors are novel ligand-operated molecular chaperones regulating a variety of signal transduction, ER stress, cellular redox, cellular survival, and synaptogenesis. Selective sigma-1 receptor ligands exert rapid antidepressant-like, anxiolytic, antinociceptive and robust neuroprotective actions in preclinical studies. The review also looks at recent studies which suggest that reactive oxygen species might play a crucial role as signal integrators at the downstream of Sig-1Rs Expert opinion The significant advances in sigma receptor research in the last decade have begun to elucidate the intracellular signal cascades upstream and downstream of sigma-1 receptors. The novel ligand-operated properties of the sigma-1 receptor chaperone may enable a variety of interventions by which stress-related cellular systems are pharmacologically controlled. PMID:21375464

  10. LiCABEDS II. Modeling of ligand selectivity for G-protein-coupled cannabinoid receptors.

    PubMed

    Ma, Chao; Wang, Lirong; Yang, Peng; Myint, Kyaw Z; Xie, Xiang-Qun

    2013-01-28

    The cannabinoid receptor subtype 2 (CB2) is a promising therapeutic target for blood cancer, pain relief, osteoporosis, and immune system disease. The recent withdrawal of Rimonabant, which targets another closely related cannabinoid receptor (CB1), accentuates the importance of selectivity for the development of CB2 ligands in order to minimize their effects on the CB1 receptor. In our previous study, LiCABEDS (Ligand Classifier of Adaptively Boosting Ensemble Decision Stumps) was reported as a generic ligand classification algorithm for the prediction of categorical molecular properties. Here, we report extension of the application of LiCABEDS to the modeling of cannabinoid ligand selectivity with molecular fingerprints as descriptors. The performance of LiCABEDS was systematically compared with another popular classification algorithm, support vector machine (SVM), according to prediction precision and recall rate. In addition, the examination of LiCABEDS models revealed the difference in structure diversity of CB1 and CB2 selective ligands. The structure determination from data mining could be useful for the design of novel cannabinoid lead compounds. More importantly, the potential of LiCABEDS was demonstrated through successful identification of newly synthesized CB2 selective compounds.

  11. Functional and phenotypic effects of AhR activation in inflammatory dendritic cells

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

    Bankoti, Jaishree; Center for Environmental Health Sciences, University of Montana, Missoula, MT; Rase, Ben

    2010-07-15

    Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immune suppression. Dendritic cells (DCs) are key antigen presenting cells governing T cell activation and differentiation. However, the consequences of AhR activation in DCs are not fully defined. We hypothesized that AhR activation alters DC differentiation and generates dysfunctional DCs. To test this hypothesis, inflammatory bone marrow-derived DCs (BMDCs) from C57Bl/6 mice were generated in the presence of vehicle or TCDD. TCDD decreased CD11c expression but increased MHC class II, CD86 and CD25 expression on the BMDCs. The effects of TCDD were strictly AhR-dependent but not exclusively DRE-mediated. Similar effects weremore » observed with two natural AhR ligands, 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid (ITE). TCDD increased LPS- and CpG-induced IL-6 and TNF-{alpha} production by BMDCs but decreased their NO production. TCDD decreased CpG-induced IL-12p70 production by BMDCs but did not affect their secretion of IL-10. TCDD downregulated LPS- and CpG-induced NF-kB p65 levels and induced a trend towards upregulation of RelB levels in the BMDCs. AhR activation by TCDD modulated BMDC uptake of both soluble and particulate antigens. Induction of indoleamine-2,3-dioxygenase (IDO) and TGF-{beta}3 has been implicated in the generation of regulatory T cells following AhR activation. TCDD increased IDO1, IDO2 and TGF-{beta}3 mRNA levels in BMDCs as compared to vehicle. Despite the induction of regulatory mediators, TCDD-treated BMDCs failed to suppress antigen-specific T cell activation. Thus, AhR activation can directly alter the differentiation and innate functions of inflammatory DCs without affecting their ability to successfully interact with T cells.« less

  12. Systematic Exploitation of Multiple Receptor Conformations for Virtual Ligand Screening

    PubMed Central

    Bottegoni, Giovanni; Rocchia, Walter; Rueda, Manuel; Abagyan, Ruben; Cavalli, Andrea

    2011-01-01

    The role of virtual ligand screening in modern drug discovery is to mine large chemical collections and to prioritize for experimental testing a comparatively small and diverse set of compounds with expected activity against a target. Several studies have pointed out that the performance of virtual ligand screening can be improved by taking into account receptor flexibility. Here, we systematically assess how multiple crystallographic receptor conformations, a powerful way of discretely representing protein plasticity, can be exploited in screening protocols to separate binders from non-binders. Our analyses encompass 36 targets of pharmaceutical relevance and are based on actual molecules with reported activity against those targets. The results suggest that an ensemble receptor-based protocol displays a stronger discriminating power between active and inactive molecules as compared to its standard single rigid receptor counterpart. Moreover, such a protocol can be engineered not only to enrich a higher number of active compounds, but also to enhance their chemical diversity. Finally, some clear indications can be gathered on how to select a subset of receptor conformations that is most likely to provide the best performance in a real life scenario. PMID:21625529

  13. Anti hyperglycaemic study of natural inhibitors for Insulin receptor

    PubMed Central

    Chatterjee, Subhojyoti; Narasimhaiah, Akshaya Lakshmi; Kundu, Sanjay; Anand, Santosh

    2012-01-01

    Diabetes is a metabolic disorder associated with either improper functioning of the beta-cells or wherein cells fail to use insulin properly. Insulin, the principal hormone regulates uptake of glucose from the blood into most of the cells except central nervous system. Therefore, deficiency of insulin or the insensitivity of its receptors plays a key role in all forms of diabetes. In the present work, attempt has been made to find out plant sources which show anti hyperglycaemic activity (AhG) (i.e. compounds that bring down the blood glucose level in the body). Ayurvedic plants showing AhG activity formed the basis of our study by using the platform of Computer Aided Drug Designing (CADD). Among 600 plants showing AhG activity, 500 compounds were selected and screened, out of which 243 compounds showed drug likeness property that can be used as therapeutic ligand/drug. Initial screening of such compounds was done based on their drug likeness or biochemical properties. Dynamic interaction of these molecules was captured through Protein-Ligand study. It also gave an insight of the binding pockets involved. Bench marking of all the parameters were done using the diabetic inhibitor drug, Glipizide. Pharmacokinetic studies of the compounds such as Aloins, Capparisine, Funiculosin and Rhein exhibited less toxicity on various levels of the body. As a conclusion these ligands can lay a foundation for a better anti-diabetic therapy. Abbreviations AhG - Anti hyperglycaemic, CADD - Computer Aided Drug Designing. PMID:23275719

  14. Anti hyperglycaemic study of natural inhibitors for Insulin receptor.

    PubMed

    Chatterjee, Subhojyoti; Narasimhaiah, Akshaya Lakshmi; Kundu, Sanjay; Anand, Santosh

    2012-01-01

    Diabetes is a metabolic disorder associated with either improper functioning of the beta-cells or wherein cells fail to use insulin properly. Insulin, the principal hormone regulates uptake of glucose from the blood into most of the cells except central nervous system. Therefore, deficiency of insulin or the insensitivity of its receptors plays a key role in all forms of diabetes. In the present work, attempt has been made to find out plant sources which show anti hyperglycaemic activity (AhG) (i.e. compounds that bring down the blood glucose level in the body). Ayurvedic plants showing AhG activity formed the basis of our study by using the platform of Computer Aided Drug Designing (CADD). Among 600 plants showing AhG activity, 500 compounds were selected and screened, out of which 243 compounds showed drug likeness property that can be used as therapeutic ligand/drug. Initial screening of such compounds was done based on their drug likeness or biochemical properties. Dynamic interaction of these molecules was captured through Protein-Ligand study. It also gave an insight of the binding pockets involved. Bench marking of all the parameters were done using the diabetic inhibitor drug, Glipizide. Pharmacokinetic studies of the compounds such as Aloins, Capparisine, Funiculosin and Rhein exhibited less toxicity on various levels of the body. As a conclusion these ligands can lay a foundation for a better anti-diabetic therapy. AhG - Anti hyperglycaemic, CADD - Computer Aided Drug Designing.

  15. Decahydrobenzoquinolin-5-one sigma receptor ligands: Divergent development of both sigma 1 and sigma 2 receptor selective examples.

    PubMed

    McLeod, Michael C; Aubé, Jeffrey; Frankowski, Kevin J

    2016-12-01

    Analogues of the decahydrobenzoquinolin-5-one class of sigma (σ) receptor ligands were used to probe the structure-activity relationship trends for this recently discovered series of σ ligands. In all, 29 representatives were tested for σ and opioid receptor affinity, leading to the identification of compounds possessing improved σ 1 selectivity and, for the first time in this series, examples possessing preferential σ 2 affinity. Several structural features associated with these selectivity trends have been identified. Two analogues of improved selectivity were evaluated in a binding panel of 43 CNS-relevant targets to confirm their sigma receptor preference. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Ligand-specific Deactivation Time Course of GluN1/GluN2D NMDA Receptors

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

    K Vance; N Simorowski; S Traynelis

    2011-12-31

    N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors that mediate a majority of excitatory synaptic transmission. One unique property of GluN1/GluN2D NMDA receptors is an unusually prolonged deactivation time course following the removal of L-glutamate. Here we show, using x-ray crystallography and electrophysiology, that the deactivation time course of GluN1/GluN2D receptors is influenced by the conformational variability of the ligand-binding domain (LBD) as well as the structure of the activating ligand. L-glutamate and L-CCG-IV induce significantly slower deactivation time courses compared with other agonists. Crystal structures of the isolated GluN2D LBD in complex with various ligands revealmore » that the binding of L-glutamate induces a unique conformation at the backside of the ligand-binding site in proximity to the region at which the transmembrane domain would be located in the intact receptors. These data suggest that the activity of the GluN1/GluN2D NMDA receptor is controlled distinctively by the endogenous neurotransmitter L-glutamate.« less

  17. International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB₁ and CB₂.

    PubMed

    Pertwee, R G; Howlett, A C; Abood, M E; Alexander, S P H; Di Marzo, V; Elphick, M R; Greasley, P J; Hansen, H S; Kunos, G; Mackie, K; Mechoulam, R; Ross, R A

    2010-12-01

    There are at least two types of cannabinoid receptors (CB(1) and CB(2)). Ligands activating these G protein-coupled receptors (GPCRs) include the phytocannabinoid Δ(9)-tetrahydrocannabinol, numerous synthetic compounds, and endogenous compounds known as endocannabinoids. Cannabinoid receptor antagonists have also been developed. Some of these ligands activate or block one type of cannabinoid receptor more potently than the other type. This review summarizes current data indicating the extent to which cannabinoid receptor ligands undergo orthosteric or allosteric interactions with non-CB(1), non-CB(2) established GPCRs, deorphanized receptors such as GPR55, ligand-gated ion channels, transient receptor potential (TRP) channels, and other ion channels or peroxisome proliferator-activated nuclear receptors. From these data, it is clear that some ligands that interact similarly with CB(1) and/or CB(2) receptors are likely to display significantly different pharmacological profiles. The review also lists some criteria that any novel "CB(3)" cannabinoid receptor or channel should fulfil and concludes that these criteria are not currently met by any non-CB(1), non-CB(2) pharmacological receptor or channel. However, it does identify certain pharmacological targets that should be investigated further as potential CB(3) receptors or channels. These include TRP vanilloid 1, which possibly functions as an ionotropic cannabinoid receptor under physiological and/or pathological conditions, and some deorphanized GPCRs. Also discussed are 1) the ability of CB(1) receptors to form heteromeric complexes with certain other GPCRs, 2) phylogenetic relationships that exist between CB(1)/CB(2) receptors and other GPCRs, 3) evidence for the existence of several as-yet-uncharacterized non-CB(1), non-CB(2) cannabinoid receptors; and 4) current cannabinoid receptor nomenclature.

  18. Biotin-transfer from a trifunctional crosslinker for identification of cell surface receptors of soluble protein ligands

    PubMed Central

    Tremblay, Tammy-Lynn; Hill, Jennifer J.

    2017-01-01

    Here we describe a novel crosslinker and its application as a biotin-transfer reagent to identify cell surface receptors of soluble protein ligands on live cells. This crosslinker contains three functional groups: an aldehyde-reactive aminooxy group, a sulfhydryl, and a biotin (ASB). It is readily synthesized via a 3-step addition reaction using standard solid-phase peptide synthesis methods and commercially available intermediates, allowing access to laboratories without specialized synthetic chemistry capabilities. For the biotin-transfer method, ASB is linked to a protein ligand through the sulfhydryl group in a two-step process that allows the introduction of a disulfide bond between the ligand and the crosslinker. Incubation of the labelled ligand with oxidized live cells leads to the formation of crosslinks with aldehyde-containing glycans on the cell surface receptor. Subsequent reduction of the disulfide bond results in biotin transfer from the ligand to the cell surface receptor. Protein biotinylation that is mediated by ligand binding to its receptor is differentiated from background biotinylation events by comparison with a similarly labelled control protein using comparative proteomic mass spectrometry to quantify streptavidin-bound proteins. Using this method, we successfully identified the cell surface receptors of a peptide hormone, a monoclonal antibody, and a single-domain antibody-Fc fusion construct. PMID:28422167

  19. Endogenous cannabinoid receptor ligand induces the migration of human natural killer cells.

    PubMed

    Kishimoto, Seishi; Muramatsu, Mayumi; Gokoh, Maiko; Oka, Saori; Waku, Keizo; Sugiura, Takayuki

    2005-02-01

    2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating which shows that 2-arachidonoylglycerol plays important physiological roles in several mammalian tissues and cells, yet the details remain ambiguous. In this study, we first examined the effects of 2-arachidonoylglycerol on the motility of human natural killer cells. We found that 2-arachidonoylglycerol induces the migration of KHYG-1 cells (a natural killer leukemia cell line) and human peripheral blood natural killer cells. The migration of natural killer cells induced by 2-arachidonoylglycerol was abolished by treating the cells with SR144528, a CB2 receptor antagonist, suggesting that the CB2 receptor is involved in the 2-arachidonoylglycerol-induced migration. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, did not induce the migration. Delta9-tetrahydrocannabinol, a major psychoactive constituent of marijuana, also failed to induce the migration; instead, the addition of delta9-tetrahydrocannabinol together with 2-arachidonoylglycerol abolished the migration induced by 2-arachidonoylglycerol. It is conceivable that the endogenous ligand for the cannabinoid receptor, that is, 2-arachidonoylglycerol, affects natural killer cell functions such as migration, thereby contributing to the host-defense mechanism against infectious viruses and tumor cells.

  20. An in silico approach to investigate the source of the controversial interpretations about the phenotypic results of the human AhR-gene G1661A polymorphism.

    PubMed

    Aftabi, Younes; Colagar, Abasalt Hosseinzadeh; Mehrnejad, Faramarz

    2016-03-21

    Aryl hydrocarbon receptor (AhR) acts as an enhancer binding ligand-activated intracellular receptor. Chromatin remodeling components and general transcription factors such as TATA-binding protein (TBP) are evoked on AhR-target genes by interaction with its flexible transactivation domain (TAD). AhR-G1661A single nucleotide polymorphism (SNP: rs2066853) causes an arginine to lysine substitution in the acidic sub-domain of TAD at position 554 (R554K). Although, numerous studies associate the SNP with some abnormalities such as cancer, other reliable investigations refuse the associations. Consequently, the interpretation of the phenotypic results of G1661A-transition has been controversial. In this study, an in silico analysis were performed to investigate the possible effects of the transition on AhR-mRNA, protein structure, interaction properties and modifications. The analysis revealed that the R554K substitution affects secondary structure and solvent accessibility of adjacent residues. Also, it causes to decreasing of the AhR stability; altering the hydropathy features of the local sequence and changing the pattern of the residues at the binding site of the TAD-acidic sub-domain. Generating of new sites for ubiquitination and acetylation for AhR-K554 variant respectively at positions 544 and 560 was predicted. Our findings intensify the idea that the AhR-G1661A transition may affects AhR-TAD interactions, especially with the TBP, which influence AhR-target genes expression. However, the previously reported flexibility of the modular TAD could act as an intervening factor, moderate the SNP effects and causes distinct outcomes in different individuals and tissues. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Tetrahydroquinoline Ring as a Versatile Bioisostere of Tetralin for Melatonin Receptor Ligands.

    PubMed

    Rivara, Silvia; Scalvini, Laura; Lodola, Alessio; Mor, Marco; Caignard, Daniel-Henri; Delagrange, Philippe; Collina, Simona; Lucini, Valeria; Scaglione, Francesco; Furiassi, Lucia; Mari, Michele; Lucarini, Simone; Bedini, Annalida; Spadoni, Gilberto

    2018-04-26

    A new family of melatonin receptor ligands, characterized by a tetrahydroquinoline (THQ) scaffold carrying an amide chain in position 3, was devised as conformationally constrained analogs of flexible N-anilinoethylamides previously developed. Molecular superposition models allowed to identify the patterns of substitution conferring high receptor binding affinity and to support the THQ ring as a suitable scaffold for the preparation of melatonin ligands. The biological activity of 3-acylamino-THQs was compared with that of the corresponding tetralin derivatives. The THQ ring proved to be a versatile scaffold for easy feasible MT 1 and MT 2 ligands, which resulted as more polar bioisosteres of their tetralin analogs. Potent partial agonists, with subnanomolar binding affinity for the MT 2 receptor, were obtained, and a new series of THQ derivatives is presented. The putative binding mode of potent THQs and tetralines was discussed on the basis of their conformational equilibria as inferred from molecular dynamics simulations and experimental NMR data.

  2. Molecular mechanism of peroxisome proliferator-activated receptor α activation by WY14643: a new mode of ligand recognition and receptor stabilization.

    PubMed

    Bernardes, Amanda; Souza, Paulo C T; Muniz, João R C; Ricci, Clarisse G; Ayers, Stephen D; Parekh, Nili M; Godoy, André S; Trivella, Daniela B B; Reinach, Peter; Webb, Paul; Skaf, Munir S; Polikarpov, Igor

    2013-08-23

    Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Novel cellular targets of AhR underlie alterations in neutrophilic inflammation and iNOS expression during influenza virus infection

    PubMed Central

    Head Wheeler, Jennifer L.; Martin, Kyle C.; Lawrence, B. Paige

    2012-01-01

    The underlying reasons for variable clinical outcomes from respiratory viral infections remain uncertain. Several studies suggest that environmental factors contribute to this variation, but limited knowledge of cellular and molecular targets of these agents hampers our ability to quantify or modify their contribution to disease and improve public health. The aryl hydrocarbon receptor (AhR) is an environment sensing transcription factor that binds many anthropogenic and natural chemicals. The immunomodulatory properties of AhR ligands are best characterized with extensive studies of changes in CD4+ T cell responses. Yet, AhR modulates other aspects of immune function. We previously showed that during influenza virus infection, AhR activation modulates neutrophil accumulation in the lung, and this contributes to increased mortality in mice. Enhanced levels of inducible nitric oxide synthase (iNOS) in infected lungs are observed during the same timeframe as AhR-mediated increased pulmonary neutrophilia. In this study, we evaluated whether these two consequences of AhR activation are causally linked. Reciprocal inhibition of AhR-mediated elevations in iNOS and pulmonary neutrophilia reveal that, although they are contemporaneous, they are not causally related. We show using Cre/loxP technology that elevated iNOS levels and neutrophil number in the infected lung result from separate, AhR-dependent signaling in endothelial and respiratory epithelial cells, respectively. Studies using mutant mice further reveal that AhR-mediated alterations in these innate responses to infection require a functional nuclear localization signal and DNA binding domain. Thus, gene targets of AhR in non-hematopoietic cells are important new considerations for understanding AhR-mediated changes in innate anti-viral immunity. PMID:23233726

  4. Pregnane X receptor regulates the AhR/Cyp1A1 pathway and protects liver cells from benzo-[α]-pyrene-induced DNA damage.

    PubMed

    Cui, Hongmei; Gu, Xinsheng; Chen, Jingshu; Xie, Ying; Ke, Sui; Wu, Jing; Golovko, Andrei; Morpurgo, Benjamin; Yan, Chunhong; Phillips, Timothy D; Xie, Wen; Luo, Jianyuan; Zhou, Zhijun; Tian, Yanan

    2017-06-05

    Pregnane X receptor (PXR) plays an important role in protecting cells from mutagenic DNA damages induced by endogenous and exogenous toxicants. This protective function is often attributed to the PXR-regulated metabolic detoxification. Here we report a novel potential mechanism that PXR reduces benzo-[α]-pyrene(BaP)-induced DNA damage through inhibiting the transcriptional activity of aryl hydrocarbon receptor (AhR) which plays a pivotal role in the bioactivation of BaP. We have utilized three well-characterized cell lines, i.e. Hepa1c1c7, AhR +/+; Bpr lacks AhR obligatory partner ARNT; Tao, lacks AhR, to analyze pivotal role of AhR/ARNT complex in mediating the BaP-induced DNA damages using comet assay (single-cell gel electrophoresis). We found that PXR activation could significantly inhibit BaP-induced DNA damage in the HepG2 cells as well as mouse hepatocytes. Using PXR-null and wild type mouse hepatocytes we showed that PXR activation by pregnenolone 16α-carbonitrile (PCN) significantly inhibited BaP-induced DNA damage and this protective effect was abolished in PXR-null hepatocytes. Mechanistically, PXR activation inhibited expression of AhR-target genes for CYP1A1, CYP1B1 and CYP1A2 that are required for BaP biotransformation in cultured liver cells, or in the livers of C57BL/6J mice. Using an AhR-responsive reporter assay as well as chromatin immunoprecipitation assay we found that PXR activation transcriptionally represses AhR-regulated gene expression. Furthermore, we found that PXR directly bound AhR at its DNA-binding domain, and this association may play a role in preventing of the AhR from binding to its target genes as shown in the ChIP assay. Taken together, our study has revealed a novel mechanism by which PXR protects liver cells from BaP-induced DNA damage through inhibiting the BaP biotransformation. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Understanding the molecular differential recognition of muramyl peptide ligands by LRR domains of human NOD receptors.

    PubMed

    Vijayrajratnam, Sukhithasri; Pushkaran, Anju Choorakottayil; Balakrishnan, Aathira; Vasudevan, Anil Kumar; Biswas, Raja; Mohan, Chethampadi Gopi

    2017-07-27

    Human nucleotide-binding oligomerization domain proteins, hNOD1 and hNOD2, are host intracellular receptors with C-terminal leucine-rich repeat (LRR) domains, which recognize specific bacterial peptidoglycan (PG) fragments as their ligands. The specificity of this recognition is dependent on the third amino acid of the stem peptide of the PG ligand, which is usually meso -diaminopimelic acid ( meso DAP) or l-lysine (l-Lys). Since the LRR domains of hNOD receptors had been experimentally shown to confer the PG ligand-sensing specificity, we developed three-dimensional structures of hNOD1-LRR and the hNOD2-LRR to understand the mechanism of differential recognition of muramyl peptide ligands by hNOD receptors. The hNOD1-LRR and hNOD2-LRR receptor models exhibited right-handed curved solenoid shape. The hot-spot residues experimentally proved to be critical for ligand recognition were located in the concavity of the NOD-LRR and formed the recognition site. Our molecular docking analyses and molecular electrostatic potential mapping studies explain the activation of hNOD-LRRs, in response to effective molecular interactions of PG ligands at the recognition site; and conversely, the inability of certain PG ligands to activate hNOD-LRRs, by deviations from the recognition site. Based on molecular docking studies using PG ligands, we propose few residues - G825, D826 and N850 in hNOD1-LRR and L904, G905, W931, L932 and S933 in hNOD2-LRR, evolutionarily conserved across different host species, which may play a major role in ligand recognition. Thus, our integrated experimental and computational approach elucidates the molecular basis underlying the differential recognition of PG ligands by hNOD receptors. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  6. Increased aryl hydrocarbon receptor expression in patients with allergic rhinitis.

    PubMed

    Wei, P; Hu, G-H; Kang, H-Y; Yao, H-B; Kou, W; Liu, H; Hong, S-L

    2014-02-01

    A predominant Th17 population is a marker of allergic rhinitis (AR). As a ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR) plays a vital role in promoting or inhibiting the development of specific Th cells. However, its role in AR remains undefined. To analyze the potential role of AhR in the pathogenesis of AR. In total, 30 AR patients and 13 healthy controls were recruited for this study and AR patients had clinical features, as demonstrated by rhinoconjunctivitis quality of life questionnaires, total symptom scores and visual analog scale scores. The expression of AhR, IL-17 and IL-22 and the presence of Th17 cells in peripheral blood mononuclear cells were measured before and after treatment with the nontoxic AhR ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Pretreatment ITE studies revealed that all AR patients had a significant increase in AhR expression compared with controls and AhR expression positively correlated with clinical parameters. After ITE intervention, a severe reduction in the differentiation of Th17 cells and the production of IL-17 and IL-22 was noted in both AR patients and normal subjects. Simultaneously, a dramatic enhancement of AhR expression was also observed in all healthy controls, but not in AR patients. The results suggested that the AhR may be one of the mechanisms underlying the Th17 response during the pathogenesis of AR and AhR levels were closely related to clinical severity in all AR patients. Additionally, ITE may represent a new drug candidate in the treatment of AR.

  7. Nicotinic acetylcholine receptor ligands; a patent review (2006-2011)

    PubMed Central

    Gündisch, Daniela; Eibl, Christoph

    2012-01-01

    Introduction Nicotinic acetylcholine receptors (nAChRs), pentameric ligand-gated cation channels, are potential targets for the development of therapeutics for a variety of disease states. Areas covered This article is reviewing recent advances in the development of small molecule ligands for diverse nAChR subtypes and is a continuation of an earlier review in this journal. Expert opinion The development of nAChR ligands with preference for α4β2 or α7 subtypes for the treatment of CNS disorders are in the most advanced developmental stage. In addition, there is a fast growing interest to generate so-called PAMs, positive allosteric modulators, to influence the channels’ functionalities. PMID:22098319

  8. Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels.

    PubMed

    Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L

    2013-03-05

    AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl(-) lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels

    PubMed Central

    Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L.

    2014-01-01

    SUMMARY AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine and phenylalanine which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate and serine. AvGluR1 LBD crystal structures reveal a novel scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, while in the alanine, methionine and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl− lowers affinity for these ligands, but not for glutamate, aspartate or for phenylalanine which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure based studies on iGluR-ligand interactions. PMID:23434404

  10. Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range

    PubMed Central

    Ventura, Alejandra C.; Bush, Alan; Vasen, Gustavo; Goldín, Matías A.; Burkinshaw, Brianne; Bhattacharjee, Nirveek; Folch, Albert; Brent, Roger; Chernomoretz, Ariel; Colman-Lerner, Alejandro

    2014-01-01

    Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general “systems level” mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step. PMID:25172920

  11. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    PubMed

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-04-28

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

  12. Sub-millisecond ligand probing of cell receptors with multiple solution exchange

    PubMed Central

    Sylantyev, Sergiy; Rusakov, Dmitri A

    2013-01-01

    The accurate knowledge of receptor kinetics is crucial to our understanding of cell signal transduction in general and neural function in particular. The classical technique of probing membrane receptors on a millisecond scale involves placing a recording micropipette with a membrane patch in front of a double-barrel (θ-glass) application pipette mounted on a piezo actuator. Driven by electric pulses, the actuator can rapidly shift the θ-glass pipette tip, thus exposing the target receptors to alternating ligand solutions. However, membrane patches survive for only a few minutes, thus normally restricting such experiments to a single-application protocol. In order to overcome this deficiency, we have introduced pressurized supply microcircuits in the θ-glass channels, thus enabling repeated replacement of application solutions within 10–15 s. this protocol, which has been validated in our recent studies and takes 20–60 min to implement, allows the characterization of ligand-receptor interactions with high sensitivity, thereby also enabling a powerful paired-sample statistical design. PMID:23744290

  13. Is the isolated ligand binding domain a good model of the domain in the native receptor?

    PubMed

    Deming, Dustin; Cheng, Qing; Jayaraman, Vasanthi

    2003-05-16

    Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.

  14. The Nuclear Receptor HIZR-1 Uses Zinc as a Ligand to Mediate Homeostasis in Response to High Zinc

    PubMed Central

    Warnhoff, Kurt; Roh, Hyun C.; Kocsisova, Zuzana; Tan, Chieh-Hsiang; Morrison, Andrew; Croswell, Damari; Schneider, Daniel L.; Kornfeld, Kerry

    2017-01-01

    Nuclear receptors were originally defined as endocrine sensors in humans, leading to the identification of the nuclear receptor superfamily. Despite intensive efforts, most nuclear receptors have no known ligand, suggesting new ligand classes remain to be discovered. Furthermore, nuclear receptors are encoded in the genomes of primitive organisms that lack endocrine signaling, suggesting the primordial function may have been environmental sensing. Here we describe a novel Caenorhabditis elegans nuclear receptor, HIZR-1, that is a high zinc sensor in an animal and the master regulator of high zinc homeostasis. The essential micronutrient zinc acts as a HIZR-1 ligand, and activated HIZR-1 increases transcription of genes that promote zinc efflux and storage. The results identify zinc as the first inorganic molecule to function as a physiological ligand for a nuclear receptor and direct environmental sensing as a novel function of nuclear receptors. PMID:28095401

  15. Effects of N-Substitutions on the Tetrahydroquinoline (THQ) Core of Mixed-Efficacy μ-Opioid Receptor (MOR)/δ-Opioid Receptor (DOR) Ligands.

    PubMed

    Harland, Aubrie A; Bender, Aaron M; Griggs, Nicholas W; Gao, Chao; Anand, Jessica P; Pogozheva, Irina D; Traynor, John R; Jutkiewicz, Emily M; Mosberg, Henry I

    2016-05-26

    N-Acetylation of the tetrahydroquinoline (THQ) core of a series of μ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist ligands increases DOR affinity, resulting in ligands with balanced MOR and DOR affinities. We report a series of N-substituted THQ analogues that incorporate various carbonyl-containing moieties to maintain DOR affinity and define the steric and electronic requirements of the binding pocket across the opioid receptors. 4h produced in vivo antinociception (ip) for 1 h at 10 mg/kg.

  16. Revisiting the Quinoxalinedione Scaffold in the Construction of New Ligands for the Ionotropic Glutamate Receptors.

    PubMed

    Demmer, Charles S; Rombach, David; Liu, Na; Nielsen, Birgitte; Pickering, Darryl S; Bunch, Lennart

    2017-11-15

    More than two decades ago, the quinoxalinedione scaffold was shown to act as an α-amino acid bioisoster. Following extensive structure-activity relationship (SAR) studies, the antagonists DNQX, CNQX, and NBQX in the ionotropic glutamate receptor field were identified. In this work, we revisit the quinoxalinedione scaffold and explore the incorporation of an acid functionality in the 6-position. The SAR studies disclose that by this strategy it was possible to tune in iGluR selectivity among the AMPA, NMDA, and KA receptors, and to some extent also obtain full receptor subtype selectivity. Highlights of the study of 44 new analogues are compound 2m being a high affinity ligand for native AMPA receptors (IC 50 = 0.48 μM), analogues 2e,f,h,k,v all displayed selectivity for native NMDA receptors, and compounds 2s,t,u are selective ligand for the GluK1 receptor. Most interestingly, compound 2w was shown to be a GluK3-preferring ligand with full selectivity over native AMPA, KA and NMDA receptors.

  17. Peroxisome Proliferator-Activated Receptor Ligands and Their Role in Chronic Myeloid Leukemia: Therapeutic Strategies.

    PubMed

    Yousefi, Bahman; Samadi, Nasser; Baradaran, Behzad; Shafiei-Irannejad, Vahid; Zarghami, Nosratollah

    2016-07-01

    Imatinib therapy remains the gold standard for treatment of chronic myeloid leukemia; however, the acquired resistance to this therapeutic agent in patients has urged the scientists to devise modalities for overcoming this chemoresistance. For this purpose, initially therapeutic agents with higher tyrosine kinase activity were introduced, which had the potential for inhibiting even mutant forms of Bcr-Abl. Furthermore, coupling imatinib with peroxisome proliferator-activated receptor ligands also showed beneficial effects in chronic myeloid leukemia cell proliferation. These combination protocols inhibited cell growth and induced apoptosis as well as differentiation in chronic myeloid leukemia cell lines. In addition, peroxisome proliferator-activated receptors ligands increased imatinib uptake by upregulating the expression of human organic cation transporter 1. Taken together, peroxisome proliferator-activated receptors ligands are currently being considered as novel promising therapeutic candidates for chronic myeloid leukemia treatment, because they can synergistically enhance the efficacy of imatinib. In this article, we reviewed the potential of peroxisome proliferator-activated receptors ligands for use in chronic myeloid leukemia treatment. The mechanism of action of these therapeutics modalities are also presented in detail. © 2016 John Wiley & Sons A/S.

  18. α2A- and α2C-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands.

    PubMed

    Sánchez-Soto, Marta; Casadó-Anguera, Verònica; Yano, Hideaki; Bender, Brian Joseph; Cai, Ning-Sheng; Moreno, Estefanía; Canela, Enric I; Cortés, Antoni; Meiler, Jens; Casadó, Vicent; Ferré, Sergi

    2018-03-18

    The poor norepinephrine innervation and high density of Gi/o-coupled α 2A - and α 2C -adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D 2 -like receptor ligands, such as the D 3 receptor agonist 7-OH-PIPAT and the D 4 receptor agonist RO-105824, to α 2 -adrenoceptors in cortical and striatal tissue, which express α 2A -adrenoceptors and both α 2A - and α 2C -adrenoceptors, respectively. The affinity of dopamine for α 2 -adrenoceptors was found to be similar to that for D 1 -like and D 2 -like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α 2A - and α 2C -adrenoceptors. Their ability to activate Gi/o proteins through α 2A - and α 2C -adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α 2 -adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α 2A - and α 2C -adrenoceptors was nearly identical to its binding to the crystallized D 3 receptor. Therefore, we provide conclusive evidence that α 2A - and α 2C -adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D 2 -like receptor ligands, which calls for revisiting previous studies with those ligands.

  19. Exploring Flexibility of Progesterone Receptor Ligand Binding Domain Using Molecular Dynamics

    PubMed Central

    Zheng, Liangzhen; Mu, Yuguang

    2016-01-01

    Progesterone receptor (PR), a member of nuclear receptor (NR) superfamily, plays a vital role for female reproductive tissue development, differentiation and maintenance. PR ligand, such as progesterone, induces conformation changes in PR ligand binding domain (LBD), thus mediates subsequent gene regulation cascades. PR LBD may adopt different conformations upon an agonist or an antagonist binding. These different conformations would trigger distinct transcription events. Therefore, the dynamics of PR LBD would be of general interest to biologists for a deep understanding of its structure-function relationship. However, no apo-form (non-ligand bound) of PR LBD model has been proposed either by experiments or computational methods so far. In this study, we explored the structural dynamics of PR LBD using molecular dynamics simulations and advanced sampling tools in both ligand-bound and the apo-forms. Resolved by the simulation study, helix 11, helix 12 and loop 895–908 (the loop between these two helices) are quite flexible in antagonistic conformation. Several residues, such as Arg899 and Glu723, could form salt-bridging interaction between helix 11 and helix 3, and are important for the PR LBD dynamics. And we also propose that helix 12 in apo-form PR LBD, not like other NR LBDs, such as human estrogen receptor α (ERα) LBD, may not adopt a totally extended conformation. With the aid of umbrella sampling and metadynamics simulations, several stable conformations of apo-form PR LBD have been sampled, which may work as critical structural models for further large scale virtual screening study to discover novel PR ligands for therapeutic application. PMID:27824891

  20. Sliding tethered ligands add topological interactions to the toolbox of ligand–receptor design

    PubMed Central

    Bauer, Martin; Kékicheff, Patrick; Iss, Jean; Fajolles, Christophe; Charitat, Thierry; Daillant, Jean; Marques, Carlos M.

    2015-01-01

    Adhesion in the biological realm is mediated by specific lock-and-key interactions between ligand–receptor pairs. These complementary moieties are ubiquitously anchored to substrates by tethers that control the interaction range and the mobility of the ligands and receptors, thus tuning the kinetics and strength of the binding events. Here we add sliding anchoring to the toolbox of ligand–receptor design by developing a family of tethered ligands for which the spacer can slide at the anchoring point. Our results show that this additional sliding degree of freedom changes the nature of the adhesive contact by extending the spatial range over which binding may sustain a significant force. By introducing sliding tethered ligands with self-regulating length, this work paves the way for the development of versatile and reusable bio-adhesive substrates with potential applications for drug delivery and tissue engineering. PMID:26350224

  1. The heterodimeric sweet taste receptor has multiple potential ligand binding sites.

    PubMed

    Cui, Meng; Jiang, Peihua; Maillet, Emeline; Max, Marianna; Margolskee, Robert F; Osman, Roman

    2006-01-01

    The sweet taste receptor is a heterodimer of two G protein coupled receptors, T1R2 and T1R3. This discovery has increased our understanding at the molecular level of the mechanisms underlying sweet taste. Previous experimental studies using sweet receptor chimeras and mutants show that there are at least three potential binding sites in this heterodimeric receptor. Receptor activity toward the artificial sweeteners aspartame and neotame depends on residues in the amino terminal domain of human T1R2. In contrast, receptor activity toward the sweetener cyclamate and the sweet taste inhibitor lactisole depends on residues within the transmembrane domain of human T1R3. Furthermore, receptor activity toward the sweet protein brazzein depends on the cysteine rich domain of human T1R3. Although crystal structures are not available for the sweet taste receptor, useful homology models can be developed based on appropriate templates. The amino terminal domain, cysteine rich domain and transmembrane helix domain of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic glutamate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. We have used homology models of the sweet taste receptors, molecular docking of sweet ligands to the receptors, and site-directed mutagenesis of the receptors to identify potential ligand binding sites of the sweet taste receptor. These studies have led to a better understanding of the structure and function of this heterodimeric receptor, and can act as a guide for rational structure-based design of novel non-caloric sweeteners, which can be used in the fighting against obesity and diabetes.

  2. Morphine-induced antinociception in the rat: supra-additive interactions with imidazoline I₂ receptor ligands.

    PubMed

    Li, Jun-Xu; Zhang, Yanan; Winter, Jerrold C

    2011-11-01

    Pain remains a significant clinical challenge and currently available analgesics are not adequate to meet clinical needs. Emerging evidence suggests the role of imidazoline I(2) receptors in pain modulation primarily from studies of the non-selective imidazoline receptor ligand, agmatine. However, little is known of the generality of the effect to selective I(2) receptor ligands. This study examined the antinociceptive effects of two selective I(2) receptor ligands 2-BFI and BU224 (>2000-fold selectivity for I(2) receptors over α(2) adrenoceptors) in a hypertonic (5%) saline-induced writhing test and analyzed their interaction with morphine using a dose-addition analysis. Morphine, 2-BFI and BU224 but not agmatine produced a dose-dependent antinociceptive effect. Both composite additive curve analyses and isobolographical plots revealed a supra-additive interaction between morphine and 2-BFI or BU224, whereas the interaction between 2-BFI and BU224 was additive. The antinociceptive effect of 2-BFI and BU224 was attenuated by the I(2) receptor antagonist/α(2) adrenoceptor antagonist idazoxan but not by the selective α(2) adrenoceptor antagonist yohimbine, suggesting an I(2) receptor-mediated mechanism. Agmatine enhanced the antinociceptive effect of morphine, 2-BFI and BU224 and the enhancement was prevented by yohimbine, suggesting that the effect was mediated by α(2) adrenoceptors. Taken together, these data represent the first report that selective I(2) receptor ligands have substantial antinociceptive activity and produce antinociceptive synergy with opioids in a rat model of acute pain. These data suggest that drugs acting on imidazoline I(2) receptors may be useful either alone or in combination with opioids for the treatment of pain. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Ligand-Receptor Interaction-Mediated Transmembrane Transport of Dendrimer-like Soft Nanoparticles: Mechanisms and Complicated Diffusive Dynamics.

    PubMed

    Liang, Junshi; Chen, Pengyu; Dong, Bojun; Huang, Zihan; Zhao, Kongyin; Yan, Li-Tang

    2016-05-09

    Nearly all nanomedical applications of dendrimer-like soft nanoparticles rely on the functionality of attached ligands. Understanding how the ligands interact with the receptors in cell membrane and its further effect on the cellular uptake of dendrimer-like soft nanoparticles is thereby a key issue for their better application in nanomedicine. However, the essential mechanism and detailed kinetics for the ligand-receptor interaction-mediated transmembrane transport of such unconventional nanoparticles remain poorly elucidated. Here, using coarse-grained simulations, we present the very first study of molecular mechanism and kinetics behaviors for the transmembrane transport of dendrimer-like soft nanoparticles conjugated with ligands. A phase diagram of interaction states is constructed through examining ligand densities and membrane tensions that allows us to identify novel endocytosis mechanisms featured by the direct wrapping and the penetration-extraction vesiculation. The results provide an in-depth insight into the diffusivity of receptors and dendrimer in the membrane plane and demonstrate how the ligand density influences receptor diffusion and uptake kinetics. It is interesting to find that the ligand-conjugated dendrimers present superdiffusive behaviors on a membrane, which is revealed to be driven by the random fluctuation dynamics of the membrane. The findings facilitate our understanding of some recent experimental observations and could establish fundamental principles for the future development of such important nanomaterials for widespread nanomedical applications.

  4. Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor.

    PubMed

    Lane-Serff, Harriet; MacGregor, Paula; Lowe, Edward D; Carrington, Mark; Higgins, Matthew K

    2014-12-12

    The haptoglobin-haemoglobin receptor (HpHbR) of African trypanosomes allows acquisition of haem and provides an uptake route for trypanolytic factor-1, a mediator of innate immunity against trypanosome infection. In this study, we report the structure of Trypanosoma brucei HpHbR in complex with human haptoglobin-haemoglobin (HpHb), revealing an elongated ligand-binding site that extends along its membrane distal half. This contacts haptoglobin and the β-subunit of haemoglobin, showing how the receptor selectively binds HpHb over individual components. Lateral mobility of the glycosylphosphatidylinositol-anchored HpHbR, and a ∼50° kink in the receptor, allows two receptors to simultaneously bind one HpHb dimer. Indeed, trypanosomes take up dimeric HpHb at significantly lower concentrations than monomeric HpHb, due to increased ligand avidity that comes from bivalent binding. The structure therefore reveals the molecular basis for ligand and innate immunity factor uptake by trypanosomes and identifies adaptations that allow efficient ligand uptake in the context of the complex trypanosome cell surface.

  5. Ligand-Dependent Activation and Deactivation of the Human Adenosine A2A Receptor

    PubMed Central

    Li, Jianing; Jonsson, Amanda L.; Beuming, Thijs; Shelley, John C.; Voth, Gregory A.

    2013-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins with critical functions in cellular signal transduction, representing a primary class of drug targets. Acting by direct binding, many drugs modulate GPCR activity and influence the signaling pathways associated with numerous diseases. However, complete details of ligand-dependent GPCR activation/deactivation are difficult to obtain from experiments. Therefore, it remains unclear how ligands modulate a GPCR’s activity. To elucidate the ligand-dependent activation/deactivation mechanism of the human adenosine A2A receptor (AA2AR), a member of the class A GPCRs, we performed large-scale unbiased molecular dynamics and metadynamics simulations of the receptor embedded in a membrane. At the atomic level, we have observed distinct structural states that resemble the active and inactive states. In particular we noted key structural elements changing in a highly concerted fashion during the conformational transitions, including six conformational states of a tryptophan (Trp2466.48). Our findings agree with a previously proposed view, that during activation, this tryptophan residue undergoes a rotameric transition that may be coupled to a series of coherent conformational changes, resulting in the opening of the G protein-binding site. Further, metadynamics simulations provide quantitative evidence for this mechanism, suggesting how ligand binding shifts the equilibrium between the active and inactive states. Our analysis also proposes that a few specific residues are associated with agonism/antagonism, affinity and selectivity, and suggests that the ligand-binding pocket can be thought of as having three distinct regions, providing dynamic features for structure-based design. Additional simulations with AA2AR bound to a novel ligand are consistent with our proposed mechanism. Generally, our study provides insights into the ligand-dependent AA2AR activation/deactivation in addition to what has been found in crystal

  6. Ligand-dependent activation and deactivation of the human adenosine A(2A) receptor.

    PubMed

    Li, Jianing; Jonsson, Amanda L; Beuming, Thijs; Shelley, John C; Voth, Gregory A

    2013-06-12

    G-protein-coupled receptors (GPCRs) are membrane proteins with critical functions in cellular signal transduction, representing a primary class of drug targets. Acting by direct binding, many drugs modulate GPCR activity and influence the signaling pathways associated with numerous diseases. However, complete details of ligand-dependent GPCR activation/deactivation are difficult to obtain from experiments. Therefore, it remains unclear how ligands modulate a GPCR's activity. To elucidate the ligand-dependent activation/deactivation mechanism of the human adenosine A2A receptor (AA2AR), a member of the class A GPCRs, we performed large-scale unbiased molecular dynamics and metadynamics simulations of the receptor embedded in a membrane. At the atomic level, we have observed distinct structural states that resemble the active and inactive states. In particular, we noted key structural elements changing in a highly concerted fashion during the conformational transitions, including six conformational states of a tryptophan (Trp246(6.48)). Our findings agree with a previously proposed view that, during activation, this tryptophan residue undergoes a rotameric transition that may be coupled to a series of coherent conformational changes, resulting in the opening of the G-protein binding site. Further, metadynamics simulations provide quantitative evidence for this mechanism, suggesting how ligand binding shifts the equilibrium between the active and inactive states. Our analysis also proposes that a few specific residues are associated with agonism/antagonism, affinity, and selectivity, and suggests that the ligand-binding pocket can be thought of as having three distinct regions, providing dynamic features for structure-based design. Additional simulations with AA2AR bound to a novel ligand are consistent with our proposed mechanism. Generally, our study provides insights into the ligand-dependent AA2AR activation/deactivation in addition to what has been found in

  7. The Role of AhR in Autoimmune Regulation and Its Potential as a Therapeutic Target against CD4 T Cell Mediated Inflammatory Disorder

    PubMed Central

    Zhu, Conghui; Xie, Qunhui; Zhao, Bin

    2014-01-01

    AhR has recently emerged as a critical physiological regulator of immune responses affecting both innate and adaptive systems. Since the AhR signaling pathway represents an important link between environmental stimulators and immune-mediated inflammatory disorder, it has become the object of great interest among researchers recently. The current review discusses new insights into the mechanisms of action of a select group of inflammatory autoimmune diseases and the ligand-activated AhR signaling pathway. Representative ligands of AhR, both exogenous and endogenous, are also reviewed relative to their potential use as tools for understanding the role of AhR and as potential therapeutics for the treatment of various inflammatory autoimmune diseases, with a focus on CD4 helper T cells, which play important roles both in self-immune tolerance and in inflammatory autoimmune diseases. Evidence indicating the potential use of these ligands in regulating inflammation in various diseases is highlighted, and potential mechanisms of action causing immune system effects mediated by AhR signaling are also discussed. The current review will contribute to a better understanding of the role of AhR and its signaling pathway in CD4 helper T cell mediated inflammatory disorder. Considering the established importance of AhR in immune regulation and its potential as a therapeutic target, we also think that both further investigation into the molecular mechanisms of immune regulation that are mediated by the ligand-specific AhR signaling pathway, and integrated research and development of new therapeutic drug candidates targeting the AhR signaling pathway should be pursued urgently. PMID:24905409

  8. Multi-functional norrin is a ligand for the LGR4 receptor

    PubMed Central

    Deng, Cheng; Reddy, Pradeep; Cheng, Yuan; Luo, Ching-Wei; Hsiao, Chih-Lun; Hsueh, Aaron J. W.

    2013-01-01

    Summary Mammalian LGR4, 5 and 6 are seven-transmembrane receptors that are important for diverse physiological processes. These receptors are orthologous to DLGR2, a Drosophila receptor activated by the burs/pburs heterodimer important for morphogenesis. Although recent studies indicated that four R-spondin proteins are cognate ligands for LGR4, 5 and 6 receptors, several BMP antagonists in vertebrates have been postulated to be orthologous to burs and pburs. Using newly available genome sequences, we showed that norrin is a vertebrate ortholog for insect burs and pburs and stimulates Wnt signaling mediated by LGR4, but not by LGR5 and 6, in mammalian cells. Although norrin could only activate LGR4, binding studies suggested interactions between norrin and LGR4, 5 and 6. Norrin, the Norrie disease gene product, is also capable of activating Wnt signaling mediated by the Frizzled4 receptor and serves as a BMP antagonist. Mutagenesis studies indicated that different norrin mutations found in patients with Norrie disease can be categorized into subgroups according to defects for signaling through the three distinct binding proteins. Thus, norrin is a rare ligand capable of binding three receptors/binding proteins that are important for BMP and Wnt signaling pathways. PMID:23444378

  9. Multi-functional norrin is a ligand for the LGR4 receptor.

    PubMed

    Deng, Cheng; Reddy, Pradeep; Cheng, Yuan; Luo, Ching-Wei; Hsiao, Chih-Lun; Hsueh, Aaron J W

    2013-05-01

    Mammalian LGR4, 5 and 6 are seven-transmembrane receptors that are important for diverse physiological processes. These receptors are orthologous to DLGR2, a Drosophila receptor activated by the burs/pburs heterodimer important for morphogenesis. Although recent studies indicated that four R-spondin proteins are cognate ligands for LGR4, 5 and 6 receptors, several BMP antagonists in vertebrates have been postulated to be orthologous to burs and pburs. Using newly available genome sequences, we showed that norrin is a vertebrate ortholog for insect burs and pburs and stimulates Wnt signaling mediated by LGR4, but not by LGR5 and 6, in mammalian cells. Although norrin could only activate LGR4, binding studies suggested interactions between norrin and LGR4, 5 and 6. Norrin, the Norrie disease gene product, is also capable of activating Wnt signaling mediated by the Frizzled4 receptor and serves as a BMP antagonist. Mutagenesis studies indicated that different norrin mutations found in patients with Norrie disease can be categorized into subgroups according to defects for signaling through the three distinct binding proteins. Thus, norrin is a rare ligand capable of binding three receptors/binding proteins that are important for BMP and Wnt signaling pathways.

  10. 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

  11. Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

    PubMed

    Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

    2017-03-15

    Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor.

    PubMed

    Vandenbulcke, F; Nouel, D; Vincent, J P; Mazella, J; Beaudet, A

    2000-09-01

    The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30-45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.

  13. Activin receptor ligand traps in chronic kidney disease.

    PubMed

    Jelkmann, Wolfgang

    2018-05-29

    Sotatercept and luspatercept are recombinant soluble activin type-II receptor-IgG-Fc fusion proteins that are tested in clinical trials for the treatment of various types of anemias, including renal anemia. The mechanism of the action of the novel drugs is incompletely understood, but it seems to be based on the inactivation of soluble proteins of the transforming growth factor-ß (TGFß) family. This review considers pros and cons of the clinical use of the drugs in reference to the current therapy with recombinant erythropoiesis-stimulating agents (ESAs). One or more activin type-II receptor (ActRII) ligands appear to inhibit erythroid precursors, for example growth and differentiation factor 11. Trapping of these ligands by the recombinant ActRII fusion proteins, sotatercept and luspatercept increases red blood cell numbers and hemoglobin levels in humans. Reportedly, the novel compounds were well tolerated in trials on healthy volunteers and patients suffering from anemia due to chronic kidney disease or malignancies. On approval, the drugs may prove particularly useful in patients suffering from ineffective erythropoiesis, such as in myelodysplastic syndrome, multiple myeloma or ß-thalassemia, where ESAs are of little use. Independent of their effect on erythropoiesis, ActRII ligand traps were found to exert beneficial effects on renal tissue in experimental animals. ESAs are likely to remain standard of care in renal anemia. There is a need for a better understanding of the effects of ActRII ligand traps on TGFß-like proteins. The novel drugs have not been approved for sale as therapeutics so far. Their long-term efficacy and safety still needs to be proven, particularly with respect to immunogenicity. Antifibrotic effects may be worthy to be investigated in humans.

  14. Synthesis and biological activity of small peptides as NOP and opioid receptors' ligands: view on current developments.

    PubMed

    Naydenova, Emilia; Todorov, Petar; Zamfirova, Rositza

    2015-01-01

    The heptadecapeptide nociceptin, also called orphanin FQ (N/OFQ), is the endogenous agonist of the N/OFQ peptide receptor (NOP receptor) and is involved in several central nervous system pathways, such as nociception, reward, tolerance, and feeding. The discovery of small molecule ligands for NOP is being actively pursued for several therapeutic applications. This review presents overview of the several recently reported NOP ligands (agonists and antagonists), with an emphasis of the structural features that may be important for modulating the intrinsic activity of these ligands. In addition, a brief account on the characterization of newly synthesized ligands of NOP receptor with aminophosphonate moiety and β-tryptophan analogues will be presented. © 2015 Elsevier Inc. All rights reserved.

  15. Dose- and time-dependent expression of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) in PCB-, B[a]P-, and TBT-exposed intertidal copepod Tigriopus japonicus.

    PubMed

    Kim, Bo-Mi; Rhee, Jae-Sung; Hwang, Un-Ki; Seo, Jung Soo; Shin, Kyung-Hoon; Lee, Jae-Seong

    2015-02-01

    The aryl hydrocarbon receptor (AhR) and aryl hydrocarbon nuclear translocator (ARNT) genes from the copepod Tigriopus japonicus (Tj) were cloned to examine their potential functions in the invertebrate putative AhR-CYP signaling pathway. The amino acid sequences encoded by the Tj-AhR and Tj-ARNT genes showed high similarity to homologs of Daphnia and Drosophila, ranging from 68% and 70% similarity for the AhR genes to 56% for the ARNT genes. To determine whether Tj-AhR and Tj-ARNT are modulated by environmental pollutants, transcriptional expression of Tj-AhR and Tj-ARNT was analyzed in response to exposure to five concentrations of polychlorinated biphenyl (PCB 126) (control, 10, 50, 100, 500 μg L(-1)), benzo[a]pyrene (B[a]P) (control, 5, 10, 50, 100 μg L(-1)), and tributyltin (TBT) (control, 1, 5, 10, 20 μg L(-1)) 24h after exposure. A time-course experiment (0, 3, 6, 12, 24h) was performed to analyze mRNA expression patterns after exposure to PCB, B[a]P, and TBT. T. japonicus exhibited dose-dependent and time-dependent upregulation of Tj-AhR and Tj-ARNT in response to pollutant exposure, and the degree of expression was dependent on the pollutant, suggesting that pollutants such as PCB, B[a]P, and TBT modulate expression of Tj-AhR and Tj-ARNT genes in the putative AhR-CYP signaling pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Differential expression of VEGF ligands and receptors in prostate cancer.

    PubMed

    Woollard, David J; Opeskin, Kenneth; Coso, Sanja; Wu, Di; Baldwin, Megan E; Williams, Elizabeth D

    2013-05-01

    Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer. The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer. Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue. These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer. Copyright © 2012 Wiley Periodicals, Inc.

  17. Serotonin is an endogenous regulator of intestinal CYP1A1 via AhR.

    PubMed

    Manzella, Christopher; Singhal, Megha; Alrefai, Waddah A; Saksena, Seema; Dudeja, Pradeep K; Gill, Ravinder K

    2018-04-17

    Aryl hydrocarbon receptor (AhR) is a nuclear receptor that controls xenobiotic detoxification via induction of cytochrome P450 1A1 (CYP1A1) and regulates immune responses in the intestine. Metabolites of L-tryptophan activate AhR, which confers protection against intestinal inflammation. We tested the hypothesis that serotonin (5-HT) is an endogenous activator of AhR in intestinal epithelial cells. Treatment of Caco-2 monolayers with 5-HT induced CYP1A1 mRNA in a time- and concentration-dependent manner and also stimulated CYP1A1 activity. CYP1A1 induction by 5-HT was dependent upon uptake via serotonin transporter (SERT). Antagonism of AhR and knockdown of AhR and its binding partner aryl hydrocarbon receptor nuclear translocator (ARNT) attenuated CYP1A1 induction by 5-HT. Activation of AhR was evident by its nuclear translocation after 5-HT treatment and by induction of an AhR-responsive luciferase reporter. In vivo studies showed a dramatic decrease in CYP1A1 expression and other AhR target genes in SERT KO ileal mucosa by microarray analysis. These results suggest that intracellular accumulation of 5-HT via SERT induces CYP1A1 expression via AhR in intestinal epithelial cells, and SERT deficiency in vivo impairs activation of AhR. Our studies provide a novel link between the serotonergic and AhR pathways which has implications in xenobiotic metabolism and intestinal inflammation.

  18. Trace metals in the brain: allosteric modulators of ligand-gated receptor channels, the case of ATP-gated P2X receptors.

    PubMed

    Huidobro-Toro, J Pablo; Lorca, Ramón A; Coddou, Claudio

    2008-03-01

    Zinc and copper are indispensable trace metals for life with a recognized role as catalysts in enzyme actions. We now review evidence supporting the role of trace metals as novel allosteric modulators of ionotropic receptors: a new and fundamental physiological role for zinc and copper in neuronal and brain excitability. The review is focussed on ionotropic receptor channels including nucleotide receptors, in particular the P2X receptor family. Since zinc and copper are stored within synaptic vesicles in selected brain regions, and released to the synaptic cleft upon electrical nerve ending depolarization, it is plausible that zinc and copper reach concentrations in the synapse that profoundly affect ligand-gated ionic channels, including the ATP-gated currents of P2X receptors. The identification of key P2X receptor amino acids that act as ligands for trace metal coordination, carves the structural determinants underlying the allosteric nature of the trace metal modulation. The recognition that the identified key residues such as histidines, aspartic and glutamic acids or cysteines in the extracellular domain are different for each P2X receptor subtype and may be different for each metal, highlights the notion that each P2X receptor subtype evolved independent strategies for metal coordination, which form upon the proper three-dimensional folding of the receptor channels. The understanding of the molecular mechanism of allosteric modulation of ligand-operated ionic channels by trace metals is a new contribution to metallo-neurobiology.

  19. Ligand-Independent Epidermal Growth Factor Receptor Overexpression Correlates with Poor Prognosis in Colorectal Cancer.

    PubMed

    Yun, Sumi; Kwak, Yoonjin; Nam, Soo Kyung; Seo, An Na; Oh, Heung-Kwon; Kim, Duck-Woo; Kang, Sung-Bum; Lee, Hye Seung

    2018-01-17

    Molecular treatments targeting epidermal growth factor receptors (EGFRs) are important strategies for advanced colorectal cancer (CRC). However, clinicopathologic implications of EGFRs and EGFR ligand signaling have not been fully evaluated. We evaluated the expression of EGFR ligands and correlation with their receptors, clinicopathologic factors, and patients' survival with CRC. The expression of EGFR ligands, including heparin binding epidermal growth factor like growth factor (HBEGF), transforming growth factor (TGF), betacellulin, and epidermal growth factor (EGF), were evaluated in 331 consecutive CRC samples using mRNA in situ hybridization (ISH). We also evaluated the expression status of EGFR, human epidermal growth factor receptor 2 (HER2), HER3, and HER4 using immunohistochemistry and/or silver ISH. Unlike low incidences of TGF (38.1%), betacellulin (7.9%), and EGF (2.1%), HBEGF expression was noted in 62.2% of CRC samples. However, the expression of each EGFR ligand did not reveal significant correlations with survival. The combined analyses of EGFR ligands and EGFR expression indicated that the ligands‒/EGFR+ group showed a significant association with the worst disease-free survival (DFS, p=0.018) and overall survival (OS, p=0.005). It was also an independent, unfavorable prognostic factor for DFS (p=0.026) and OS (p=0.007). Additionally, HER4 nuclear expression, regardless of ligand expression, was an independent, favorable prognostic factor for DFS (p=0.034) and OS (p=0.049), by multivariate analysis. Ligand-independent EGFR overexpression was suggested to have a significant prognostic impact; thus, the expression status of EGFR ligands, in addition to EGFR, might be necessary for predicting patients' outcome in CRC.

  20. Aqueous and Ethanolic Valeriana officinalis Extracts Change the Binding of Ligands to Glutamate Receptors.

    PubMed

    Del Valle-Mojica, Lisa M; Cordero-Hernández, José M; González-Medina, Giselle; Ramos-Vélez, Igmeris; Berríos-Cartagena, Nairimer; Torres-Hernández, Bianca A; Ortíz, José G

    2011-01-01

    The effects of two valerian extracts (aqueous and hydroalcoholic) were investigated through [(3)H]Glutamate ([(3)H]Glu) and [(3)H]Fluorowillardine ([(3)H]FW) receptor binding assays using rat synaptic membranes in presence of different receptor ligands. In addition, the extract stability was monitored spectrophotometrically. Both extracts demonstrated interaction with ionotropic glutamate receptors (iGluRs). However, the extracts displayed considerable differences in receptor selectivity. The hydroalcoholic extract selectively interacted with quisqualic acid (QA), group I metabotropic glutamate receptor (mGluR) ligand, while the aqueous extract did not alter the binding of QA. The stability of the extracts was examined during several weeks. Freshly prepared extract inhibited 38-60% of [(3)H]FW binding (AMPA). After 10 days, the aqueous extract inhibited 85% of [(3)H]FW binding while the hydroalcoholic extract markedly potentiated (200%) [(3)H]FW binding to AMPA receptors. Thus, our results showed that factors such as extraction solvent and extract stability determine the selectivity for glutamate receptor (GluR) interactions.

  1. Ligand-dependent nucleo-cytoplasmic shuttling of peroxisome proliferator-activated receptors, PPARα and PPARγ.

    PubMed

    Umemoto, Tomoe; Fujiki, Yukio

    2012-07-01

    Peroxisome proliferator-activated receptors (PPARs) play important roles in diverse biological processes including metabolisms of sugars and lipids and differentiation of cells such as adipocytes. PPARs are transcription factors belonging to the ligand-dependent hormone receptor group. To function as transcription factors, PPARs translocate into nucleus where they associate with transcription apparatus. However, mechanisms underlying nuclear transport of PPARs remain enigmatic. We show here that PPARα and PPARγ dynamically shuttle between nucleus and cytoplasm, although they constitutively and predominantly appear in nucleus. With a series of truncation mutants, we identify that PPAR nuclear transport is mediated by at least two nuclear localization signals (NLSs) in DNA-binding domain (DBD)-hinge and activation function 1 (AF1) regions and their respective receptors including importinα/β, importin 7, and an unidentified receptor. PPARs also harbor two nuclear export signals in DBD and ligand-binding domain regions that are recognized by distinct export receptors, calreticulin and CRM1. Moreover, we show that nuclear-cytoplasmic shuttling of PPARs is regulated by respective PPAR ligands and Ca2+ concentration. Taken together, we suggest that the multiple pathways for the nuclear-cytoplasmic transport of PPARs regulate the biological functions of PPARs in response to external signals. © 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  2. Optimizing Ligand Efficiency of Selective Androgen Receptor Modulators (SARMs)

    PubMed Central

    2015-01-01

    A series of selective androgen receptor modulators (SARMs) containing the 1-(trifluoromethyl)benzyl alcohol core have been optimized for androgen receptor (AR) potency and drug-like properties. We have taken advantage of the lipophilic ligand efficiency (LLE) parameter as a guide to interpret the effect of structural changes on AR activity. Over the course of optimization efforts the LLE increased over 3 log units leading to a SARM 43 with nanomolar potency, good aqueous kinetic solubility (>700 μM), and high oral bioavailability in rats (83%). PMID:26819671

  3. The aryl hydrocarbon receptor: multitasking in the immune system.

    PubMed

    Stockinger, Brigitta; Di Meglio, Paola; Gialitakis, Manolis; Duarte, João H

    2014-01-01

    The aryl hydrocarbon receptor (AhR), for many years almost exclusively studied by the pharmacology/toxicology field for its role in mediating the toxicity of xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has more recently attracted the attention of immunologists. The evolutionary conservation of this transcription factor and its widespread expression in the immune system point to important physiological functions that are slowly being unraveled. In particular, the emphasis is now shifting from the role of AhR in the xenobiotic pathway toward its mode of action in response to physiological ligands. In this article, we review the current understanding of the molecular interactions and functions of AhR in the immune system in steady state and in the presence of infection and inflammation, with a focus on barrier organs such as the skin, the gut, and the lung.

  4. General Linker Diversification Approach to Bivalent Ligand Assembly: Generation of an Array of Ligands for the Cation-Independent Mannose 6-Phosphate Receptor.

    PubMed

    Fei, Xiang; Zavorka, Megan E; Malik, Guillaume; Connelly, Christopher M; MacDonald, Richard G; Berkowitz, David B

    2017-08-18

    A generalized strategy is presented for the rapid assembly of a set of bivalent ligands with a variety of linking functionalities from a common monomer. Herein, an array of phosphatase-inert mannose-6-phosphonate-presenting ligands for the cation-independent-mannose 6-phosphate receptor (CI-MPR) is constructed. Receptor binding affinity varies with linking functionality-the simple amide and 1,5-triazole(tetrazole) being preferred over the 1,4-triazole. This approach is expected to find application across chemical biology, particularly in glycoscience, wherein multivalency often governs molecular recognition.

  5. Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor.

    PubMed

    Freeman, Spencer A; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E; Wong, Harikesh S; Abraham, Libin; Graves, Marcia L; Coombs, Daniel; Roskelley, Calvin D; Das, Raibatak; Grinstein, Sergio; Gold, Michael R

    2015-02-03

    Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection.

  6. Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor

    PubMed Central

    Freeman, Spencer A.; Jaumouillé, Valentin; Choi, Kate; Hsu, Brian E.; Wong, Harikesh S.; Abraham, Libin; Graves, Marcia L.; Coombs, Daniel; Roskelley, Calvin D.; Das, Raibatak; Grinstein, Sergio; Gold, Michael R.

    2015-01-01

    Integrating signals from multiple receptors allows cells to interpret the physiological context in which a signal is received. Here we describe a mechanism for receptor crosstalk in which receptor-induced increases in actin dynamics lower the threshold for signalling by another receptor. We show that the Toll-like receptor ligands lipopolysaccharide and CpG DNA, which are conserved microbial molecules, enhance signalling by the B-cell antigen receptor (BCR) by activating the actin-severing protein cofilin. Single-particle tracking reveals that increased severing of actin filaments reduces the spatial confinement of the BCR within the plasma membrane and increases BCR mobility. This allows more frequent collisions between BCRs and greater signalling in response to low densities of membrane-bound antigen. These findings implicate actin dynamics as a means of tuning receptor signalling and as a mechanism by which B cells distinguish inert antigens from those that are accompanied by indicators of microbial infection. PMID:25644899

  7. Characterization and Evaluation of Two Novel Fluorescent Sigma-2 Receptor Ligands as Proliferation Probes

    PubMed Central

    Zeng, Chenbo; Vangveravong, Suwanna; Jones, Lynne A.; Hyrc, Krzysztof; Chang, Katherine C.; Xu, Jinbin; Rothfuss, Justin M.; Goldberg, Mark P.; Hotchkiss, Richard S.; Mach, Robert H.

    2015-01-01

    We synthesized and characterized two novel fluorescent sigma-2 receptor selective ligands, SW120 and SW116, and evaluated these ligands as potential probes for imaging cell proliferation. Both ligands are highly selective for sigma-2 receptors versus sigma-1 receptors. SW120 and SW116 were internalized into MDA-MB-435 cells, and 50% of the maximum fluorescent intensity was reached in 11 and 24 minutes, respectively. In vitro studies showed that 50% of SW120 or SW116 washed out of cells in 1 hour. The internalization of SW120 was reduced ≈30% by phenylarsine oxide, an inhibitor of endocytosis, suggesting that sigma-2 ligands are internalized, in part, by an endocytotic pathway. Subcellular localization studies using confocal and two-photon microscopy showed that SW120 and SW116 partially colocalized with fluorescent markers of mitochondria, endoplasmic reticulum, lysosomes, and the plasma membrane, suggesting that sigma-2 receptors localized to the cytoplasmic organelles and plasma membrane. SW120 did not colocalize with the nuclear dye 4′,6-diamidino-2-phenylindole. In vivo studies showed that the uptake of SW120 in solid tumors and peripheral blood mononuclear cells of mice positively correlated with the expression level of the cell proliferation marker Ki-67, suggesting that sigma-2 fluorescent probes may be used to image cell proliferation in mice. PMID:22201533

  8. Novel Somatostatin Receptor Ligands Therapies for Acromegaly

    PubMed Central

    Paragliola, Rosa Maria; Salvatori, Roberto

    2018-01-01

    Surgery is considered the treatment of choice in acromegaly, but patients with persistent disease after surgery or in whom surgery cannot be considered require medical therapy. Somatostatin receptor ligands (SRLs) octreotide (OCT), lanreotide, and the more recently approved pasireotide, characterized by a broader receptor ligand binding profile, are considered the mainstay in the medical management of acromegaly. However, in the attempt to offer a more efficacious and better tolerated medical approach, recent research has been aimed to override some limitations related to the use of currently approved drugs and novel SRLs therapies, with potential attractive features, have been proposed. These include both new formulation of older molecules and new molecules. Novel OCT formulations are aimed in particular to improve patients’ compliance and to reduce injection discomfort. They include an investigational ready-to-use subcutaneous depot OCT formulation (CAM2029), delivered via prefilled syringes and oral OCT that uses a “transient permeability enhancer” technology, which allows for OCT oral absorption. Another new delivery system is a long-lasting OCT implant (VP-003), which provide stable doses of OCT throughout a period of several months. Finally, a new SRL DG3173 (somatoprim) seems to be more selective for GH secretion, suggesting possible advantages in the presence of hyperglycemia or diabetes. How much these innovations will actually be beneficial to acromegaly patients in real clinical practice remains to be seen. PMID:29563895

  9. International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid Receptors and Their Ligands: Beyond CB1 and CB2

    PubMed Central

    Howlett, A. C.; Abood, M. E.; Alexander, S. P. H.; Di Marzo, V.; Elphick, M. R.; Greasley, P. J.; Hansen, H. S.; Kunos, G.; Mackie, K.; Mechoulam, R.; Ross, R. A.

    2010-01-01

    There are at least two types of cannabinoid receptors (CB1 and CB2). Ligands activating these G protein-coupled receptors (GPCRs) include the phytocannabinoid Δ9-tetrahydrocannabinol, numerous synthetic compounds, and endogenous compounds known as endocannabinoids. Cannabinoid receptor antagonists have also been developed. Some of these ligands activate or block one type of cannabinoid receptor more potently than the other type. This review summarizes current data indicating the extent to which cannabinoid receptor ligands undergo orthosteric or allosteric interactions with non-CB1, non-CB2 established GPCRs, deorphanized receptors such as GPR55, ligand-gated ion channels, transient receptor potential (TRP) channels, and other ion channels or peroxisome proliferator-activated nuclear receptors. From these data, it is clear that some ligands that interact similarly with CB1 and/or CB2 receptors are likely to display significantly different pharmacological profiles. The review also lists some criteria that any novel “CB3” cannabinoid receptor or channel should fulfil and concludes that these criteria are not currently met by any non-CB1, non-CB2 pharmacological receptor or channel. However, it does identify certain pharmacological targets that should be investigated further as potential CB3 receptors or channels. These include TRP vanilloid 1, which possibly functions as an ionotropic cannabinoid receptor under physiological and/or pathological conditions, and some deorphanized GPCRs. Also discussed are 1) the ability of CB1 receptors to form heteromeric complexes with certain other GPCRs, 2) phylogenetic relationships that exist between CB1/CB2 receptors and other GPCRs, 3) evidence for the existence of several as-yet-uncharacterized non-CB1, non-CB2 cannabinoid receptors; and 4) current cannabinoid receptor nomenclature. PMID:21079038

  10. Ligand-receptor co-evolution shaped the jasmonate pathway in land plants.

    PubMed

    Monte, Isabel; Ishida, Sakiko; Zamarreño, Angel M; Hamberg, Mats; Franco-Zorrilla, José M; García-Casado, Gloria; Gouhier-Darimont, Caroline; Reymond, Philippe; Takahashi, Kosaku; García-Mina, José M; Nishihama, Ryuichi; Kohchi, Takayuki; Solano, Roberto

    2018-05-01

    The phytohormone jasmonoyl-isoleucine (JA-Ile) regulates defense, growth and developmental responses in vascular plants. Bryophytes have conserved sequences for all JA-Ile signaling pathway components but lack JA-Ile. We show that, in spite of 450 million years of independent evolution, the JA-Ile receptor COI1 is functionally conserved between the bryophyte Marchantia polymorpha and the eudicot Arabidopsis thaliana but COI1 responds to different ligands in each species. We identified the ligand of Marchantia MpCOI1 as two isomeric forms of the JA-Ile precursor dinor-OPDA (dinor-cis-OPDA and dinor-iso-OPDA). We demonstrate that AtCOI1 functionally complements Mpcoi1 mutation and confers JA-Ile responsiveness and that a single-residue substitution in MpCOI1 is responsible for the evolutionary switch in ligand specificity. Our results identify the ancestral bioactive jasmonate and clarify its biosynthetic pathway, demonstrate the functional conservation of its signaling pathway, and show that JA-Ile and COI1 emergence in vascular plants required co-evolution of hormone biosynthetic complexity and receptor specificity.

  11. Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands

    PubMed Central

    Chilmonczyk, Zdzisław; Bojarski, Andrzej Jacek; Pilc, Andrzej; Sylte, Ingebrigt

    2015-01-01

    Serotonin (5-HT) is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT) inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems), which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies. PMID:26262615

  12. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    ERIC Educational Resources Information Center

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  13. Ligand and coactivator identity determines the requirement of the charge clamp for coactivation of the peroxisome proliferator-activated receptor gamma.

    PubMed

    Wu, Yifei; Chin, William W; Wang, Yong; Burris, Thomas P

    2003-03-07

    The activation function 2 (AF-2)-dependent recruitment of coactivator is essential for gene activation by nuclear receptors. We show that the peroxisome proliferator-activated receptor gamma (PPARgamma) (NR1C3) coactivator-1 (PGC-1) requires both the intact AF-2 domain of PPARgamma and the LXXLL domain of PGC-1 for ligand-dependent and ligand-independent interaction and coactivation. Although the AF-2 domain of PPARgamma is absolutely required for PGC-1-mediated coactivation, this coactivator displayed a unique lack of requirement for the charge clamp of the ligand-binding domain of the receptor that is thought to be essential for LXXLL motif recognition. The mutation of a single serine residue adjacent to the core LXXLL motif of PGC-1 led to restoration of the typical charge clamp requirement. Thus, the unique structural features of the PGC-1 LXXLL motif appear to mediate an atypical mode of interaction with PPARgamma. Unexpectedly, we discovered that various ligands display variability in terms of their requirement for the charge clamp of PPARgamma for coactivation by PGC-1. This ligand-selective variable requirement for the charge clamp was coactivator-specific. Thus, distinct structural determinants, which may be unique for a particular ligand, are utilized by the receptor to recognize the coactivator. Our data suggest that even subtle differences in ligand structure are perceived by the receptor and translated into a unique display of the coactivator-binding surface of the ligand-binding domain, allowing for differential recognition of coactivators that may underlie distinct pharmacological profiles observed for ligands of a particular nuclear receptor.

  14. Dysregulation of Notch and ERα signaling in AhR−/− male mice

    PubMed Central

    Huang, Bo; Butler, Ryan; Miao, Yifei; Dai, Yubing; Wu, Wanfu; Su, Wen; Fujii-Kuriyama, Yoshiaki; Warner, Margaret; Gustafsson, Jan-Åke

    2016-01-01

    The aryl hydrocarbon receptor (AhR) is now recognized as an important physiological regulator in the immune and reproductive systems, and in the development of the liver and vascular system. AhR regulates cell cycle, cell proliferation, and differentiation through interacting with other signaling pathways, like estrogen receptor α (ERα), androgen receptor (AR), and Notch signaling. In the present study, we investigated Notch and estrogen signaling in AhR−/− mice. We found low fertility with degenerative changes in the testes, germ cell apoptosis, and a reduced number of early spermatids. There was no change in aromatase, AR, ERα, or ERβ expression in the testis and no detectable change in serum estrogen levels. However, expression of Notch receptors (Notch1 and Notch3) and their target Hairy and Enhancer of Split homolog 1 (HES1) was reduced. In addition, the testosterone level was slightly reduced in the serum. In the mammary fat pad, AhR appeared to regulate estrogen signaling because, in AhR−/− males, there was significant growth of the mammary ducts with high expression of ERα in the ductal epithelium. The enhanced mammary ductal growth appears to be related to overexpression of ERα accompanied by a high proliferation index, whereas the reduced fertility appears to be related defects in Notch signaling that leads to reduced expression of HES1 and, consequently, early maturation of spermatocytes and a depletion of primary spermatids. Previous reports have indicated that AhR pathway is associated with infertility in men. Our results provide a mechanistic explanation for this defect. PMID:27688768

  15. Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation*

    PubMed Central

    Macdonald-Obermann, Jennifer L.; Pike, Linda J.

    2014-01-01

    The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers. PMID:25086039

  16. Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor-ligand interactions

    PubMed Central

    Kueng, Hans J.; Manta, Calin; Haiderer, Daniela; Leb, Victoria M.; Schmetterer, Klaus G.; Neunkirchner, Alina; Byrne, Ruth A.; Scheinecker, Clemens; Steinberger, Peter; Seed, Brian; Pickl, Winfried F.

    2010-01-01

    We describe for the first time fluorescent virus-like particles decorated with biologically active mono- and multisubunit immune receptors of choice and the basic application of such fluorosomes (FSs) to visualize and target immune receptor-ligand interactions. For that purpose, human embryonic kidney (HEK)-293 cells were stably transfected with Moloney murine leukemia virus (MoMLV) matrix protein (MA) GFP fusion constructs. To produce FSs, interleukins (ILs), IL-receptors (IL-Rs), and costimulatory molecules were fused to the glycosyl phosphatidyl inositol anchor acceptor sequence of CD16b and coexpressed along with MoMLV group-specific antigen-polymerase (gag-pol) in MA::GFP+ HEK-293 cells. We show that IL-2 decorated but not control-decorated FSs specifically identify normal and malignant IL-2 receptor-positive (IL-2R+) lymphocytes by flow cytometry. In addition to cytokines and costimulatory molecules, FSs were also successfully decorated with the heterotrimeric IL-2Rs, allowing identification of IL-2+ target cells. Specificity of binding was proven by complete inhibition with nonlabeled, soluble ligands. Moreover, IL-2R FSs efficiently neutralized soluble IL-2 and thus induced unresponsiveness of T cells receiving full activation stimuli via T-cell antigen receptor and CD28. FSs are technically simple, multivalent tools for assessing and blocking mono- and multisubunit immune receptor-ligand interactions with natural constituents in a plasma membrane context.—Kueng, H. J., Manta, C., Haiderer, D., Leb, V. M., Schmetterer, K. G., Neunkirchner, A., Byrne, R. A., Scheinecker, C., Steinberger, P., Seed, B., Pickl, W. F. Fluorosomes: a convenient new reagent to detect and block multivalent and complex receptor-ligand interactions. PMID:20056716

  17. NMR spectroscopy of the ligand binding core of ionotropic glutamate receptor 2 bound to 5-substituted willardiine partial agonists

    PubMed Central

    Fenwick, Michael K.; Oswald, Robert E.

    2008-01-01

    Glutamate receptors mediate neuronal intercommunication in the central nervous system by coupling extracellular neurotransmitter-receptor interactions to ion channel conductivity. To gain insight into structural and dynamical factors that underlie this coupling, solution NMR experiments were performed on the bi-lobed ligand-binding core of glutamate receptor 2 in complexes with a set of willardiine partial agonists. These agonists are valuable for studying structure-function relationships because their 5-position substituent size is correlated with ligand efficacy and extent of receptor desensitization whereas the substituent electronegativity is correlated with ligand potency. NMR results show that the protein backbone amide chemical shift deviations correlate mainly with efficacy and extent of desensitization. Pronounced deviations occur at specific residues in the ligand-binding site and in the two helical segments that join the lobes by a disulfide bond. Experiments detecting conformational exchange show that micro- to millisecond timescale motions also occur near the disulfide bond and vary largely with efficacy and extent of desensitization. These results thus identify regions displaying structural and dynamical dissimilarity arising from differences in ligand-protein interactions and lobe closure which may play a critical role in receptor response. Furthermore, measures of line broadening and conformational exchange for a portion of the ligand-binding site correlate with ligand EC50 data. These results do not have any correlate in the currently available crystal structures and thus provide a novel view of ligand-binding events that may be associated with agonist potency differences. PMID:18387631

  18. S2RSLDB: a comprehensive manually curated, internet-accessible database of the sigma-2 receptor selective ligands.

    PubMed

    Nastasi, Giovanni; Miceli, Carla; Pittalà, Valeria; Modica, Maria N; Prezzavento, Orazio; Romeo, Giuseppe; Rescifina, Antonio; Marrazzo, Agostino; Amata, Emanuele

    2017-01-01

    Sigma (σ) receptors are accepted as a particular receptor class consisting of two subtypes: sigma-1 (σ 1 ) and sigma-2 (σ 2 ). The two receptor subtypes have specific drug actions, pharmacological profiles and molecular characteristics. The σ 2 receptor is overexpressed in several tumor cell lines, and its ligands are currently under investigation for their role in tumor diagnosis and treatment. The σ 2 receptor structure has not been disclosed, and researchers rely on σ 2 receptor radioligand binding assay to understand the receptor's pharmacological behavior and design new lead compounds. Here we present the sigma-2 Receptor Selective Ligands Database (S2RSLDB) a manually curated database of the σ 2 receptor selective ligands containing more than 650 compounds. The database is built with chemical structure information, radioligand binding affinity data, computed physicochemical properties, and experimental radioligand binding procedures. The S2RSLDB is freely available online without account login and having a powerful search engine the user may build complex queries, sort tabulated results, generate color coded 2D and 3D graphs and download the data for additional screening. The collection here reported is extremely useful for the development of new ligands endowed of σ 2 receptor affinity, selectivity, and appropriate physicochemical properties. The database will be updated yearly and in the near future, an online submission form will be available to help with keeping the database widely spread in the research community and continually updated. The database is available at http://www.researchdsf.unict.it/S2RSLDB.

  19. 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

  20. Control of scavenger receptor-mediated endocytosis by novel ligands of different length.

    PubMed

    Guaderrama-Díaz, Margarita; Solís, Carlos F; Velasco-Loyden, Gabriela; Laclette, Juan P; Mas-Oliva, Jaime

    2005-03-01

    The scavenger receptor recognized as a multiligand family of receptors falls in the group that is internalised through endocytosis. In this report we used several recombinant fragments of the tapeworm protein paramyosin, known to form filamentous dimers that bind collagenous structures as ligands of different length for the class A type I scavenger receptor (SR-AI). While native CHO cells are unresponsive to any of the recombinant fragments, it is shown that CHO cells transfected with this receptor efficiently internalise recombinant fragments that correspond to two thirds of the full-length paramyosin. In contrast, recombinant products corresponding to one-third of the full-length paramyiosin are not internalised. It is also shown that important molecules in the organization of the coated pit, are enriched when the two-thirds long paramyosin fragments were bound and internalised through the SR-AI. Moreover, internalisation of these fragments trigger a classical apoptotic pathway shown by the presence of TUNEL positive cells and the appearance of apoptotic bodies. We report paramyosin as a new ligand for the scavenger receptor and provide evidence supporting the notion that these receptors upon the formation of arrays with length-specific molecules, not only trigger endocytosis but also seem to regulate the synthesis of molecules involved in the organization of coated pits.

  1. Integrated in silico and in vivo approaches to investigate effects of BDE-99 mediated by the nuclear receptors on developing zebrafish.

    PubMed

    Zhang, Li; Jin, Yaru; Han, Zhihua; Liu, Hongling; Shi, Laihao; Hua, Xiaoxue; Doering, Jon A; Tang, Song; Giesy, John P; Yu, Hongxia

    2018-03-01

    One of the most abundant polybrominated diphenyl ethers (PBDEs) is 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), which persists and potentially bioaccumulates in aquatic wildlife. Previous studies in mammals have shown that BDE-99 affects development and disrupts certain endocrine functions through signaling pathways mediated by nuclear receptors. However, fewer studies have investigated the potential of BDE-99 to interact with nuclear receptors in aquatic vertebrates such as fish. In the present study, interactions between BDE-99 and nuclear receptors were investigated by in silico and in vivo approaches. This PBDE was able to dock into the ligand-binding domain of zebrafish aryl hydrocarbon receptor 2 (AhR2) and pregnane X receptor (PXR). It had a significant effect on the transcriptional profiles of genes associated with AhR or PXR. Based on the developed cytoscape of all zebrafish genes, it was also inferred that AhR and PXR could interact via cross-talk. In addition, both the in silico and in vivo approaches found that BDE-99 affected peroxisome proliferator-activated receptor alpha (PPARα), glucocorticoid receptor, and thyroid receptor. Collectively, our results demonstrate for the first time detailed in silico evidence that BDE-99 can bind to and interact with zebrafish AhR and PXR. These findings can be used to elaborate the molecular mechanism of BDE-99 and guide more objective environmental risk assessments. Environ Toxicol Chem 2018;37:780-787. © 2017 SETAC. © 2017 SETAC.

  2. Structure-Based Virtual Screening for Dopamine D2 Receptor Ligands as Potential Antipsychotics.

    PubMed

    Kaczor, Agnieszka A; Silva, Andrea G; Loza, María I; Kolb, Peter; Castro, Marián; Poso, Antti

    2016-04-05

    Structure-based virtual screening using a D2 receptor homology model was performed to identify dopamine D2 receptor ligands as potential antipsychotics. From screening a library of 6.5 million compounds, 21 were selected and were subjected to experimental validation. From these 21 compounds tested, ten D2 ligands were identified (47.6% success rate, among them D2 receptor antagonists, as expected) that have additional affinity for other receptors tested, in particular 5-HT2A receptors. The affinity (Ki values) of the compounds ranged from 58 nm to about 24 μM. Similarity and fragment analysis indicated a significant degree of structural novelty among the identified compounds. We found one D2 receptor antagonist that did not have a protonatable nitrogen atom, which is a key structural element of the classical D2 pharmacophore model necessary for interaction with the conserved Asp(3.32) residue. This compound exhibited greater than 20-fold binding selectivity for the D2 receptor over the D3 receptor. We provide additional evidence that the amide hydrogen atom of this compound forms a hydrogen bond with Asp(3.32), as determined by tests of its derivatives that cannot maintain this interaction. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Bio-nanocapsule-based scaffold improves the sensitivity and ligand-binding capacity of mammalian receptors on the sensor chip.

    PubMed

    Iijima, Masumi; Yoshimoto, Nobuo; Niimi, Tomoaki; Maturana, Andrés D; Kuroda, Shun'ichi

    2016-06-01

    Mammalian receptors are recognized as target molecules for drug discovery, and chemical libraries have been screened for both potential antagonists and agonists mainly by ligand-binding assays using immobilized receptors. A bio-nanocapsule (BNC) of approximately 30 nm that displays a tandem form of the protein A-derived immunoglobulin G (IgG) Fc-binding Z domains (denoted as ZZ-BNC) has been developed for both clustering and oriented immobilization of IgGs on the solid phase of immunosensors. In this study, human IgG1 Fc-fused vascular endothelial growth factor (VEGF) receptor was immobilized through ZZ-BNC on the sensor chip of quartz crystal microbalance (ZZ-BNC-coating). When compared with direct adsorption and protein A-coating, the sensor chip showed higher sensitivity (∽46- and ∽165-fold, respectively) and larger ligand-binding capacity (∽4- and ∽18-fold, respectively). Furthermore, the number of VEGF molecules bound to its receptor increased from 0.20 (direct adsorption) to 2.06 by ZZ-BNC-coating, strongly suggesting that ZZ-BNC reduced the steric hindrance near ligand recognition sites through oriented immobilization. Similarly, the sensitivity and ligand-binding capacity of leptin and prolactin receptors were both enhanced at a level comparable to that observed for the VEGF receptor. Thus, the combination of ZZ-BNC and Fc-fused receptors could significantly improve the function of ligand-binding assays. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Aqueous and Ethanolic Valeriana officinalis Extracts Change the Binding of Ligands to Glutamate Receptors

    PubMed Central

    Del Valle-Mojica, Lisa M.; Cordero-Hernández, José M.; González-Medina, Giselle; Ramos-Vélez, Igmeris; Berríos-Cartagena, Nairimer; Torres-Hernández, Bianca A.; Ortíz, José G.

    2011-01-01

    The effects of two valerian extracts (aqueous and hydroalcoholic) were investigated through [3H]Glutamate ([3H]Glu) and [3H]Fluorowillardine ([3H]FW) receptor binding assays using rat synaptic membranes in presence of different receptor ligands. In addition, the extract stability was monitored spectrophotometrically. Both extracts demonstrated interaction with ionotropic glutamate receptors (iGluRs). However, the extracts displayed considerable differences in receptor selectivity. The hydroalcoholic extract selectively interacted with quisqualic acid (QA), group I metabotropic glutamate receptor (mGluR) ligand, while the aqueous extract did not alter the binding of QA. The stability of the extracts was examined during several weeks. Freshly prepared extract inhibited 38–60% of [3H]FW binding (AMPA). After 10 days, the aqueous extract inhibited 85% of [3H]FW binding while the hydroalcoholic extract markedly potentiated (200%) [3H]FW binding to AMPA receptors. Thus, our results showed that factors such as extraction solvent and extract stability determine the selectivity for glutamate receptor (GluR) interactions. PMID:21151614

  5. Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells

    PubMed Central

    Tone, Masahide; Tone, Yukiko; Adams, Elizabeth; Yates, Stephen F.; Frewin, Mark R.; Cobbold, Stephen P.; Waldmann, Herman

    2003-01-01

    Recently, agonist antibodies to glucocorticoid-induced tumor necrosis factor receptor (GITR) (tumor necrosis factor receptor superfamily 18) have been shown to neutralize the suppressive activity of CD4+CD25+ regulatory T cells. It was anticipated that this would be the role of the physiological ligand. We have identified and expressed the gene for mouse GITR ligand and have confirmed that its interaction with GITR reverses suppression by CD4+CD25+ T cells. It also, however, provides a costimulatory signal for the antigen-driven proliferation of naïve T cells and polarized T helper 1 and T helper 2 clones. RT-PCR and mAb staining revealed mouse GITR ligand expression in dendritic cells, macrophages, and B cells. Expression was controlled by the transcription factor NF-1 and potentially by alternative splicing of mRNA destabilization sequences. PMID:14608036

  6. Structural basis of ligand interaction with atypical chemokine receptor 3

    NASA Astrophysics Data System (ADS)

    Gustavsson, Martin; Wang, Liwen; van Gils, Noortje; Stephens, Bryan S.; Zhang, Penglie; Schall, Thomas J.; Yang, Sichun; Abagyan, Ruben; Chance, Mark R.; Kufareva, Irina; Handel, Tracy M.

    2017-01-01

    Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally driven models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.

  7. Growth suppression of Leydig TM3 cells mediated by aryl hydrocarbon receptor

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

    Iseki, Minoru; Department of Regulation Biology, Faculty of Science, Saitama University, Saitama; Ikuta, Togo

    2005-06-17

    Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental toxicity in reproductive organs. To elucidate the function of AhR, we generated stable transformants of TM3 cells overexpressing wild-type aryl hydrocarbon receptor (AhR) or its mutants which carried mutations in nuclear localization signal or nuclear export signal. In the presence of 3-methylcholanthrene (MC), proliferation of the cells transfected with wild-type AhR was completely suppressed, whereas cells expressing AhR mutants proliferated in a manner equivalent to control TM3 cells, suggesting AhR-dependent growth inhibition. The suppression was associated with up-regulation of cyclin-dependent kinase inhibitor p21{sup Cip1}, which was abolished by pretreatment with actinomycin D. A p38 MAPKmore » specific inhibitor, SB203580, blocked the increase of p21{sup Cip1} mRNA in response to MC. Treatment with indigo, another AhR ligand, failed to increase of p21{sup Cip1} mRNA, although up-regulation of mRNA for CYP1A1 was observed. These data suggest AhR in Leydig cells mediates growth inhibition by inducing p21{sup Cip1}.« less

  8. Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits.

    PubMed

    Suga, Hinako; Haga, Tatsuya

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.

  9. Structural Insights into Selective Ligand-Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists.

    PubMed

    Cai, Minying; Marelli, Udaya Kiran; Mertz, Blake; Beck, Johannes G; Opperer, Florian; Rechenmacher, Florian; Kessler, Horst; Hruby, Victor J

    2017-08-15

    Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His 6 -d-Nal(2') 7 -NMe-Arg 8 -Trp 9 -Lys]-NH 2 (15) and Ac-Nle-c[Asp-His 6 -d-Nal(2') 7 -NMe-Arg 8 -NMe-Trp 9 -NMe-Lys]-NH 2 (17). It is known that the pharmacophore (His 6 -DNal 7 -Arg 8 -Trp 9 ) of the SHU-9119 peptides occupies a β II-turn-like region with the turn centered about DNal 7 -Arg 8 . The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp 9 side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg 8 and Trp 9 side chains are involved in a majority of the interactions with the receptor. While Arg 8 forms polar contacts with D154 and D158 of hMC3R, Trp 9 utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp 9 -hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.

  10. Variable ligand- and receptor-binding hot spots in key strains of influenza neuraminidase

    PubMed Central

    Votapka, Lane; Demir, Özlem; Swift, Robert V; Walker, Ross C; Amaro, Rommie E

    2012-01-01

    Influenza A continues to be a major public health concern due to its ability to cause epidemic and pandemic disease outbreaks in humans. Computational investigations of structural dynamics of the major influenza glycoproteins, especially the neuraminidase (NA) enzyme, are able to provide key insights beyond what is currently accessible with standard experimental techniques. In particular, all-atom molecular dynamics simulations reveal the varying degrees of flexibility for such enzymes. Here we present an analysis of the relative flexibility of the ligand- and receptor-binding area of three key strains of influenza A: highly pathogenic H5N1, the 2009 pandemic H1N1, and a human N2 strain. Through computational solvent mapping, we investigate the various ligand- and receptor-binding “hot spots” that exist on the surface of NA which interacts with both sialic acid receptors on the host cells and antiviral drugs. This analysis suggests that the variable cavities found in the different strains and their corresponding capacities to bind ligand functional groups may play an important role in the ability of NA to form competent reaction encounter complexes with other species of interest, including antiviral drugs, sialic acid receptors on the host cell surface, and the hemagglutinin protein. Such considerations may be especially useful for the prediction of how such complexes form and with what binding capacity. PMID:22872804

  11. Unprecedented genomic diversity of AhR1 and AhR2 genes in Atlantic salmon (Salmo salar L.).

    PubMed

    Hansson, Maria C; Wittzell, Håkan; Persson, Kerstin; von Schantz, Torbjörn

    2004-06-24

    Aryl hydrocarbon receptor (AhR) genes encode proteins involved in mediating the toxic responses induced by several environmental pollutants. Here, we describe the identification of the first two AhR1 (alpha and beta) genes and two additional AhR2 (alpha and beta) genes in the tetraploid species Atlantic salmon (Salmo salar L.) from a cosmid library screening. Cosmid clones containing genomic salmon AhR sequences were isolated using a cDNA clone containing the coding region of the Atlantic salmon AhR2gamma as a probe. Screening revealed 14 positive clones, from which four were chosen for further analyses. One of the cosmids contained genomic AhR sequences that were highly similar to the rainbow trout (Oncorhynchus mykiss) AhR2alpha and beta genes. SMART RACE amplified two complete, highly similar but not identical AhR type 2 sequences from salmon cDNA, which from phylogenetic analyses were determined as the rainbow trout AhR2alpha and beta orthologs. The salmon AhR2alpha and beta encode proteins of 1071 and 1058 residues, respectively, and encompass characteristic AhR sequence elements like a basic-helix-loop-helix (bHLH) and two PER-ARNT-SIM (PAS) domains. Both genes are transcribed in liver, spleen and muscle tissues of adult salmon. A second cosmid contained partial sequences, which were identical to the previously characterized AhR2gamma gene. The last two cosmids contained partial genomic AhR sequences, which were more similar to other AhR type 1 fish genes than the four characterized salmon AhR2 genes. However, attempts to amplify the corresponding complete cDNA sequences of the inserts proved very difficult, suggesting that these genes are non-functional or very weakly transcribed in the examined tissues. Phylogenetic analyses of the conserved regions did, however, clearly indicate that these two AhRs belong to the AhR type 1 clade and have been assigned as the Atlantic salmon AhR1alpha and AhR1beta genes. Taken together, these findings demonstrate that

  12. Purinergic receptor ligands stimulate pro-opiomelanocortin gene expression in AtT-20 pituitary corticotroph cells.

    PubMed

    Zhao, L-F; Iwasaki, Y; Oki, Y; Tsugita, M; Taguchi, T; Nishiyama, M; Takao, T; Kambayashi, M; Hashimoto, K

    2006-04-01

    Although recent studies have suggested that purinergic receptors are expressed in the anterior pituitary gland, their involvement in the regulation of pituitary hormone gene expression is not completely understood. In the present study, we examined the expression of purinergic receptors and the effects of purinergic receptor ligands on pro-opiomelanocortin (POMC) gene expression, in AtT20 mouse corticotroph cells. We identified the expression of most of the purinergic receptor subtypes (A1, A2, P2X1, 3-7, P2Y1, 2, 4) mRNAs, analysed by the reverse transcriptase-polymerase chain reaction. We also found that adenosine and ATP, two representative and endogenous agonists of A1-3 and P2X/P2Y receptors, respectively, stimulated the 5'-promoter activity of the POMC gene in a dose- and time-related manner. When these ligands were simultaneously used with corticotrophin-releasing hormone (CRH), effects that were more than additive were observed, suggesting an enhancing role of these compounds in CRH-mediated adrenocorticotrophic hormone (ACTH) synthesis. These ligands also stimulated the expression of transcription factors involved in the regulation of the POMC gene, but did not enhance ACTH secretion. Finally, the positive effect of adenosine as well as CRH was completely inhibited by the protein kinase A inhibitor H89, whereas that of ATP was not influenced, indicating that different intracellular signalling pathways mediate these effects. Altogether, our results suggest a stimulatory role for these purinergic receptor ligands in the regulation of POMC gene expression in corticotroph cells. Because adenosine and ATP are known to be produced within the pituitary gland, it is possible they may be acting in an autocrine/paracrine fashion.

  13. Synthesis and biological evaluation of bivalent cannabinoid receptor ligands based on hCB₂R selective benzimidazoles reveal unexpected intrinsic properties.

    PubMed

    Nimczick, Martin; Pemp, Daniela; Darras, Fouad H; Chen, Xinyu; Heilmann, Jörg; Decker, Michael

    2014-08-01

    The design of bivalent ligands targeting G protein-coupled receptors (GPCRs) often leads to the development of new, highly selective and potent compounds. To date, no bivalent ligands for the human cannabinoid receptor type 2 (hCB₂R) of the endocannabinoid system (ECS) are described. Therefore, two sets of homobivalent ligands containing as parent structure the hCB2R selective agonist 13a and coupled at different attachment positions were synthesized. Changes of the parent structure at these positions have a crucial effect on the potency and efficacy of the ligands. However, we discovered that bivalency has an influence on the effect at both cannabinoid receptors. Moreover, we found out that the spacer length and the attachment position altered the efficacy of the bivalent ligands at the receptors by turning agonists into antagonists and inverse agonists. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes.

    PubMed

    Schwartzkopff, Franziska; Petersen, Frank; Grimm, Tobias Alexander; Brandt, Ernst

    2012-02-01

    During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.

  15. Biased mu-opioid receptor ligands: a promising new generation of pain therapeutics.

    PubMed

    Siuda, Edward R; Carr, Richard; Rominger, David H; Violin, Jonathan D

    2017-02-01

    Opioid chemistry and biology occupy a pivotal place in the history of pharmacology and medicine. Morphine offers unmatched efficacy in alleviating acute pain, but is also associated with a host of adverse side effects. The advent of biased agonism at G protein-coupled receptors has expanded our understanding of intracellular signaling and highlighted the concept that certain ligands are able to differentially modulate downstream pathways. The ability to target one pathway over another has allowed for the development of biased ligands with robust clinical efficacy and fewer adverse events. In this review we summarize these concepts with an emphasis on biased mu opioid receptor pharmacology and highlight how far opioid pharmacology has evolved. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Interaction study between synthetic glycoconjugate ligands and endocytic receptors using flow cytometry.

    PubMed

    Yura, Hirofumi; Ishihara, Masayuki; Kanatani, Yasuhiro; Takase, Bonpei; Hattori, Hidemi; Suzuki, Shinya; Kawakami, Mitsuyuki; Matsui, Takemi

    2006-04-01

    Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17alpha-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry.

  17. AhR-dependent secretion of PDGF-BB by human classically activated macrophages exposed to DEP extracts stimulates lung fibroblast proliferation

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

    Jaguin, Marie; Fardel, Olivier; Pôle Biologie, Centre Hospitalier Universitaire

    2015-06-15

    Lung diseases are aggravated by exposure to diesel exhaust particles (DEPs) found in air pollution. Macrophages are thought to play a crucial role in lung immune response to these pollutants, even if the mechanisms involved remain incompletely characterized. In the present study, we demonstrated that classically and alternative human macrophages (MΦ) exhibited increased secretion of PDGF-B in response to DEP extract (DEPe). This occurred via aryl hydrocarbon receptor (AhR)-activation because DEPe-induced PDGF-B overexpression was abrogated after AhR expression knock-down by RNA interference, in both M1 and M2 polarizing MΦ. In addition, TCDD and benzo(a)pyrene, two potent AhR ligands, also significantlymore » increased mRNA expression of PDGF-B in M1 MΦ, whereas some weak ligands of AhR did not. We next evaluated the impact of conditioned media (CM) from MΦ culture exposed to DEPe or of recombinant PDGF-B onto lung fibroblast proliferation. The tyrosine kinase inhibitor, AG-1295, prevents phosphorylations of PDGF-Rβ, AKT and ERK1/2 and the proliferation of MRC-5 fibroblasts induced by recombinant PDGF-B and by CM from M1 polarizing MΦ, strongly suggesting that the PDGF-BB secreted by DEPe-exposed MΦ is sufficient to activate the PDGF-Rβ pathway of human lung fibroblasts. In conclusion, we demonstrated that human MΦ, whatever their polarization status, secrete PDGF-B in response to DEPe and that PDGF-B is a target gene of AhR. Therefore, induction of PDGF-B by DEP may participate in the deleterious effects towards human health triggered by such environmental urban contaminants. - Highlights: • PDGF-B expression and secretion are increased by DEPe exposure in human M1 and M2 MΦ. • DEPe-induced PDGF-B expression is aryl-hydrocarbon-dependent. • DEPe-exposed M1 MΦ secrete sufficient PDGF-B to increase lung fibroblast proliferation.« less

  18. Development of 7TM receptor-ligand complex models using ligand-biased, semi-empirical helix-bundle repacking in torsion space: application to the agonist interaction of the human dopamine D2 receptor.

    PubMed

    Malo, Marcus; Persson, Ronnie; Svensson, Peder; Luthman, Kristina; Brive, Lars

    2013-03-01

    Prediction of 3D structures of membrane proteins, and of G-protein coupled receptors (GPCRs) in particular, is motivated by their importance in biological systems and the difficulties associated with experimental structure determination. In the present study, a novel method for the prediction of 3D structures of the membrane-embedded region of helical membrane proteins is presented. A large pool of candidate models are produced by repacking of the helices of a homology model using Monte Carlo sampling in torsion space, followed by ranking based on their geometric and ligand-binding properties. The trajectory is directed by weak initial restraints to orient helices towards the original model to improve computation efficiency, and by a ligand to guide the receptor towards a chosen conformational state. The method was validated by construction of the β1 adrenergic receptor model in complex with (S)-cyanopindolol using bovine rhodopsin as template. In addition, models of the dopamine D2 receptor were produced with the selective and rigid agonist (R)-N-propylapomorphine ((R)-NPA) present. A second quality assessment was implemented by evaluating the results from docking of a library of 29 ligands with known activity, which further discriminated between receptor models. Agonist binding and recognition by the dopamine D2 receptor is interpreted using the 3D structure model resulting from the approach. This method has a potential for modeling of all types of helical transmembrane proteins for which a structural template with sequence homology sufficient for homology modeling is not available or is in an incorrect conformational state, but for which sufficient empirical information is accessible.

  19. Tuning the allosteric regulation of artificial muscarinic and dopaminergic ligand-gated potassium channels by protein engineering of G protein-coupled receptors

    PubMed Central

    Moreau, Christophe J.; Revilloud, Jean; Caro, Lydia N.; Dupuis, Julien P.; Trouchet, Amandine; Estrada-Mondragón, Argel; Nieścierowicz, Katarzyna; Sapay, Nicolas; Crouzy, Serge; Vivaudou, Michel

    2017-01-01

    Ligand-gated ion channels enable intercellular transmission of action potential through synapses by transducing biochemical messengers into electrical signal. We designed artificial ligand-gated ion channels by coupling G protein-coupled receptors to the Kir6.2 potassium channel. These artificial channels called ion channel-coupled receptors offer complementary properties to natural channels by extending the repertoire of ligands to those recognized by the fused receptors, by generating more sustained signals and by conferring potassium selectivity. The first artificial channels based on the muscarinic M2 and the dopaminergic D2L receptors were opened and closed by acetylcholine and dopamine, respectively. We find here that this opposite regulation of the gating is linked to the length of the receptor C-termini, and that C-terminus engineering can precisely control the extent and direction of ligand gating. These findings establish the design rules to produce customized ligand-gated channels for synthetic biology applications. PMID:28145461

  20. AhR-deficiency as a cause of demyelinating disease and inflammation.

    PubMed

    Juricek, Ludmila; Carcaud, Julie; Pelhaitre, Alice; Riday, Thorfinn T; Chevallier, Aline; Lanzini, Justine; Auzeil, Nicolas; Laprévote, Olivier; Dumont, Florent; Jacques, Sebastien; Letourneur, Frank; Massaad, Charbel; Agulhon, Cendra; Barouki, Robert; Beraneck, Mathieu; Coumoul, Xavier

    2017-08-29

    The Aryl hydrocarbon Receptor(AhR) is among the most important receptors which bind pollutants; however it also regulates signaling pathways independently of such exposure. We previously demonstrated that AhR is expressed during development of the central nervous system(CNS) and that its deletion leads to the occurrence of a congenital nystagmus. Objectives of the present study are to decipher the origin of these deficits, and to identify the role of the AhR in the development of the CNS. We show that the AhR-knockout phenotype develops during early infancy together with deficits in visual-information-processing which are associated with an altered optic nerve myelin sheath, which exhibits modifications in its lipid composition and in the expression of myelin-associated-glycoprotein(MAG), a cell adhesion molecule involved in myelin-maintenance and glia-axon interaction. In addition, we show that the expression of pro-inflammatory cytokines is increased in the impaired optic nerve and confirm that inflammation is causally related with an AhR-dependent decreased expression of MAG. Overall, our findings demonstrate the role of the AhR as a physiological regulator of myelination and inflammatory processes in the developing CNS. It identifies a mechanism by which environmental pollutants might influence CNS myelination and suggest AhR as a relevant drug target for demyelinating diseases.

  1. Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions in living specimens.

    PubMed

    Dörlich, René M; Chen, Qing; Niklas Hedde, Per; Schuster, Vittoria; Hippler, Marc; Wesslowski, Janine; Davidson, Gary; Nienhaus, G Ulrich

    2015-05-07

    Cellular communication in multi-cellular organisms is mediated to a large extent by a multitude of cell-surface receptors that bind specific ligands. An in-depth understanding of cell signaling networks requires quantitative information on ligand-receptor interactions within living systems. In principle, fluorescence correlation spectroscopy (FCS) based methods can provide such data, but live-cell applications have proven extremely challenging. Here, we have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Absolute ligand and receptor concentrations and their diffusion coefficients within the cell membrane can be quantified without the need to perform additional calibration experiments. We also determine the concentration of ligands diffusing in the medium outside the cell within the same experiment by using a raster image correlation spectroscopy (RICS) based analysis. We have applied this robust technique to study the interactions of two Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), to their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of living HEK293T cells. We obtained significantly lower affinities than previously reported using in vitro studies, underscoring the need to measure such data on living cells or tissues.

  2. On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain.

    PubMed

    Speranskiy, Kirill; Kurnikova, Maria

    2005-08-30

    Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.

  3. Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

    PubMed Central

    Vandenberg, R J; French, C R; Barry, P H; Shine, J; Schofield, P R

    1992-01-01

    The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily. PMID:1311851

  4. Identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex

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

    Andersen, Jacob Lauwring, E-mail: jla@mb.au.dk; Schrøder, Tenna Juul; Christensen, Søren

    2014-02-01

    The identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex are reported. Sortilin is a type I membrane glycoprotein belonging to the vacuolar protein sorting 10 protein (Vps10p) family of sorting receptors and is most abundantly expressed in the central nervous system. Sortilin has emerged as a key player in the regulation of neuronal viability and has been implicated as a possible therapeutic target in a range of disorders. Here, the identification of AF40431, the first reported small-molecule ligand of sortilin, is reported. Crystals of the sortilin–AF40431 complex were obtained bymore » co-crystallization and the structure of the complex was solved to 2.7 Å resolution. AF40431 is bound in the neurotensin-binding site of sortilin, with the leucine moiety of AF40431 mimicking the binding mode of the C-terminal leucine of neurotensin and the 4-methylumbelliferone moiety of AF40431 forming π-stacking with a phenylalanine.« less

  5. International Union of Basic and Clinical Pharmacology. XC. multisite pharmacology: recommendations for the nomenclature of receptor allosterism and allosteric ligands.

    PubMed

    Christopoulos, Arthur; Changeux, Jean-Pierre; Catterall, William A; Fabbro, Doriano; Burris, Thomas P; Cidlowski, John A; Olsen, Richard W; Peters, John A; Neubig, Richard R; Pin, Jean-Philippe; Sexton, Patrick M; Kenakin, Terry P; Ehlert, Frederick J; Spedding, Michael; Langmead, Christopher J

    2014-10-01

    Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties. U.S. Government work not protected by U.S. copyright.

  6. Toxic ligand conjugates as tools in the study of receptor-ligand interactions.

    PubMed

    Herschman, H R; Simpson, D L; Cawley, D B

    1982-01-01

    We have constructed hybrid proteins in which the toxic A chains of ricin or diptheria toxin have been linked to either asialofetuin, fetuin, or epidermal growth factor (EGF). Both ASF-RTA and ASF-DTA are potent toxins on cultured rat hepatocytes, cells that display the asialoglycoprotein receptor. Toxicity of these two compounds is restricted to hepatocytes and can be blocked by asialoglycoproteins but not the native glycoproteins or asialoagalactoglycoprotein derivatives, indicating that the toxicity of the conjugates is mediated by the hepatic asialoglycoprotein receptor. The EGF-RTA conjugate is an extremely potent toxin on cells that can bind the hormone, but is only poorly effective on cells that are unable to bind EGF. The EGF-DTA conjugate, in contrast, is unable to kill 3T3 cells and is at least two orders of magnitude less effective than EGF-RTA on A431 cells, a cell line with 1-2 X 10(6) EGF receptors per cell. However, when EGF-RTA and EGF-DTA were tested on primary liver hepatocyte cultures, which were susceptible to both ASF-RTA and ASF-DTA, both EGF conjugates were potent toxins. Sensitivity of the hepatocyte cultures to ricin toxicity increases slightly during a 52-hr culture period. In contrast, sensitivity to EGF-RTA and ASF-RTA decline dramatically during this period. Receptors for both ligands remain plentiful on the cell surface during this time.

  7. Interfaces and hydrophobic interactions in receptor-ligand systems: A level-set variational implicit solvent approach.

    PubMed

    Cheng, Li-Tien; Wang, Zhongming; Setny, Piotr; Dzubiella, Joachim; Li, Bo; McCammon, J Andrew

    2009-10-14

    A model nanometer-sized hydrophobic receptor-ligand system in aqueous solution is studied by the recently developed level-set variational implicit solvent model (VISM). This approach is compared to all-atom computer simulations. The simulations reveal complex hydration effects within the (concave) receptor pocket, sensitive to the distance of the (convex) approaching ligand. The ligand induces and controls an intermittent switching between dry and wet states of the hosting pocket, which determines the range and magnitude of the pocket-ligand attraction. In the level-set VISM, a geometric free-energy functional of all possible solute-solvent interfaces coupled to the local dispersion potential is minimized numerically. This approach captures the distinct metastable states that correspond to topologically different solute-solvent interfaces, and thereby reproduces the bimodal hydration behavior observed in the all-atom simulation. Geometrical singularities formed during the interface relaxation are found to contribute significantly to the energy barrier between different metastable states. While the hydration phenomena can thus be explained by capillary effects, the explicit inclusion of dispersion and curvature corrections seems to be essential for a quantitative description of hydrophobically confined systems on nanoscales. This study may shed more light onto the tight connection between geometric and energetic aspects of biomolecular hydration and may represent a valuable step toward the proper interpretation of experimental receptor-ligand binding rates.

  8. Conformational Rearrangement Within the Soluble Domains of the CD4 Receptor is Ligand-Specific

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

    Ashish,F.; Juncadella, I.; Garg, R.

    2008-01-01

    Ligand binding induces shape changes within the four modular ectodomains (D1-D4) of the CD4 receptor, an important receptor in immune signaling. Small angle x-ray scattering (SAXS) on both a two-domain and a four-domain construct of the soluble CD4 (sCD4) is consistent with known crystal structures demonstrating a bilobal and a semi-extended tetralobal Z conformation in solution, respectively. Detection of conformational changes within sCD4 as a result of ligand binding was followed by SAXS on sCD4 bound to two different glycoprotein ligands: the tick saliva immunosuppressor Salp15 and the HIV-1 envelope protein gp120. Ab initio modeling of these data showed thatmore » both Salp15 and gp120 bind to the D1 domain of sCD4 and yet induce drastically different structural rearrangements. Upon binding, Salp15 primarily distorts the characteristic lobal architecture of the sCD4 without significantly altering the semi-extended shape of the sCD4 receptor. In sharp contrast, the interaction of gp120 with sCD4 induces a shape change within sCD4 that can be described as a Z-to-U bi-fold closure of the four domains across its flexible D2-D3 linker. Placement of known crystal structures within the boundaries of the SAXS-derived models suggests that the ligand-induced shape changes could be a result of conformational changes within this D2-D3 linker. Functionally, the observed shape changes in CD4 receptor causes dissociation of lymphocyte kinase from the cytoplasmic domain of Salp15-bound CD4 and facilitates an interaction between the exposed V3 loops of CD4-bound gp120 molecule to the extracellular loops of its co-receptor, a step essential for HIV-1 viral entry.« less

  9. Analysis of TNF-related apoptosis-inducing ligand and receptors and implications in thymus biology and myasthenia gravis.

    PubMed

    Kanatli, Irem; Akkaya, Bahar; Uysal, Hilmi; Kahraman, Sevim; Sanlioglu, Ahter Dilsad

    2017-02-01

    Myasthenia Gravis is an autoantibody-mediated, neuromuscular junction disease, and is usually associated with thymic abnormalities presented as thymic tumors (~10%) or hyperplastic thymus (~65%). The exact role of thymus in Myasthenia Gravis development is not clear, yet many patients benefit from thymectomy. The apoptotic ligand TNF-Related Apoptosis-Inducing Ligand is thought to be involved in the regulation of thymocyte counts, although conflicting results are reported. We investigated differential expression profiles of TNF-Related Apoptosis-Inducing Ligand and its transmembrane receptors, Nuclear Factor-kB activation status, and apoptotic cell counts in healthy thymic tissue and pathological thymus from Myasthenia Gravis patients. All tissues expressed TNF-Related Apoptosis-Inducing Ligand and its receptors, with hyperplastic tissue having the highest expression levels of death receptors DR4 and DR5. No detectable Nuclear Factor-kB activation, at least via the canonical Protein Kinase A-mediated p65 Ser276 phosphorylation, was evident in any of the tissues studied. Apoptotic cell counts were higher in MG-associated tissue compared to the normal thymus. Possible use of the TNF-Related Apoptosis-Inducing Ligand within the concept of an apoptotic ligand-mediated medical thymectomy in thymoma- or thymic hyperplasia-associated Myasthenia Gravis is also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Dopamine D3 receptor ligands for drug addiction treatment: update on recent findings.

    PubMed

    Le Foll, Bernard; Collo, Ginetta; Rabiner, Eugenii A; Boileau, Isabelle; Merlo Pich, Emilio; Sokoloff, Pierre

    2014-01-01

    The dopamine D3 receptor is located in the limbic area and apparently mediates selective effects on motivation to take drugs and drug-seeking behaviors, so that there has been considerable interest on the possible use of D3 receptor ligands to treat drug addiction. However, only recently selective tools allowing studying this receptor have been developed. This chapter presents an overview of findings that were presented at a symposium on the conference Dopamine 2013 in Sardinia in May 2013. Novel neurobiological findings indicate that drugs of abuse can lead to significant structural plasticity in rodent brain and that this is dependent on the availability of functional dopamine D3 autoreceptor, whose activation increased phosphorylation in the ERK pathway and in the Akt/mTORC1 pathway indicating the parallel engagement of a series of intracellular signaling pathways all involved in cell growth and survival. Preclinical findings using animal models of drug-seeking behaviors confirm that D3 antagonists have a promising profile to treat drug addiction across drugs of abuse type. Imaging the D3 is now feasible in human subjects. Notably, the development of (+)-4-propyl-9-hydroxynaphthoxazine ligand used in positron emission tomography (PET) studies in humans allows to measure D3 and D2 receptors based on the area of the brain under study. This PET ligand has been used to confirm up-regulation of D3 sites in psychostimulant users and to reveal that tobacco smoking produces elevation of dopamine at the level of D3 sites. There are now novel antagonists being developed, but also old drugs such as buspirone, that are available to test the D3 hypothesis in humans. The first results of clinical investigations are now being provided. Overall, those recent findings support further exploration of D3 ligands to treat drug addiction. © 2014 Elsevier B.V. All rights reserved.

  11. Antidiabetic actions of a phosphatidylcholine ligand for nuclear receptor LRH-1

    PubMed Central

    Lee, Jae Man; Lee, Yoon Kwang; Mamrosh, Jennifer L.; Busby, Scott A.; Griffin, Patrick R.; Pathak, Manish C.; Ortlund, Eric A.; Moore, David D.

    2011-01-01

    Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (NR5A2) regulates bile acid biosynthesis1,2. Structural studies have identified phospholipids as potential LRH-1 ligands3–5, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine, DLPC) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signaling pathway that regulates bile acid metabolism and glucose homeostasis. PMID:21614002

  12. Structural basis of ligand interaction with atypical chemokine receptor 3

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

    Gustavsson, Martin; Wang, Liwen; van Gils, Noortje

    2017-01-18

    Chemokines drive cell migration through their interactions with seven-transmembrane (7TM) chemokine receptors on cell surfaces. The atypical chemokine receptor 3 (ACKR3) binds chemokines CXCL11 and CXCL12 and signals exclusively through β-arrestin-mediated pathways, without activating canonical G-protein signalling. This receptor is upregulated in numerous cancers making it a potential drug target. Here we collected over 100 distinct structural probes from radiolytic footprinting, disulfide trapping, and mutagenesis to map the structures of ACKR3:CXCL12 and ACKR3:small-molecule complexes, including dynamic regions that proved unresolvable by X-ray crystallography in homologous receptors. The data are integrated with molecular modelling to produce complete and cohesive experimentally drivenmore » models that confirm and expand on the existing knowledge of the architecture of receptor:chemokine and receptor:small-molecule complexes. Additionally, we detected and characterized ligand-induced conformational changes in the transmembrane and intracellular regions of ACKR3 that elucidate fundamental structural elements of agonism in this atypical receptor.« less

  13. Atypical Signaling and Functional Desensitization Response of MAS Receptor to Peptide Ligands

    PubMed Central

    Tirupula, Kalyan C.; Desnoyer, Russell; Speth, Robert C.; Karnik, Sadashiva S.

    2014-01-01

    MAS is a G protein-coupled receptor (GPCR) implicated in multiple physiological processes. Several physiological peptide ligands such as angiotensin-(1–7), angiotensin fragments and neuropeptide FF (NPFF) are reported to act on MAS. Studies of conventional G protein signaling and receptor desensitization upon stimulation of MAS with the peptide ligands are limited so far. Therefore, we systematically analyzed G protein signals activated by the peptide ligands. MAS-selective non-peptide ligands that were previously shown to activate G proteins were used as controls for comparison on a common cell based assay platform. Activation of MAS by the non-peptide agonist (1) increased intracellular calcium and D-myo-inositol-1-phosphate (IP1) levels which are indicative of the activation of classical Gαq-phospholipase C signaling pathways, (2) decreased Gαi mediated cAMP levels and (3) stimulated Gα12-dependent expression of luciferase reporter. In all these assays, MAS exhibited strong constitutive activity that was inhibited by the non-peptide inverse agonist. Further, in the calcium response assay, MAS was resistant to stimulation by a second dose of the non-peptide agonist after the first activation has waned suggesting functional desensitization. In contrast, activation of MAS by the peptide ligand NPFF initiated a rapid rise in intracellular calcium with very weak IP1 accumulation which is unlike classical Gαq-phospholipase C signaling pathway. NPFF only weakly stimulated MAS-mediated activation of Gα12 and Gαi signaling pathways. Furthermore, unlike non-peptide agonist-activated MAS, NPFF-activated MAS could be readily re-stimulated the second time by the agonists. Functional assays with key ligand binding MAS mutants suggest that NPFF and non-peptide ligands bind to overlapping regions. Angiotensin-(1–7) and other angiotensin fragments weakly potentiated an NPFF-like calcium response at non-physiological concentrations (≥100 µM). Overall, our data

  14. The active analog approach applied to the pharmacophore identification of benzodiazepine receptor ligands

    NASA Astrophysics Data System (ADS)

    Tebib, Souhail; Bourguignon, Jean-Jacques; Wermuth, Camille-Georges

    1987-07-01

    Applied to seven potent benzodiazepine-receptor ligands belonging to chemically different classes, the active analog approach allowed the stepwise identification of the pharmacophoric pattern associated with the recognition by the benzodiazepine receptor. A unique pharmacophore model was derived which involves six critical zones: (a) a π-electron rich aromatic (PAR) zone; (b) two electron-rich zones δ1 and δ2 placed at 5.0 and 4.5 Å respectively from the reference centroid in the PAR zone; (c) a freely rotating aromatic ring (FRA) region; (d) an out-of-plane region (OPR), strongly associated with agonist properties; and (e) an additional hydrophobic region (AHR). The model accommodates all presently known ligands of the benzodiazepine receptor, identifies sensitivity to steric hindrance close to the δ1 zone, accounts for R and S differential affinities and distinguishes requirements for agonist versus non-agonist activity profiles.

  15. Multitarget-directed tricyclic pyridazinones as G protein-coupled receptor ligands and cholinesterase inhibitors.

    PubMed

    Pau, Amedeo; Catto, Marco; Pinna, Giovanni; Frau, Simona; Murineddu, Gabriele; Asproni, Battistina; Curzu, Maria M; Pisani, Leonardo; Leonetti, Francesco; Loza, Maria Isabel; Brea, José; Pinna, Gérard A; Carotti, Angelo

    2015-06-01

    By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein-coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone-based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5-HT1A, adrenergic α1A, and dopaminergic D2 receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5-HT1A because of its involvement in neuronal deficits typical of Alzheimer's and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α1A and 5-HT1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Development of Protein Degradation Inducers of Androgen Receptor by Conjugation of Androgen Receptor Ligands and Inhibitor of Apoptosis Protein Ligands.

    PubMed

    Shibata, Norihito; Nagai, Katsunori; Morita, Yoko; Ujikawa, Osamu; Ohoka, Nobumichi; Hattori, Takayuki; Koyama, Ryokichi; Sano, Osamu; Imaeda, Yasuhiro; Nara, Hiroshi; Cho, Nobuo; Naito, Mikihiko

    2018-01-25

    Targeted protein degradation using small molecules is a novel strategy for drug development. We have developed hybrid molecules named specific and nongenetic inhibitor of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) that recruit IAP ubiquitin ligases to degrade target proteins. Here, we show novel SNIPERs capable of inducing proteasomal degradation of the androgen receptor (AR). Through derivatization of the SNIPER(AR) molecule at the AR ligand and IAP ligand and linker, we developed 42a (SNIPER(AR)-51), which shows effective protein knockdown activity against AR. Consistent with the degradation of the AR protein, 42a inhibits AR-mediated gene expression and proliferation of androgen-dependent prostate cancer cells. In addition, 42a efficiently induces caspase activation and apoptosis in prostate cancer cells, which was not observed in the cells treated with AR antagonists. These results suggest that SNIPER(AR)s could be leads for an anticancer drug against prostate cancers that exhibit AR-dependent proliferation.

  17. What Can Crystal Structures of Aminergic Receptors Tell Us about Designing Subtype-Selective Ligands?

    PubMed Central

    Michino, Mayako; Beuming, Thijs; Donthamsetti, Prashant; Newman, Amy Hauck; Javitch, Jonathan A.

    2015-01-01

    G protein–coupled receptors (GPCRs) are integral membrane proteins that represent an important class of drug targets. In particular, aminergic GPCRs interact with a significant portion of drugs currently on the market. However, most drugs that target these receptors are associated with undesirable side effects, which are due in part to promiscuous interactions with close homologs of the intended target receptors. Here, based on a systematic analysis of all 37 of the currently available high-resolution crystal structures of aminergic GPCRs, we review structural elements that contribute to and can be exploited for designing subtype-selective compounds. We describe the roles of secondary binding pockets (SBPs), as well as differences in ligand entry pathways to the orthosteric binding site, in determining selectivity. In addition, using the available crystal structures, we have identified conformational changes in the SBPs that are associated with receptor activation and explore the implications of these changes for the rational development of selective ligands with tailored efficacy. PMID:25527701

  18. Ligand-Induced Modulation of the Free-Energy Landscape of G Protein-Coupled Receptors Explored by Adaptive Biasing Techniques

    PubMed Central

    Provasi, Davide; Artacho, Marta Camacho; Negri, Ana; Mobarec, Juan Carlos; Filizola, Marta

    2011-01-01

    Extensive experimental information supports the formation of ligand-specific conformations of G protein-coupled receptors (GPCRs) as a possible molecular basis for their functional selectivity for signaling pathways. Taking advantage of the recently published inactive and active crystal structures of GPCRs, we have implemented an all-atom computational strategy that combines different adaptive biasing techniques to identify ligand-specific conformations along pre-determined activation pathways. Using the prototypic GPCR β2-adrenergic receptor as a suitable test case for validation, we show that ligands with different efficacies (either inverse agonists, neutral antagonists, or agonists) modulate the free-energy landscape of the receptor by shifting the conformational equilibrium towards active or inactive conformations depending on their elicited physiological response. Notably, we provide for the first time a quantitative description of the thermodynamics of the receptor in an explicit atomistic environment, which accounts for the receptor basal activity and the stabilization of different active-like states by differently potent agonists. Structural inspection of these metastable states reveals unique conformations of the receptor that may have been difficult to retrieve experimentally. PMID:22022248

  19. Covalent modification of proteins by ligands of steroid hormone receptors.

    PubMed Central

    Takahashi, N; Breitman, T R

    1992-01-01

    Retinoylation, acylation with retinoic acid (RA), is a covalent modification of proteins occurring in a variety of eukaryotic cell lines. In this study, we found that proteins in HL-60 cells were labeled by 17 beta-[3H]estradiol (E2), [3H]progesterone (Pg), 1 alpha,25-dihydroxy[3H]vitamin D3 [1,25(OH)2D3], [125I]triiodothyronine (T3), [125I]thyroxine (T4), and [3H]prostaglandin E2 (PGE2). All of these hormones, except PGE2, are ligands of the steroid hormone receptor family. Addition to the growth medium of 5 microM ketoconazole, an inhibitor of cytochrome P450-dependent enzymes, increased about 2-fold the labeling of proteins by T3, T4, 1,25(OH)2D3, and PGE2. In contrast, ketoconazole did not change markedly the extent of labeling by RA, E2, or Pg. Alkaline methanolysis, which cleaves ester bonds, released variable percentages of the radioactive ligands bound to protein. These values were about 80% for RA and PGE2; 50% for T3, T4, and Pg; and 20% for E2 and 1,25(OH)2D3. Treatment with thioether-cleavage reagents, iodomethane or Raney nickel catalyst, released < 2% of the covalently bound ligands. Two-dimensional polyacrylamide gel electrophoresis patterns of labeled proteins were unique for each ligand. Proteins of M(r) 47,000 and 51,000 were labeled by RA, E2, T3, and T4. These proteins had the same mobilities as RI and RII, the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases. 1,25(OH)2D3 also bound to proteins of M(r) 47,000 and 51,000. However, these proteins had pI values different from those of RI or RII. These results suggest that some activities of ligands of the steroid hormone receptor family and of PGE2 may be mediated by their covalent modification of proteins. Images PMID:1438281

  20. Covalent modification of proteins by ligands of steroid hormone receptors.

    PubMed

    Takahashi, N; Breitman, T R

    1992-11-15

    Retinoylation, acylation with retinoic acid (RA), is a covalent modification of proteins occurring in a variety of eukaryotic cell lines. In this study, we found that proteins in HL-60 cells were labeled by 17 beta-[3H]estradiol (E2), [3H]progesterone (Pg), 1 alpha,25-dihydroxy[3H]vitamin D3 [1,25(OH)2D3], [125I]triiodothyronine (T3), [125I]thyroxine (T4), and [3H]prostaglandin E2 (PGE2). All of these hormones, except PGE2, are ligands of the steroid hormone receptor family. Addition to the growth medium of 5 microM ketoconazole, an inhibitor of cytochrome P450-dependent enzymes, increased about 2-fold the labeling of proteins by T3, T4, 1,25(OH)2D3, and PGE2. In contrast, ketoconazole did not change markedly the extent of labeling by RA, E2, or Pg. Alkaline methanolysis, which cleaves ester bonds, released variable percentages of the radioactive ligands bound to protein. These values were about 80% for RA and PGE2; 50% for T3, T4, and Pg; and 20% for E2 and 1,25(OH)2D3. Treatment with thioether-cleavage reagents, iodomethane or Raney nickel catalyst, released < 2% of the covalently bound ligands. Two-dimensional polyacrylamide gel electrophoresis patterns of labeled proteins were unique for each ligand. Proteins of M(r) 47,000 and 51,000 were labeled by RA, E2, T3, and T4. These proteins had the same mobilities as RI and RII, the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases. 1,25(OH)2D3 also bound to proteins of M(r) 47,000 and 51,000. However, these proteins had pI values different from those of RI or RII. These results suggest that some activities of ligands of the steroid hormone receptor family and of PGE2 may be mediated by their covalent modification of proteins.

  1. The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

    PubMed Central

    Lou, Meizhen; Garrett, Thomas P. J.; McKern, Neil M.; Hoyne, Peter A.; Epa, V. Chandana; Bentley, John D.; Lovrecz, George O.; Cosgrove, Leah J.; Frenkel, Maurice J.; Ward, Colin W.

    2006-01-01

    The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1–CR–L2) of human IR at 2.3 Å resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) β-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more α-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R. PMID:16894147

  2. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

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

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M.

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast,more » amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.« less

  3. Dietary Polyphenols Increase Paraoxonase 1 Gene Expression by an Aryl Hydrocarbon Receptor-Dependent Mechanism

    PubMed Central

    Gouédard, Cédric; Barouki, Robert; Morel, Yannick

    2004-01-01

    Human paraoxonase 1 (PON-1) is a serum high-density lipoprotein-associated enzyme mainly secreted by the liver. It has endogenous and exogenous substrates and displays protective properties with respect to cardiovascular disease and organophosphate intoxication. In the HuH7 human hepatoma cell line, PON-1 activity and mRNA levels were increased by dietary polyphenolic compounds such as quercetin but also by toxic ligands of the aryl hydrocarbon receptor (AhR) such as 3-methylcholanthrene (3-MC). However, the 2,3,7,8-tetrachlorobenzo(p)dioxin (TCDD) was a poor inducer. Transient and stable transfection assays indicated that these compounds increased the PON-1 gene promoter activity in an AhR-dependent manner, since their effect was inhibited by 7-keto-cholesterol and AhR-directed short interfering RNA. Deletions and mutations studies showed that a xenobiotic responsive element (XRE)-like sequence within the PON-1 promoter mediated the effect of 3-MC and quercetin. In contrast with consensus XREs from the cytochrome P450 1A1 gene, the PON-1 XRE-like element mediated preferentially the effect of quercetin compared to the results seen with TCDD. Furthermore, AhR binding to this element was preferentially activated by quercetin. These observations provide a molecular mechanism for the regulation of the cardioprotective enzyme PON-1 by polyphenols. They suggest also that AhR ligands may differentially regulate gene expression depending on the DNA target sequence. PMID:15169886

  4. Synthesis and Characterization of PEGylated Toll Like Receptor 7 Ligands

    PubMed Central

    Chan, Michael; Hayashi, Tomoko; Mathewson, Richard D.; Yao, Shiyin; Gray, Christine; Tawatao, Rommel; Kalenian, Kevin; Zhang, Yanmei; Hayashi, Yuki; Lao, Fitzgerald S.; Cottam, Howard B.; Carson, Dennis A.

    2011-01-01

    Toll like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid or polyethylene glycol (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in existing biologics and some low molecular weight compounds. PEGylation could be a feasible method to alter the pharmacokinetics and pharmacodynamics of TLR7 ligands. In this study, we systematically studied the influence of PEG chain length on the in vitro and in vivo properties of potent TLR7 ligands. PEGylation increased solubility of the TLR7 ligands and modulated protein binding. Adding a 6–10 length PEG to the TLR7 ligand reduced its potency toward induction of interleukin (IL)-6 by murine macrophages in vitro and IL-6 and tumor necrosis factor (TNF) in vivo. However, PEGylation with 18 or longer chain restored, and even enhanced, the agonistic activity of the drug. In human peripheral blood mononuclear cells, similar effects of PEGylation were observed for secretion of proinflammatory cytokines, IL-6, IL-12, TNF-α, IL-1β and type 1 interferon, as well for B cell proliferation. In summary, these studies demonstrate that conjugation of PEG chains to a synthetic TLR ligand can impact its potency for cytokine induction depending on the size of the PEG moiety. Thus, PEGylation may be a feasible approach to regulate the pharmacological properties of TLR7 ligands. PMID:21338093

  5. Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors

    PubMed Central

    Boursalian, Tamar E.; Fink, Pamela J.

    2003-01-01

    Fas ligand, best known as a death-inducer, is also a costimulatory molecule required for maximal proliferation of mature antigen-specific CD4+ and CD8+ T cells. We now extend the role of Fas ligand by showing that it can also influence thymocyte development. T cell maturation in some, but not all, strains of TCR transgenic mice is severely impaired in thymocytes expressing mutant Fas ligand incapable of interacting with Fas. Mutant Fas ligand inhibits neither negative selection nor death by neglect. Instead, it appears to modulate positive selection of thymocytes expressing both class I– and class II–restricted T cell receptors of moderate affinity for their positively selecting ligands. Fas ligand is therefore an inducer of death, a costimulator of peripheral T cell activation, and an accessory molecule in positive selection. PMID:12860933

  6. Collagen type I as a ligand for receptor-mediated signaling

    NASA Astrophysics Data System (ADS)

    Boraschi-Diaz, Iris; Wang, Jennifer; Mort, John S.; Komarova, Svetlana V.

    2017-05-01

    Collagens form the fibrous component of the extracellular matrix in all multi-cellular animals. Collagen type I is the most abundant collagen present in skin, tendons, vasculature, as well as the organic portion of the calcified tissue of bone and teeth. This review focuses on numerous receptors for which collagen acts as a ligand, including integrins, discoidin domain receptors DDR1 and 2, OSCAR, GPVI, G6b-B and Lair-1 of the leukocyte receptor complex and mannose family receptor uPARAP/Endo 180. We explore the process of collagen production and self-assembly, as well as its degradation by collagenases and gelatinases in order to predict potential temporal and spatial sites of action of different collagen receptors. While the interactions of the mature collagen matrix with integrins and DDR are well-appreciated, potential signals from immature matrix as well as collagen degradation products are possible but not yet described. The role of multiple collagen receptors in physiological processes and their contribution to pathophysiology of diseases affecting collagen homeostasis require further studies.

  7. Similarities among receptor pockets and among compounds: analysis and application to in silico ligand screening.

    PubMed

    Fukunishi, Yoshifumi; Mikami, Yoshiaki; Nakamura, Haruki

    2005-09-01

    We developed a new method to evaluate the distances and similarities between receptor pockets or chemical compounds based on a multi-receptor versus multi-ligand docking affinity matrix. The receptors were classified by a cluster analysis based on calculations of the distance between receptor pockets. A set of low homologous receptors that bind a similar compound could be classified into one cluster. Based on this line of reasoning, we proposed a new in silico screening method. According to this method, compounds in a database were docked to multiple targets. The new docking score was a slightly modified version of the multiple active site correction (MASC) score. Receptors that were at a set distance from the target receptor were not included in the analysis, and the modified MASC scores were calculated for the selected receptors. The choice of the receptors is important to achieve a good screening result, and our clustering of receptors is useful to this purpose. This method was applied to the analysis of a set of 132 receptors and 132 compounds, and the results demonstrated that this method achieves a high hit ratio, as compared to that of a uniform sampling, using a receptor-ligand docking program, Sievgene, which was newly developed with a good docking performance yielding 50.8% of the reconstructed complexes at a distance of less than 2 A RMSD.

  8. Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability

    PubMed Central

    Zago, Michela; Sheridan, Jared A.; Traboulsi, Hussein; Hecht, Emelia; Zhang, Yelu; Guerrina, Necola; Matthews, Jason; Nair, Parameswaran; Eidelman, David H.; Hamid, Qutayba

    2017-01-01

    Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients. PMID:28749959

  9. Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability.

    PubMed

    Zago, Michela; Sheridan, Jared A; Traboulsi, Hussein; Hecht, Emelia; Zhang, Yelu; Guerrina, Necola; Matthews, Jason; Nair, Parameswaran; Eidelman, David H; Hamid, Qutayba; Baglole, Carolyn J

    2017-01-01

    Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.

  10. Similarity- and Substructure-Based Development of β2-Adrenergic Receptor Ligands Based on Unusual Scaffolds

    PubMed Central

    2017-01-01

    The β2-adrenergic receptor (β2AR) is a G protein-coupled receptor (GPCR) and a well-explored target. Here, we report the discovery of 13 ligands, ten of which are novel, of this particular GPCR. They have been identified by similarity- and substructure-based searches using multiple ligands, which were described in an earlier study, as starting points. Of note, two of the molecules used as queries here distinguish themselves from other β2AR antagonists by their unique scaffold. The molecules described in this work allow us to explore the ligand space around the previously reported molecules in greater detail, leading to insights into their structure–activity relationship. We also report experimental binding and selectivity data and putative binding modes for the novel molecules. PMID:28523097

  11. Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis

    PubMed Central

    Pillon, Arnaud; Boussioux, Anne-Marie; Escande, Aurélie; Aït-Aïssa, Sélim; Gomez, Elena; Fenet, Hélène; Ruff, Marc; Moras, Dino; Vignon, Françoise; Duchesne, Marie-Josèphe; Casellas, Claude; Nicolas, Jean-Claude; Balaguer, Patrick

    2005-01-01

    Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-αligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-αand whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-αto separate ligands for ER and AhR that are present in river sediments. Immobilized ER-α, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis. PMID:15743715

  12. Design of ligands for the nicotinic acetylcholine receptors: the quest for selectivity.

    PubMed

    Bunnelle, William H; Dart, Michael J; Schrimpf, Michael R

    2004-01-01

    In the last decade, nicotinic acetylcholine receptors (nAChRs) have emerged as important targets for drug discovery. The therapeutic potential of nicotinic agonists depends substantially on the ability to selectively activate certain receptor subtypes that mediate beneficial effects. The design of such compounds has proceeded in spite of a general shortage of data pertaining to subtype selectivity. Medicinal chemistry efforts have been guided principally by binding affinities to the alpha4beta2 and/or alpha7 subtypes, even though these are not predictive of agonist activity at either subtype. Nevertheless, a diverse family of nAChR ligands has been developed, and several analogs with promising therapeutic potential have now advanced to human clinical trials. This paper provides an overview of the structure-affinity relationships that continue to drive development of new nAChR ligands.

  13. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  14. Molecular Basis of Ligand Dissociation in β-Adrenergic Receptors

    PubMed Central

    González, Angel; Perez-Acle, Tomas; Pardo, Leonardo; Deupi, Xavier

    2011-01-01

    The important and diverse biological functions of β-adrenergic receptors (βARs) have promoted the search for compounds to stimulate or inhibit their activity. In this regard, unraveling the molecular basis of ligand binding/unbinding events is essential to understand the pharmacological properties of these G protein-coupled receptors. In this study, we use the steered molecular dynamics simulation method to describe, in atomic detail, the unbinding process of two inverse agonists, which have been recently co-crystallized with β1 and β2ARs subtypes, along four different channels. Our results indicate that this type of compounds likely accesses the orthosteric binding site of βARs from the extracellular water environment. Importantly, reconstruction of forces and energies from the simulations of the dissociation process suggests, for the first time, the presence of secondary binding sites located in the extracellular loops 2 and 3 and transmembrane helix 7, where ligands are transiently retained by electrostatic and Van der Waals interactions. Comparison of the residues that form these new transient allosteric binding sites in both βARs subtypes reveals the importance of non-conserved electrostatic interactions as well as conserved aromatic contacts in the early steps of the binding process. PMID:21915263

  15. Binding constants of membrane-anchored receptors and ligands depend strongly on the nanoscale roughness of membranes.

    PubMed

    Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

    2013-09-17

    Cell adhesion and the adhesion of vesicles to the membranes of cells or organelles are pivotal for immune responses, tissue formation, and cell signaling. The adhesion processes depend sensitively on the binding constant of the membrane-anchored receptor and ligand proteins that mediate adhesion, but this constant is difficult to measure in experiments. We have investigated the binding of membrane-anchored receptor and ligand proteins with molecular dynamics simulations. We find that the binding constant of the anchored proteins strongly decreases with the membrane roughness caused by thermally excited membrane shape fluctuations on nanoscales. We present a theory that explains the roughness dependence of the binding constant for the anchored proteins from membrane confinement and that relates this constant to the binding constant of soluble proteins without membrane anchors. Because the binding constant of soluble proteins is readily accessible in experiments, our results provide a useful route to compute the binding constant of membrane-anchored receptor and ligand proteins.

  16. Ligands for SPECT and PET imaging of muscarinic-cholinergic receptors of the heart and brain

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

    Knapp, F.F. Jr.; McPherson, D.W.; Luo, H.

    1995-06-01

    Interest in the potential use of cerebral SPECT and PET imaging for determination of the density and activity of muscarinic-cholinergic receptors (mAChR) has been stimulated by the changes in these receptors which occur in many neurological diseases. In addition, the important involvement of mAChR in modulating negative inotropic cardiac activity suggests that such receptor ligands may have important applications in evaluation of changes which may occur in cardiac disease. In this paper, the properties of several key muscarinic receptor ligands being developed or which have been used for clinical SPECT and PET are discussed. In addition, the ORNL development ofmore » the new iodinated IQNP ligand based on QNB and the results of in vivo biodistribution studies in rats, in vitro competitive binding studies and ex vivo autoradiographic experiments are described. The use of radioiodinated IQNP may offer several advantages in comparison to IQNB because of its easy and high yield preparation and high brain uptake and the potential usefulness of the {open_quotes}partial{close_quotes} subtype selective IONP isomers. We also describe the development of new IQNP-type analogues which offer the opportunity for radiolabeling with positron-emitting radioisotopes (carbon-11, fluorine-18 and bromine-76) for potential use with PET.« less

  17. Ligand binding affinity and changes in the lateral diffusion of receptor for advanced glycation endproducts (RAGE).

    PubMed

    Syed, Aleem; Zhu, Qiaochu; Smith, Emily A

    2016-12-01

    The effect of ligand on the lateral diffusion of receptor for advanced glycation endproducts (RAGE), a receptor involved in numerous pathological conditions, remains unknown. Single particle tracking experiments that use quantum dots specifically bound to hemagglutinin (HA)-tagged RAGE (HA-RAGE) are reported to elucidate the effect of ligand binding on HA-RAGE diffusion in GM07373 cell membranes. The ligand used in these studies is methylglyoxal modified-bovine serum albumin (MGO-BSA) containing advanced glycation end products modifications. The binding affinity between soluble RAGE and MGO-BSA increases by 1.8 to 9.7-fold as the percent primary amine modification increases from 24 to 74% and with increasing negative charge on the MGO-BSA. Ligand incubation affects the HA-RAGE diffusion coefficient, the radius of confinement, and duration of confinement. There is, however, no correlation between MGO-BSA ligand binding affinity with soluble RAGE and the extent of the changes in HA-RAGE lateral diffusion. The ligand induced changes to HA-RAGE lateral diffusion do not occur when cholesterol is depleted from the cell membrane, indicating the mechanism for ligand-induced changes to HA-RAGE diffusion is cholesterol dependent. The results presented here serve as a first step in unraveling how ligand influences RAGE lateral diffusion. Copyright © 2016. Published by Elsevier B.V.

  18. Design of chimeric peptide ligands to galanin receptors and substance P receptors.

    PubMed

    Langel, U; Land, T; Bartfai, T

    1992-06-01

    Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Peroxisome Proliferator-Activated Receptor γ (PPARγ) and Ligand Choreography: Newcomers Take the Stage.

    PubMed

    Garcia-Vallvé, Santiago; Guasch, Laura; Tomas-Hernández, Sarah; del Bas, Josep Maria; Ollendorff, Vincent; Arola, Lluís; Pujadas, Gerard; Mulero, Miquel

    2015-07-23

    Thiazolidinediones (TZDs), such as rosiglitazone and pioglitazone, are peroxisome proliferator-activated receptor γ (PPARγ) full agonists that have been widely used in the treatment of type 2 diabetes mellitus. Despite the demonstrated beneficial effect of reducing glucose levels in the plasma, TZDs also induce several adverse effects. Consequently, the search for new compounds with potent antidiabetic effects but fewer undesired effects is an active field of research. Interestingly, the novel proposed mechanisms for the antidiabetic activity of PPARγ agonists, consisting of PPARγ Ser273 phosphorylation inhibition, ligand and receptor mutual dynamics, and the presence of an alternate binding site, have recently changed the view regarding the optimal characteristics for the screening of novel PPARγ ligands. Furthermore, transcriptional genomics could bring essential information about the genome-wide effects of PPARγ ligands. Consequently, facing the new mechanistic scenario proposed for these compounds is essential for resolving the paradoxes among their agonistic function, antidiabetic activities, and side effects and should allow the rational development of better and safer PPARγ-mediated antidiabetic drugs.

  20. The Impact of CB2 Receptor Ligands on the MK-801-Induced Hyperactivity in Mice.

    PubMed

    Kruk-Slomka, Marta; Banaszkiewicz, Izabela; Biala, Grazyna

    2017-04-01

    It has been known that there is a relationship between cannabis use and schizophrenia-related symptoms; however, it can be a subject of controversy. The involvement of CB1 receptor ligands in the schizophrenia has already been revealed and confirmed. However, there is still lack of information concerning the role of CB2 receptors in the psychosis-like effects in mice and the further studies are needed.The aim of the present research was to study the role of the CB2 receptor ligands in the symptoms typical for schizophrenia. We provoked hyperlocomotion in mice which is analogous to positive psychosis-like effects in humans, by an acute administration of a NMDA receptor antagonist, MK-801 (0.3 and 0.6 mg/kg), a pharmacological model of schizophrenia. An acute administration of MK-801 induced the increase in locomotor activity (hyperactivity) in rodents, measured in actimeters.We revealed that an acute injection of CB2 receptor agonist JWH 133 at the dose range (0.05-1.0 mg/kg) and CB2 receptor antagonist, AM 630 at the dose range (0.1-1.0 mg/kg) decreased locomotion of mice. An acute injection of JWH 133 (2.0 mg/kg) and AM 630 (2.0 mg/kg) had no statistical significant influence on the locomotor activity of mice. However, an acute injection of both CB2 receptor ligands (agonist and antagonist), JWH 133, at the non-effective dose of 2.0 mg/kg and AM 630 at the non-effective dose of 2.0 mg/kg, potentiated the MK-801-induced hyperactivity.The present findings have confirmed that endocannabinoid system, not only via CB1, but also via CB2 receptors, may be involved in the schizophrenia-like responses, including hyperlocomotion in mice.

  1. Highly Pathogenic Influenza A(H5Nx) Viruses with Altered H5 Receptor-Binding Specificity

    PubMed Central

    Guo, Hongbo; de Vries, Erik; McBride, Ryan; Dekkers, Jojanneke; Peng, Wenjie; Bouwman, Kim M.; Nycholat, Corwin; Verheije, M. Helene; Paulson, James C.; van Kuppeveld, Frank J.M.

    2017-01-01

    Emergence and intercontinental spread of highly pathogenic avian influenza A(H5Nx) virus clade 2.3.4.4 is unprecedented. H5N8 and H5N2 viruses have caused major economic losses in the poultry industry in Europe and North America, and lethal human infections with H5N6 virus have occurred in Asia. Knowledge of the evolution of receptor-binding specificity of these viruses, which might affect host range, is urgently needed. We report that emergence of these viruses is accompanied by a change in receptor-binding specificity. In contrast to ancestral clade 2.3.4 H5 proteins, novel clade 2.3.4.4 H5 proteins bind to fucosylated sialosides because of substitutions K222Q and S227R, which are unique for highly pathogenic influenza virus H5 proteins. North American clade 2.3.4.4 virus isolates have retained only the K222Q substitution but still bind fucosylated sialosides. Altered receptor-binding specificity of virus clade 2.3.4.4 H5 proteins might have contributed to emergence and spread of H5Nx viruses. PMID:27869615

  2. Integration of Genome-Wide Computation DRE Search, AhR ChIP-chip and Gene Expression Analyses of TCDD-Elicited Responses in the Mouse Liver

    PubMed Central

    2011-01-01

    Background The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor (TF) that mediates responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Integration of TCDD-induced genome-wide AhR enrichment, differential gene expression and computational dioxin response element (DRE) analyses further elucidate the hepatic AhR regulatory network. Results Global ChIP-chip and gene expression analyses were performed on hepatic tissue from immature ovariectomized mice orally gavaged with 30 μg/kg TCDD. ChIP-chip analysis identified 14,446 and 974 AhR enriched regions (1% false discovery rate) at 2 and 24 hrs, respectively. Enrichment density was greatest in the proximal promoter, and more specifically, within ± 1.5 kb of a transcriptional start site (TSS). AhR enrichment also occurred distal to a TSS (e.g. intergenic DNA and 3' UTR), extending the potential gene expression regulatory roles of the AhR. Although TF binding site analyses identified over-represented DRE sequences within enriched regions, approximately 50% of all AhR enriched regions lacked a DRE core (5'-GCGTG-3'). Microarray analysis identified 1,896 number of TCDD-responsive genes (|fold change| ≥ 1.5, P1(t) > 0.999). Integrating this gene expression data with our ChIP-chip and DRE analyses only identified 625 differentially expressed genes that involved an AhR interaction at a DRE. Functional annotation analysis of differentially regulated genes associated with AhR enrichment identified overrepresented processes related to fatty acid and lipid metabolism and transport, and xenobiotic metabolism, which are consistent with TCDD-elicited steatosis in the mouse liver. Conclusions Details of the AhR regulatory network have been expanded to include AhR-DNA interactions within intragenic and intergenic genomic regions. Moreover, the AhR can interact with DNA independent of a DRE core suggesting there are alternative mechanisms of AhR-mediated gene regulation. PMID:21762485

  3. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

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

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the nativemore » ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.« less

  4. Structural Analysis of Chemokine Receptor–Ligand Interactions

    PubMed Central

    2017-01-01

    This review focuses on the construction and application of structural chemokine receptor models for the elucidation of molecular determinants of chemokine receptor modulation and the structure-based discovery and design of chemokine receptor ligands. A comparative analysis of ligand binding pockets in chemokine receptors is presented, including a detailed description of the CXCR4, CCR2, CCR5, CCR9, and US28 X-ray structures, and their implication for modeling molecular interactions of chemokine receptors with small-molecule ligands, peptide ligands, and large antibodies and chemokines. These studies demonstrate how the integration of new structural information on chemokine receptors with extensive structure–activity relationship and site-directed mutagenesis data facilitates the prediction of the structure of chemokine receptor–ligand complexes that have not been crystallized. Finally, a review of structure-based ligand discovery and design studies based on chemokine receptor crystal structures and homology models illustrates the possibilities and challenges to find novel ligands for chemokine receptors. PMID:28165741

  5. Unravelling intrinsic efficacy and ligand bias at G protein coupled receptors: A practical guide to assessing functional data.

    PubMed

    Stott, Lisa A; Hall, David A; Holliday, Nicholas D

    2016-02-01

    Stephenson's empirical definition of an agonist, as a ligand with binding affinity and intrinsic efficacy (the ability to activate the receptor once bound), underpins classical receptor pharmacology. Quantifying intrinsic efficacy using functional concentration response relationships has always presented an experimental challenge. The requirement for realistic determination of efficacy is emphasised by recent developments in our understanding of G protein coupled receptor (GPCR) agonists, with recognition that some ligands stabilise different active conformations of the receptor, leading to pathway-selective, or biased agonism. Biased ligands have potential as therapeutics with improved selectivity and clinical efficacy, but there are also pitfalls to the identification of pathway selective effects. Here we explore the basics of concentration response curve analysis, beginning with the need to distinguish ligand bias from other influences of the functional system under study. We consider the different approaches that have been used to quantify and compare biased ligands, many of which are based on the Black and Leff operational model of agonism. Some of the practical issues that accompany these analyses are highlighted, with opportunities to improve estimates in future, particularly in the separation of true agonist intrinsic efficacy from the contributions of system dependent coupling efficiency. Such methods are by their nature practical approaches, and all rely on Stephenson's separation of affinity and efficacy parameters, which are interdependent at the mechanistic level. Nevertheless, operational analysis methods can be justified by mechanistic models of GPCR activation, and if used wisely are key elements to biased ligand identification. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Evaluation of AhR-agonists and AhR-agonist activity in sediments of Liaohe River protected areas, China.

    PubMed

    Zhang, Yun; Ke, Xin; Gui, Shaofeng; Wu, Xiaoqiong; Wang, Chunyong; Zhang, Haijun

    2017-02-15

    A total of 9 sediment samples of Liaohe River protected areas were collected to evaluate aryl hydrocarbon receptor agonists (AhR-agonists) and AhR-agonist activity via chemical analysis and in vitro H4IIE cell bioassay. Results indicated that bioassay-derived 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (Bio-TEQs) ranged from 89.1 to 251.1pg/g dry weight. Concentrations of 16 EPA polycyclic aromatic hydrocarbons (PAHs), 12 dioxin-like polychlorinated biphenyls (PCBs), and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) ranged from 256.8 to 560.1ng/g, 79.2 to 416.2pg/g, and 199.6 to 538.4pg/g, respectively. According to potency balance analysis, TEQ chem s based on PAHs, PCBs, and PCDD/Fs could contribute 16.56% to 26.11% of Bio-TEQs. This could be explained by the potential existence of unidentified AhR-agonists and the potential non-additive interactions among AhR-agonists in sediment extracts. Through the different contributions to Bio-TEQs, this study confirms that PCDD/Fs were the main pollutants that induced significantly AhR-agonist activity in sediments of Liaohe River protected areas. Copyright © 2016. Published by Elsevier Ltd.

  7. Conformational study on cyclic melanocortin ligands and new insight into their binding mode at the MC4 receptor.

    PubMed

    Grieco, Paolo; Brancaccio, Diego; Novellino, Ettore; Hruby, Victor J; Carotenuto, Alfonso

    2011-09-01

    The melanocortin receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets to treat obesity, sexual dysfunction, etc. Understanding the basis of the ligand-receptor interactions is crucial for the design of potent and selective ligands for these receptors. The conformational preferences of the cyclic melanocortin ligands MTII (Ac-Nle(4)-c[Asp(5)-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)) and SHU9119 (Ac-Nle(4)-c[Asp(5)-His(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)), which show agonist and antagonist activity at the h-MC4R, respectively, were comprehensively investigated by solution NMR spectroscopy in different environments. In particular, water and water/DMSO (8:2) solutions were used as isotropic solutions and an aqueous solution of DPC (dodecylphosphocholine) micelles was used as a membrane mimetic environment. NMR-derived conformations of these two ligands were docked within h-MC4R models. NMR and docking studies revealed intriguing differences which can help explain the different activities of these two ligands. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  8. DEVELOPMENTAL EXPRESSION OF TWO MEMBERS OF A NEW CLASS OF TRANSCRIPTION FACTORS: II. EXPRESSION OF ARYL HYDROCARBON RECEPTOR NUCLEAR TRANSLOCATOR IN THE C57BL/6N MOUSE EMBRYO

    EPA Science Inventory

    The Aryl hydrocarbon receptor (AhR) is a ligand-activated, transcription factor with a basic region/helix (bHLH) motif. hR has been sequenced and the functional domains defined and there is information on the formation of complexes with other peptides and interactions with DNA, a...

  9. Oculomotor Deficits in Aryl Hydrocarbon Receptor Null Mouse

    PubMed Central

    Chevallier, Aline; Mialot, Antoine; Petit, Jean-Maurice; Fernandez-Salguero, Pedro; Barouki, Robert

    2013-01-01

    The Aryl hydrocarbon Receptor or AhR, a ligand-activated transcription factor, is known to mediate the toxic and carcinogenic effects of various environmental pollutants such as 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). Recent studies in Caenorhabditis elegans and Drosophila melanogaster show that the orthologs of the AhR are expressed exclusively in certain types of neurons and are implicated in the development and the homeostasis of the central nervous system. While physiological roles of the AhR were demonstrated in the mammalian heart, liver and gametogenesis, its ontogenic expression and putative neural functions remain elusive. Here, we report that the constitutive absence of the AhR in adult mice (AhR−/−) leads to abnormal eye movements in the form of a spontaneous pendular horizontal nystagmus. To determine if the nystagmus is of vestibular, visual, or cerebellar origin, gaze stabilizing reflexes, namely vestibulo-ocular and optokinetic reflexes (VOR and OKR), were investigated. The OKR is less effective in the AhR−/− mice suggesting a deficit in the visuo-motor circuitry, while the VOR is mildly affected. Furthermore, the AhR is expressedin the retinal ganglion cells during the development, however electroretinograms revealed no impairment of retinal cell function. The structure of the cerebellum of the AhR−/− mice is normal which is compatible with the preserved VOR adaptation, a plastic process dependent on cerebellar integrity. Finally, intoxication with TCDD of control adults did not lead to any abnormality of the oculomotor control. These results demonstrate that the absence of the AhR leads to acquired central nervous system deficits in the adults. Given the many common features between both AhR mouse and human infantile nystagmus syndromes, the AhR−/− mice might give insights into the developmental mechanisms which lead to congenital eye disorders. PMID:23301081

  10. Sigma receptor ligand N,N'-di-(ortho-tolyl)guanidine inhibits release of acetylcholine in the guinea pig ileum.

    PubMed

    Cambell, B G; Keana, J F; Weber, E

    1991-11-26

    The inhibition of stimulated contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation by sigma receptor ligands has been previously described. In this study, the stimulated release of [3H]acetylcholine from cholinergic nerve terminals in this same preparation was monitored in the presence and absence of sigma receptor ligands. N,N'-Di-(orthotolyl)guanidine (DTG) and other compounds selective for the sigma receptor inhibited stimulated [3H]acetylcholine release. These results suggest that their inhibition of stimulated contractions in this preparation was mediated by inhibition of acetylcholine release.

  11. Ligand binding and dynamics of the monomeric epidermal growth factor receptor ectodomain

    PubMed Central

    Loeffler, Hannes H; Winn, Martyn D

    2013-01-01

    The ectodomain of the human epidermal growth factor receptor (hEGFR) controls input to several cell signalling networks via binding with extracellular growth factors. To gain insight into the dynamics and ligand binding of the ectodomain, the hEGFR monomer was subjected to molecular dynamics simulation. The monomer was found to be substantially more flexible than the ectodomain dimer studied previously. Simulations where the endogeneous ligand EGF binds to either Subdomain I or Subdomain III, or where hEGFR is unbound, show significant differences in dynamics. The molecular mechanics Poisson–Boltzmann surface area method has been used to derive relative free energies of ligand binding, and we find that the ligand is capable of binding either subdomain with a slight preference for III. Alanine-scanning calculations for the effect of selected ligand mutants on binding reproduce the trends of affinity measurements. Taken together, these results emphasize the possible role of the ectodomain monomer in the initial step of ligand binding, and add details to the static picture obtained from crystal structures. Proteins 2013; 81:1931–1943. © 2013 The Authors. Proteins published by Wiley Periodicals, Inc. PMID:23760854

  12. Differential modulation of endothelin ligand-induced contraction in isolated tracheae from endothelin B (ETB) receptor knockout mice

    PubMed Central

    Hay, Douglas W P; Douglas, Stephen A; Ao, Zhaohui; Moesker, Rodney M; Self, Glenn J; Rigby, Paul J; Luttmann, Mark A; Goldie, Roy G

    2001-01-01

    The role of endothelin B (ETB) receptors in mediating ET ligand-induced contractions in mouse trachea was examined in ETB receptor knockout animals.Autoradiographic binding studies, using [125I]-ET-1, confirmed the presence of ETA receptors in tracheal and bronchial airway smooth muscle from wild-type (+/+) and homozygous recessive (−/−) ETB receptor knockout mice. In contrast, ETB receptors were not detected in airway tissues from (−/−) mice.In tracheae from (+/+) mice, the rank order of potencies of the ET ligands was sarafotoxin (Stx) S6c>ET-1>ET-3; Stx S6c had a lower efficacy than ET-1 or ET-3. In tissues from (−/−) mice there was no response to Stx S6c (up to 0.1 μM), whereas the maximum responses and potencies of ET-1 and ET-3 were similar to those in (+/+) tracheae. ET-3 concentration-response curve was biphasic in (+/+) tissues (via ETA and ETB receptor activation), and monophasic in (−/−) preparations (via stimulation of only ETA receptors).In (+/+) preparations SB 234551 (1 nM), an ETA receptor-selective antagonist, inhibited the secondary phase, but not the first phase, of the ET-3 concentration-response curve, whereas A192621 (100 nM), an ETB receptor-selective antagonist, had the opposite effect. In (−/−) tissues SB 234551 (1 nM), but not A192621 (100 nM), produced a rightward shift in ET-3 concentration-response curves.The results confirm the significant influence of both ETA and ETB receptors in mediating ET-1-induced contractions in mouse trachea. Furthermore, the data do not support the hypothesis of atypical ETB receptors. In this preparation ET-3 is not an ETB receptor-selective ligand, producing contractions via activation of both ETA and ETB receptors. PMID:11309263

  13. A photoaffinity ligand for dopamine D2 receptors: azidoclebopride.

    PubMed

    Niznik, H B; Guan, J H; Neumeyer, J L; Seeman, P

    1985-02-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind [3H]spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol. The degree of D2 dopamine receptor photoinduced inactivation by azidoclebopride was not significantly affected by scavengers such as p-aminobenzoic acid and dithiothreitol. Furthermore, irradiation of striatal membranes with a concentration of azidoclebopride sufficient to inactivate dopamine D2 receptors by 60% did not significantly reduce dopamine D1, serotonin (S2), benzodiazepine, alpha 1- or beta-noradrenergic receptors. This study describes the use of a novel and selective photoaffinity ligand for brain dopamine D2 receptors. The molecule, in radiolabeled form, may aid in the

  14. Dimeric Arrangement of the Parathyroid Hormone Receptor and a Structural Mechanism for Ligand-induced Dissociation

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

    Pioszak, Augen A.; Harikumar, Kaleeckal G.; Parker, Naomi R.

    2010-06-25

    The parathyroid hormone receptor (PTH1R) is a class B G protein-coupled receptor that is activated by parathyroid hormone (PTH) and PTH-related protein (PTHrP). Little is known about the oligomeric state of the receptor and its regulation by hormone. The crystal structure of the ligand-free PTH1R extracellular domain (ECD) reveals an unexpected dimer in which the C-terminal segment of both ECD protomers forms an {alpha}-helix that mimics PTH/PTHrP by occupying the peptide binding groove of the opposing protomer. ECD-mediated oligomerization of intact PTH1R was confirmed in living cells by bioluminescence and fluorescence resonance energy transfer experiments. As predicted by the structure,more » PTH binding disrupted receptor oligomerization. A receptor rendered monomeric by mutations in the ECD retained wild-type PTH binding and cAMP signaling ability. Our results are consistent with the hypothesis that PTH1R forms constitutive dimers that are dissociated by ligand binding and that monomeric PTH1R is capable of activating G protein.« less

  15. The future of type 1 cannabinoid receptor allosteric ligands.

    PubMed

    Alaverdashvili, Mariam; Laprairie, Robert B

    2018-02-01

    Allosteric modulation of the type 1 cannabinoid receptor (CB1R) holds great therapeutic potential. This is because allosteric modulators do not possess intrinsic efficacy, but instead augment (positive allosteric modulation) or diminish (negative allosteric modulation) the receptor's response to endogenous ligand. Consequently, CB1R allosteric modulators have an effect ceiling which allows for the tempering of CB1R signaling without the desensitization, tolerance, dependence, and psychoactivity associated with orthosteric compounds. Pain, movement disorders, epilepsy, obesity are all potential therapeutic targets for CB1R allosteric modulation. Several challenges exist for the development of CB1R allosteric modulators, such as receptor subtype specificity, translation to in vivo systems, and mixed allosteric/agonist/inverse agonist activity. Despite these challenges, elucidation of crystal structures of CB1R and compound design based on structure-activity relationships will advance the field. In this review, we will cover recent progress for CB1R allosteric modulators and discuss the future promise of this research.

  16. Dibutyl Phthalate (DBP)-Induced Apoptosis and Neurotoxicity are Mediated via the Aryl Hydrocarbon Receptor (AhR) but not by Estrogen Receptor Alpha (ERα), Estrogen Receptor Beta (ERβ), or Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) in Mouse Cortical Neurons.

    PubMed

    Wójtowicz, Anna K; Szychowski, Konrad A; Wnuk, Agnieszka; Kajta, Małgorzata

    2017-01-01

    Dibutyl phthalate (di-n-butyl phthalate, DBP) is one of the most commonly used phthalate esters. DBP is widely used as a plasticizer in a variety of household industries and consumer products. Because phthalates are not chemically bound to products, they can easily leak out to enter the environment. DBP can pass through the placental and blood-brain barriers due to its chemical structure, but little is known about its mechanism of action in neuronal cells. This study demonstrated the toxic and apoptotic effects of DBP in mouse neocortical neurons in primary cultures. DBP stimulated caspase-3 and LDH activities as well as ROS formation in a concentration (10 nM-100 µM) and time-dependent (3-48 h) manner. DBP induced ROS formation at nanomolar concentrations, while it activated caspase-3 and LDH activities at micromolar concentrations. The biochemical effects of DBP were accompanied by decreased cell viability and induction of apoptotic bodies. Exposure to DBP reduced Erα and Pparγ mRNA expression levels, which were inversely correlated with protein expression of the receptors. Treatment with DBP enhanced Ahr mRNA expression, which was reflected by the increased AhR protein level observed at 3 h after exposure. ERα, ERβ, and PPARγ antagonists stimulated DBP-induced caspase-3 and LDH activities. AhR silencing demonstrated that DBP-induced apoptosis and neurotoxicity are mediated by AhR, which is consistent with the results from DBP-induced enhancement of AhR mRNA and protein expression. Our study showed that AhR is involved in DBP-induced apoptosis and neurotoxicity, while the ERs and PPARγ signaling pathways are impaired by the phthalate.

  17. The neuraminidases of MDCK grown human influenza A(H3N2) viruses isolated since 1994 can demonstrate receptor binding.

    PubMed

    Mohr, Peter G; Deng, Yi-Mo; McKimm-Breschkin, Jennifer L

    2015-04-22

    The neuraminidases (NAs) of MDCK passaged human influenza A(H3N2) strains isolated since 2005 are reported to have dual functions of cleavage of sialic acid and receptor binding. NA agglutination of red blood cells (RBCs) can be inhibited by neuraminidase inhibitors (NAIs), thus distinguishing it from haemagglutinin (HA) binding. We wanted to know if viruses prior to 2005 can demonstrate this property. Pairs of influenza A(H3N2) isolates ranging from 1993-2008 passaged in parallel only in eggs or in MDCK cells were tested for inhibition of haemagglutination by various NAIs. Only viruses isolated since 1994 and cultured in MDCK cells bound chicken RBCs solely through their NA. NAI inhibition of agglutination of turkey RBCs was seen for some, but not all of these same MDCK grown viruses. Efficacy of inhibition of enzyme activity and haemagglutination differed between NAIs. For many viruses lower concentrations of oseltamivir could inhibit agglutination compared to zanamivir, although they could both inhibit enzyme activity at comparable concentrations. An E119V mutation reduced sensitivity to oseltamivir and 4-aminoDANA for both the enzyme assay and inhibition of agglutination. Sequence analysis of the NAs and HAs of some paired viruses revealed mutations in the haemagglutinin of all egg passaged viruses. For many of the paired egg and MDCK cultured viruses we found no differences in their NA sequences by Sanger sequencing. However, deep sequencing of MDCK grown isolates revealed low levels of variant populations with mutations at either D151 or T148 in the NA, suggesting mutations at either site may be able to confer this property. The NA active site of MDCK cultured human influenza A(H3N2) viruses isolated since 1994 can express dual enzyme and receptor binding functions. Binding correlated with either D151 or T148 mutations. The catalytic and receptor binding sites do not appear to be structurally identical since relative concentrations of the NAIs to inhibit

  18. Crystal Structure of Oligomeric β1-Adrenergic G Protein- Coupled Receptors in Ligand-Free Basal State

    PubMed Central

    Huang, Jianyun; Chen, Shuai; Zhang, J. Jillian; Huang, Xin-Yun

    2013-01-01

    G protein-coupled receptors (GPCRs) mediate transmembrane signaling. Before ligand binding, GPCRs exist in a basal state. Crystal structures of several GPCRs bound with antagonists or agonists have been solved. However, the crystal structure of the ligand-free basal state of a GPCR, the starting point of GPCR activation and function, has not been determined. Here we report the X-ray crystal structure of the first ligand-free basal state of a GPCR in a lipid membrane-like environment. Oligomeric turkey β1-adrenergic receptors display two alternating dimer interfaces. One interface involves the transmembrane domain (TM) 1, TM2, the C-terminal H8, and the extracellular loop 1. The other interface engages residues from TM4, TM5, the intracellular loop 2 and the extracellular loop 2. Structural comparisons show that this ligand-free state is in an inactive conformation. This provides the structural information regarding GPCR dimerization and oligomerization. PMID:23435379

  19. Unconventional ligands and modulators of nicotinic receptors.

    PubMed

    Pereira, Edna F R; Hilmas, Corey; Santos, Mariton D; Alkondon, Manickavasagom; Maelicke, Alfred; Albuquerque, Edson X

    2002-12-01

    Evidence gathered from epidemiologic and behavioral studies have indicated that neuronal nicotinic receptors (nAChRs) are intimately involved in the pathogenesis of a number of neurologic disorders, including Alzheimer's disease, Parkinson's disease, and schizophrenia. In the mammalian brain, neuronal nAChRs, in addition to mediating fast synaptic transmission, modulate fast synaptic transmission mediated by the major excitatory and inhibitory neurotransmitters glutamate and GABA, respectively. Of major interest, however, is the fact that the activity of the different subtypes of neuronal nAChR is also subject to modulation by substances of endogenous origin such as choline, the tryptophan metabolite kynurenic acid, neurosteroids, and beta-amyloid peptides and by exogenous substances, including the so-called nicotinic allosteric potentiating ligands, of which galantamine is the prototype, and psychotomimetic drugs such as phencyclidine and ketamine. The present article reviews and discusses the effects of unconventional ligands on nAChR activity and briefly describes the potential benefits of using some of these compounds in the treatment of neuropathologic conditions in which nAChR function/expression is known to be altered. Copyright 2002 Wiley Periodicals, Inc.

  20. Ligand Recognition by A-Class Eph Receptors: Crystal Structures of the EphA2 Ligand-Binding Domain and the EphA2/ephrin-A1 Complex

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

    Himanen, J.; Goldgur, Y; Miao, H

    2009-01-01

    Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B-class receptors. Here, we present the crystal structures of an A-class complex between EphA2 and ephrin-A1 and of unbound EphA2. Although these structures are similar overall to their B-class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A-class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a 'lock-and-key'-type binding mechanism, in contrast to the 'induced fit' mechanism defining the B-class molecules.more » This model is supported by structure-based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell-based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2-specific homotypic cell adhesion interactions.« less

  1. Effect of peripheral benzodiazepine receptor ligands on lipopolysaccharide-induced tumor necrosis factor activity in thioglycolate-treated mice.

    PubMed Central

    Matsumoto, T; Ogata, M; Koga, K; Shigematsu, A

    1994-01-01

    To investigate the effect of peripheral and central benzodiazepine receptor ligands on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) activity in mouse macrophages, three types of ligands, 4'-chlorodiazepam (pure peripheral), midazolam (mixed), and clonazepam (pure central), were compared. Midazolam and 4'-chlorodiazepam significantly suppressed LPS (1-microgram/ml)-induced TNF activity in thioglycolate-elicited mouse macrophages. In every concentration examined (0.001 to 100 microM), 4'-chlorodiazepam was the most effective agent, clonazepam was the least effective agent, and midazolam had an effect intermediate between those of the other two ligands. The peripheral benzodiazepine receptor ligands had a dose-dependent suppressive effect, and the 50% inhibitory concentrations were 0.01 microM for 4'-chlorodiazepam and 5 microM for midazolam. Concomitant use of PK 11195 (10 microM), an antagonist of the peripheral benzodiazepine receptor, reversed this suppressive effect with 4'-chlorodiazepam (10 microM) or midazolam (10 microM). PK 11195 showed this antagonistic effect in a dose-dependent manner. Intravenous 4'-chlorodiazepam (5 mg/kg of body weight) significantly suppressed LPS (100-micrograms)-induced TNF activity of sera (2 h postchallenge with LPS) from thioglycolate-treated mice. The present findings suggest that the peripheral benzodiazepine receptor plays an important role in modulating LPS-induced TNF activity in mouse macrophages. PMID:8031051

  2. Kinase cascades and ligand-directed signaling at the kappa opioid receptor.

    PubMed

    Bruchas, Michael R; Chavkin, Charles

    2010-06-01

    The dynorphin/kappa opioid receptor (KOR) system has been implicated as a critical component of the stress response. Stress-induced activation of dynorphin-KOR is well known to produce analgesia, and more recently, it has been implicated as a mediator of stress-induced responses including anxiety, depression, and reinstatement of drug seeking. Drugs selectively targeting specific KOR signaling pathways may prove potentially useful as therapeutic treatments for mood and addiction disorders. KOR is a member of the seven transmembrane spanning (7TM) G-protein coupled receptor (GPCR) superfamily. KOR activation of pertussis toxin-sensitive G proteins leads to Galphai/o inhibition of adenylyl cyclase production of cAMP and releases Gbetagamma, which modulates the conductances of Ca(+2) and K(+) channels. In addition, KOR agonists activate kinase cascades including G-protein coupled Receptor Kinases (GRK) and members of the mitogen-activated protein kinase (MAPK) family: ERK1/2, p38 and JNK. Recent pharmacological data suggests that GPCRs exist as dynamic, multi-conformational protein complexes that can be directed by specific ligands towards distinct signaling pathways. Ligand-induced conformations of KOR that evoke beta-arrestin-dependent p38 MAPK activation result in aversion; whereas ligand-induced conformations that activate JNK without activating arrestin produce long-lasting inactivation of KOR signaling. In this review, we discuss the current status of KOR signal transduction research and the data that support two novel hypotheses: (1) KOR selective partial agonists that do not efficiently activate p38 MAPK may be useful analgesics without producing the dysphoric or hallucinogenic effects of selective, highly efficacious KOR agonists and (2) KOR antagonists that do not activate JNK may be effective short-acting drugs that may promote stress-resilience.

  3. Interaction of ligands with the opiate receptors of brain membranes: Regulation by ions and nucleotides

    PubMed Central

    Blume, Arthur J.

    1978-01-01

    This study shows that nucleotides, as well as ions, regulate the opiate receptors of brain. GMP-P(NH)P and Na+ reduce the amount of steady-state specific [3H]dihydromorphine binding and increase the rate of dissociation of the ligand from the opiate receptor. In contrast, Mn2+ decreases the rate of ligand dissociation and antagonizes the ability of Na+ to increase dissociation. The effects of GMP-P(NH)P on steady-state binding and dissociation are not reversed by washing. Only GTP, GDP, ITP, and IMP-P(NH)P, in addition to GMP-P(NH)P, increase the rate of dihydromorphine dissociation. The site of nucleotide action appears to have high affinity: <1 μM GMP-P(NH)P produces half-maximal increases in ligand dissociation. GMP-P(NH)P- and Na+-directed increases in dissociation have also been found for the opiate agonists [3H]etorphine, [3H]Leu-enkephalin, and [3H]Met-enkephalin and the opiate antagonist [3H]naltrexone. Mn2+-directed decreases in dissociation have been found for the agonist [3H]-etorphine and the antagonist [3H]naltrexone. Although the plasma membrane receptors for a number of other neuro-transmitters and hormones are also regulated by guanine nucleotides, the opiate receptors appear unique because only they show nucleotide regulation of both agonist and antagonist binding. PMID:205867

  4. Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies.

    PubMed

    Wu, Qing-Ping; Zhang, Lei; Shao, Xiao-Xia; Wang, Jia-Hui; Gao, Yu; Xu, Zeng-Guang; Liu, Ya-Li; Guo, Zhan-Yun

    2016-04-01

    Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding assay for the relaxin-RXFP1 system to facilitate their interaction studies, such as characterization of relaxin analogues or screening novel agonists or antagonists of RXFP1.

  5. Structure-Guided Screening for Functionally Selective D2 Dopamine Receptor Ligands from a Virtual Chemical Library.

    PubMed

    Männel, Barbara; Jaiteh, Mariama; Zeifman, Alexey; Randakova, Alena; Möller, Dorothee; Hübner, Harald; Gmeiner, Peter; Carlsson, Jens

    2017-10-20

    Functionally selective ligands stabilize conformations of G protein-coupled receptors (GPCRs) that induce a preference for signaling via a subset of the intracellular pathways activated by the endogenous agonists. The possibility to fine-tune the functional activity of a receptor provides opportunities to develop drugs that selectively signal via pathways associated with a therapeutic effect and avoid those causing side effects. Animal studies have indicated that ligands displaying functional selectivity at the D 2 dopamine receptor (D 2 R) could be safer and more efficacious drugs against neuropsychiatric diseases. In this work, computational design of functionally selective D 2 R ligands was explored using structure-based virtual screening. Molecular docking of known functionally selective ligands to a D 2 R homology model indicated that such compounds were anchored by interactions with the orthosteric site and extended into a common secondary pocket. A tailored virtual library with close to 13 000 compounds bearing 2,3-dichlorophenylpiperazine, a privileged orthosteric scaffold, connected to diverse chemical moieties via a linker was docked to the D 2 R model. Eighteen top-ranked compounds that occupied both the orthosteric and allosteric site were synthesized, leading to the discovery of 16 partial agonists. A majority of the ligands had comparable maximum effects in the G protein and β-arrestin recruitment assays, but a subset displayed preference for a single pathway. In particular, compound 4 stimulated β-arrestin recruitment (EC 50 = 320 nM, E max = 16%) but had no detectable G protein signaling. The use of structure-based screening and virtual libraries to discover GPCR ligands with tailored functional properties will be discussed.

  6. Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor.

    PubMed

    Yang, Zhenlin; Han, Shuo; Keller, Max; Kaiser, Anette; Bender, Brian J; Bosse, Mathias; Burkert, Kerstin; Kögler, Lisa M; Wifling, David; Bernhardt, Guenther; Plank, Nicole; Littmann, Timo; Schmidt, Peter; Yi, Cuiying; Li, Beibei; Ye, Sheng; Zhang, Rongguang; Xu, Bo; Larhammar, Dan; Stevens, Raymond C; Huster, Daniel; Meiler, Jens; Zhao, Qiang; Beck-Sickinger, Annette G; Buschauer, Armin; Wu, Beili

    2018-04-01

    Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y 1 , Y 2 , Y 4 and Y 5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y 1 receptor (Y 1 R) 4 . A number of peptides and small-molecule compounds have been characterized as Y 1 R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y 1 R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y 1 R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y 1 R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y 1 R can enable structure-based drug discovery that targets NPY receptors.

  7. The Ligand Binding Region of the Sigma-1 Receptor: Studies Utilizing Photoaffinity Probes, Sphingosine and N-Alkylamines

    PubMed Central

    Ruoho, Arnold E.; Chu, Uyen B.; Ramachandran, Subramaniam; Fontanilla, Dominique; Mavlyutov, Timur; Hajipour, Abdol R.

    2015-01-01

    The sigma-1 receptor is a 26 kDa endoplasmic reticulum resident membrane protein that has been shown to have chaperone activity in addition to its promiscuous binding to pharmacological agents. Ligand binding domain(s) of the sigma-1 receptor have been identified using the E. coli expressed and purified receptor protein and novel radioiodinated azido photoaffinity probes followed by pro-teolytic and chemical cleavage strategies. The outcome of these experiments indicates that the sigma-1 receptor ligand binding regions are formed primarily by juxtaposition of its second and third hydrophobic domains, regions where the protein shares considerable homology with the fungal enzyme, sterol isomerase that is essential for the biosynthesis of ergosterol. Data indicate that these hydrophobic steroid binding domain like (SBDL) regions on the sigma-1 receptor are likely to interact selectively with N-alkyl amines such as the endogenous sphingolipids and with synthetic N-alkylamines and N-aralkylamines derivatives. A proposed model for the sigma-1 receptor is presented. PMID:22288412

  8. The Caenorhabditis elegans DAF-12 nuclear receptor: structure, dynamics, and interaction with ligands.

    PubMed

    Alvarez, Lautaro D; Mañez, Pau Arroyo; Estrin, Darío A; Burton, Gerardo

    2012-07-01

    A structure for the ligand binding domain (LBD) of the DAF-12 receptor from Caenorhabditis elegans was obtained from the X-ray crystal structure of the receptor LBD from Strongyloides stercoralis bound to (25R)-Δ(7)-dafachronic acid (DA) (pdb:3GYU). The model was constructed in the presence of the ligand using a combination of Modeller, Autodock, and molecular dynamics (MD) programs, and then its dynamical behavior was studied by MD. A strong ligand binding mode (LBM) was found, with the three arginines in the ligand binding pocket (LBP) contacting the C-26 carboxylate group of the DA. The quality of the ceDAF-12 model was then evaluated by constructing several ligand systems for which the experimental activity is known. Thus, the dynamical behavior of the ceDAF-12 complex with the more active (25S)-Δ(7)-DA showed two distinct binding modes, one of them being energetically more favorable compared with the 25R isomer. Then the effect of the Arg564Cys and Arg598Met mutations on the (25R)-Δ(7)-DA binding was analyzed. The MD simulations showed that in the first case the complex was unstable, consistent with the lack of transactivation activity of (25R)-Δ(7)-DA in this mutant. Instead, in the case of the Arg598Met mutant, known to produce a partial loss of activity, our model predicted smaller effects on the LBM with a more stable MD trajectory. The model also showed that removal of the C-25 methyl does not impede the simultaneous strong interaction of the carboxylate with the three arginines, predicting that 27-nor-DAs are putative ceDAF-12 ligands. Copyright © 2012 Wiley Periodicals, Inc.

  9. Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain.

    PubMed

    Runge, Steffen; Thøgersen, Henning; Madsen, Kjeld; Lau, Jesper; Rudolph, Rainer

    2008-04-25

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.

  10. How does oxygen rise drive evolution? Clues from oxygen-dependent biosynthesis of nuclear receptor ligands

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

    Jiang, Ying-Ying; Kong, De-Xin; Qin, Tao

    2010-01-08

    It is well known that oxygen rise greatly facilitated biological evolution. However, the underlying mechanisms remain elusive. Recently, Raymond and Segre revealed that molecular oxygen allows 1000 more metabolic reactions than can occur in anoxic conditions. From the novel metabolites produced in aerobic metabolism, we serendipitously found that some of the metabolites are signaling molecules that target nuclear receptors. Since nuclear signaling systems are indispensable to superior organisms, we speculated that aerobic metabolism may facilitate biological evolution through promoting the establishment of nuclear signaling systems. This hypothesis is validated by the observation that most (97.5%) nuclear receptor ligands are producedmore » by aerobic metabolism, which is further explained in terms of the chemical criteria (appropriate volume and rather high hydrophobicity) of nuclear receptor ligands that aerobic metabolites are more ready than anaerobic counterparts to satisfy these criteria.« less

  11. Single-Molecule Patch-Clamp FRET Anisotropy Microscopy Studies of NMDA Receptor Ion Channel Activation and Deactivation under Agonist Ligand Binding in Living Cells.

    PubMed

    Sasmal, Dibyendu Kumar; Yadav, Rajeev; Lu, H Peter

    2016-07-20

    N-methyl-d-aspartate (NMDA) receptor ion channel is activated by the binding of two pairs of glycine and glutamate along with the application of action potential. Binding and unbinding of ligands changes its conformation that plays a critical role in the open-close activities of NMDA receptor. Conformation states and their dynamics due to ligand binding are extremely difficult to characterize either by conventional ensemble experiments or single-channel electrophysiology method. Here we report the development of a new correlated technical approach, single-molecule patch-clamp FRET anisotropy imaging and demonstrate by probing the dynamics of NMDA receptor ion channel and kinetics of glycine binding with its ligand binding domain. Experimentally determined kinetics of ligand binding with receptor is further verified by computational modeling. Single-channel patch-clamp and four-channel fluorescence measurement are recorded simultaneously to get correlation among electrical on and off states, optically determined conformational open and closed states by FRET, and binding-unbinding states of the glycine ligand by anisotropy measurement at the ligand binding domain of GluN1 subunit. This method has the ability to detect the intermediate states in addition to electrical on and off states. Based on our experimental results, we have proposed that NMDA receptor gating goes through at least one electrically intermediate off state, a desensitized state, when ligands remain bound at the ligand binding domain with the conformation similar to the fully open state.

  12. AhV_aPA-induced vasoconstriction involves the IP₃Rs-mediated Ca²⁺ releasing.

    PubMed

    Zeng, Fuxing; Zou, Zhisong; Niu, Liwen; Li, Xu; Teng, Maikun

    2013-08-01

    AhV_aPA, the acidic PLA₂ purified from Agkistrodon halys pallas venom, was previously reported to possess a strong enzymatic activity and can remarkably induce a further contractile response on the 60 mM K⁺-induced contraction with an EC₅₀ in 369 nM on mouse thoracic aorta rings. In the present study, we found that the p-bromo-phenacyl-bromide (pBPB), which can completely inhibit the enzymatic activity of AhV_aPA, did not significantly reduce the contractile response on vessel rings induced by AhV_aPA, indicating that the vasoconstrictor effects of AhV_aPA are independent of the enzymatic activity. The inhibitor experiments showed that the contractile response induced by AhV_aPA is mainly attributed to the Ca²⁺ releasing from Ca²⁺ store, especially sarcoplasmic reticulum (SR). Detailed studies showed that the Ca²⁺ release from SR is related to the activation of inositol trisphosphate receptors (IP₃Rs) rather than ryanodine receptors (RyRs). Furthermore, the vasoconstrictor effect could be strongly reduced by pre-incubation with heparin, indicating that the basic amino acid residues on the surface of AhV_aPA may be involved in the interaction between AhV_aPA and the molecular receptors. These findings offer new insights into the functions of snake PLA₂ and provide a novel pathogenesis of A. halys pallas venom. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Cellular responses to oxidative stress: the [Ah] gene battery as a paradigm.

    PubMed Central

    Nebert, D W; Petersen, D D; Fornace, A J

    1990-01-01

    A major source of oxidative stress in animals is plant stress metabolites, also termed phytoalexins. The aromatic hydrocarbon-responsive [Ah] gene battery is considered here as a model system in which we can study metabolically coordinated enzymes that respond to phytoalexin-induced oxidative stress. In the mouse, the [Ah] battery comprises at least six genes: two Phase I genes, CYP1A1 and CYP1A2; and four Phase II genes, Nmo-1, Aldh-1, Ugt-1, and Gt-1. All six genes appear to be regulated positively by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other ligands of the Ah receptor. In the absence of foreign inducer, the control of Nmo-1 gene expression is independent of the control of CYP1A1 and CYP1A2 gene expression. The radiation deletion homozygote c14CoS/c14CoS mouse is lacking about 1.1 centiMorgans of chromosome 7. Although having no detectable CYP1A1 or CYP1A2 activation, the untreated c14CoS/c14CoS mouse exhibits markedly elevated transcripts of the Nmo-1 gene and three growth arrest- and DNA damage-inducible (gadd) genes. These data suggest that the missing region on chromosome 7 in the c14CoS/c14CoS mouse contains a gene(s), which we propose to call Nmo-1n, encoding a trans-acting factor(s) that is a negative effector of the Nmo-1 and gadd genes. The three other [Ah] battery Phase II genes behave similarly to Nmo-1 in the c14CoS/c14CoS mouse. This coordinated response to oxidative stress and DNA damage, by way of the release of a mammalian battery of genes from negative control, bears an interesting resemblance to the SOS response in bacteria. PMID:2272308

  14. Third-generation Ah receptor-responsive luciferase reporter plasmids: amplification of dioxin-responsive elements dramatically increases CALUX bioassay sensitivity and responsiveness.

    PubMed

    He, Guochun; Tsutsumi, Tomoaki; Zhao, Bin; Baston, David S; Zhao, Jing; Heath-Pagliuso, Sharon; Denison, Michael S

    2011-10-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like chemicals are widespread and persistent environmental contaminants that produce diverse toxic and biological effects through their ability to bind to and activate the Ah receptor (AhR) and AhR-dependent gene expression. The chemically activated luciferase expression (CALUX) system is an AhR-responsive recombinant luciferase reporter gene-based cell bioassay that has been used in combination with chemical extraction and cleanup methods for the relatively rapid and inexpensive detection and relative quantitation of dioxin and dioxin-like chemicals in a wide variety of sample matrices. Although the CALUX bioassay has been validated and used extensively for screening purposes, it has some limitations when screening samples with very low levels of dioxin-like chemicals or when there is only a small amount of sample matrix for analysis. Here, we describe the development of third-generation (G3) CALUX plasmids with increased numbers of dioxin-responsive elements, and stable transfection of these new plasmids into mouse hepatoma (Hepa1c1c7) cells has produced novel amplified G3 CALUX cell bioassays that respond to TCDD with a dramatically increased magnitude of luciferase induction and significantly lower minimal detection limit than existing CALUX-type cell lines. The new G3 CALUX cell lines provide a highly responsive and sensitive bioassay system for the detection and relative quantitation of very low levels of dioxin-like chemicals in sample extracts.

  15. Cell-specific targeting by heterobivalent ligands.

    PubMed

    Josan, Jatinder S; Handl, Heather L; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M; Vagner, Josef; Mash, Eugene A; Hruby, Victor J; Gillies, Robert J

    2011-07-20

    Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

  16. Cell-Specific Targeting by Heterobivalent Ligands

    PubMed Central

    Josan, Jatinder S.; Handl, Heather L.; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M.; Vagner, Josef; Mash, Eugene A.; Hruby, Victor J.; Gillies, Robert J.

    2012-01-01

    Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach—to specifically target combinations of cell-surface receptors using heteromultivalent ligands (“receptor combination approach”). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle4, DPhe7]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20–50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH2. Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging. PMID:21639139

  17. Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis

    PubMed Central

    Savas, Jeffrey N.; De Wit, Joris; Comoletti, Davide; Zemla, Roland; Ghosh, Anirvan

    2015-01-01

    Ligand-receptor interactions represent essential biological triggers which regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol which couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared to previous approaches, our analysis increases sensitivity, shortens analysis duration, and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These “ecto-Fcs” are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion trap mass spectrometers. In four working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our “Ecto-Fc MS” approach outperforms antibody-based approaches and provides a reproducible and robust framework to identify extracellular ligandreceptor interactions. PMID:25101821

  18. Dimensionality of Motion and Binding Valency Govern Receptor-Ligand Kinetics As Revealed by Agent-Based Modeling.

    PubMed

    Lehnert, Teresa; Figge, Marc Thilo

    2017-01-01

    Mathematical modeling and computer simulations have become an integral part of modern biological research. The strength of theoretical approaches is in the simplification of complex biological systems. We here consider the general problem of receptor-ligand binding in the context of antibody-antigen binding. On the one hand, we establish a quantitative mapping between macroscopic binding rates of a deterministic differential equation model and their microscopic equivalents as obtained from simulating the spatiotemporal binding kinetics by stochastic agent-based models. On the other hand, we investigate the impact of various properties of B cell-derived receptors-such as their dimensionality of motion, morphology, and binding valency-on the receptor-ligand binding kinetics. To this end, we implemented an algorithm that simulates antigen binding by B cell-derived receptors with a Y-shaped morphology that can move in different dimensionalities, i.e., either as membrane-anchored receptors or as soluble receptors. The mapping of the macroscopic and microscopic binding rates allowed us to quantitatively compare different agent-based model variants for the different types of B cell-derived receptors. Our results indicate that the dimensionality of motion governs the binding kinetics and that this predominant impact is quantitatively compensated by the bivalency of these receptors.

  19. REACTIVITY PROFILE OF LIGANDS OF MAMMALIAN RETINOIC ACID RECEPTORS: A PRELIMINARY COREPA ANALYSIS

    EPA Science Inventory

    Retinoic acid and associated derivatives comprise a class of endogenous hormones that bind to and activate different families of retinoic acid receptors (RARs, RXRs), and control many aspects of vertebrate development. Identification of potential RAR and RXR ligands is of interes...

  20. Direct Force Measurements of Receptor-Ligand Interactions on Living Cells

    NASA Astrophysics Data System (ADS)

    Eibl, Robert H.

    The characterization of cell adhesion between two living cells at the level of single receptor-ligand bonds is an experimental challenge. This chapter describes how the extremely sensitive method of atomic force microscopy (AFM) based force spectroscopy can be applied to living cells in order to probe for cell-to-cell or cell-to-substrate interactions mediated by single pairs of adhesion receptors. In addition, it is outlined how single-molecule AFM force spectroscopy can be used to detect physiologic changes of an adhesion receptor in a living cell. This force spectroscopy allows us to detect in living cells rapidly changing, chemokine SDF-1 triggered activation states of single VLA-4 receptors. This recently developed AFM application will allow for the detailed investigation of the integrin-chemokine crosstalk of integrin activation mechanisms and on how other adhesion receptors are modulated in health and disease. As adhesion molecules, living cells and even bacteria can be studied by single-molecule AFM force spectroscopy, this method is set to become a powerful tool that can not only be used in biophysics, but in cell biology as well as in immunology and cancer research.

  1. Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)*

    PubMed Central

    Chavan, Hemantkumar; Krishnamurthy, Partha

    2012-01-01

    Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics. PMID:22761424

  2. The novel sigma-2 receptor ligand SW43 stabilizes pancreas cancer progression in combination with gemcitabine

    PubMed Central

    2010-01-01

    Background Sigma-2 receptors are over-expressed in proliferating cancer cells, making an attractive target for the targeted treatment of pancreatic cancer. In this study, we investigated the role of the novel sigma-2 receptor ligand SW43 to induce apoptosis and augment standard chemotherapy. Results The binding affinity for sigma-2 ligands is high in pancreas cancer, and they induce apoptosis with a rank order of SV119 < SW43 < SRM in vitro. Combining these compounds with gemcitabine further increased apoptosis and decreased viability. Our in vivo model showed that sigma-2 ligand treatment decreased tumor volume to the same extent as gemcitabine. However, SW43 combination treatment with gemcitabine was superior to the other compounds and resulted in stabilization of tumor volume during treatment, with minimal toxicities. Conclusions This study shows that the sigma-2 ligand SW43 has the greatest capacity to augment gemcitabine in a pre-clinical model of pancreas cancer and has provided us with the rationale to move this compound forward with clinical investigations for patients with pancreatic cancer. PMID:21092190

  3. Sustained ligand-activated preconditioning via δ-opioid receptors.

    PubMed

    Peart, Jason N; Hoe, Louise E See; Gross, Garrett J; Headrick, John P

    2011-01-01

    We have previously described novel cardioprotection in response to sustained morphine exposure, efficacious in young to aged myocardium and mechanistically distinct from conventional opioid or preconditioning (PC) responses. We further investigate opioid-dependent sustained ligand-activated preconditioning (SLP), assessing duration of protection, opioid receptor involvement, additivity with conventional responses, and signaling underlying preischemic induction of the phenotype. Male C57BL/6 mice were treated with morphine (75-mg subcutaneous pellet) for 5 days followed by morphine-free periods (0, 3, 5, or 7 days) before ex vivo assessment of myocardial tolerance to 25-min ischemia/45-min reperfusion. SLP substantially reduced infarction (by ∼50%) and postischemic contractile dysfunction (eliminating contracture, doubling force development). Cardioprotection persisted for 5 to 7 days after treatment. SLP was induced specifically by δ-receptor and not κ- or μ-opioid receptor agonism, was eliminated by δ-receptor and nonselective antagonism, and was additive with adenosinergic but not acute morphine- or PC-triggered protection. Cotreatment during preischemic morphine exposure with the phosphoinositide-3 kinase (PI3K) inhibitor wortmannin, but not the protein kinase A (PKA) inhibitor myristoylated PKI-(14-22)-amide, prevented induction of SLP. This was consistent with shifts in total and phospho-Akt during the induction period. In summary, data reveal that SLP triggers sustained protection from ischemia for up to 7 days after stimulus, is δ-opioid receptor mediated, is induced in a PI3K-dependent/PKA-independent manner, and augments adenosinergic protection. Mechanisms underlying SLP may be useful targets for manipulation of ischemic tolerance in young or aged myocardium.

  4. Analysis of ligand-receptor cross-linked fragments by mass spectrometry

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

    Son, C.D.; Sargsyan, H.; Hurst, Gregory

    G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The a-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) experiments using biotinylated photoactivatable a-factor analogs. Affinity purification and enrichment of biotinylated peptides by monomeric avidin beads resulted in mass spectrometric detection of specific signals corresponding to crosslinked fragments ofmore » Ste2p. Data obtained from cyanogen bromide (CNBr) fragments of receptor cross-linked to an a-factor analog with the photoaffinity group p-benzoyl-L-phenylalanine on position 1 were in agreement with the previous results reported by our laboratory suggesting the cross-linking between position 1 of a-factor and a region of Ste2p covering residues 251 294.« less

  5. MIPs are ancestral ligands for the sex peptide receptor.

    PubMed

    Kim, Young-Joon; Bartalska, Katarina; Audsley, Neil; Yamanaka, Naoki; Yapici, Nilay; Lee, Ju-Youn; Kim, Yong-Chul; Markovic, Milica; Isaac, Elwyn; Tanaka, Yoshiaki; Dickson, Barry J

    2010-04-06

    Upon mating, females of many animal species undergo dramatic changes in their behavior. In Drosophila melanogaster, postmating behaviors are triggered by sex peptide (SP), which is produced in the male seminal fluid and transferred to female during copulation. SP modulates female behaviors via sex peptide receptor (SPR) located in a small subset of internal sensory neurons that innervate the female uterus and project to the CNS. Although required for postmating responses only in these female sensory neurons, SPR is expressed broadly in the CNS of both sexes. Moreover, SPR is also encoded in the genomes of insects that lack obvious SP orthologs. These observations suggest that SPR may have additional ligands and functions. Here, we identify myoinhibitory peptides (MIPs) as a second family of SPR ligands that is conserved across a wide range of invertebrate species. MIPs are potent agonists for Drosophila, Aedes, and Aplysia SPRs in vitro, yet are unable to trigger postmating responses in vivo. In contrast to SP, MIPs are not produced in male reproductive organs, and are not required for postmating behaviors in Drosophila females. We conclude that MIPs are evolutionarily conserved ligands for SPR, which are likely to mediate functions other than the regulation of female reproductive behaviors.

  6. Application of 3D-QSAR in the rational design of receptor ligands and enzyme inhibitors.

    PubMed

    Mor, Marco; Rivara, Silvia; Lodola, Alessio; Lorenzi, Simone; Bordi, Fabrizio; Plazzi, Pier Vincenzo; Spadoni, Gilberto; Bedini, Annalida; Duranti, Andrea; Tontini, Andrea; Tarzia, Giorgio

    2005-11-01

    Quantitative structure-activity relationships (QSARs) are frequently employed in medicinal chemistry projects, both to rationalize structure-activity relationships (SAR) for known series of compounds and to help in the design of innovative structures endowed with desired pharmacological actions. As a difference from the so-called structure-based drug design tools, they do not require the knowledge of the biological target structure, but are based on the comparison of drug structural features, thus being defined ligand-based drug design tools. In the 3D-QSAR approach, structural descriptors are calculated from molecular models of the ligands, as interaction fields within a three-dimensional (3D) lattice of points surrounding the ligand structure. These descriptors are collected in a large X matrix, which is submitted to multivariate analysis to look for correlations with biological activity. Like for other QSARs, the reliability and usefulness of the correlation models depends on the validity of the assumptions and on the quality of the data. A careful selection of compounds and pharmacological data can improve the application of 3D-QSAR analysis in drug design. Some examples of the application of CoMFA and CoMSIA approaches to the SAR study and design of receptor or enzyme ligands is described, pointing the attention to the fields of melatonin receptor ligands and FAAH inhibitors.

  7. Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators

    PubMed Central

    Lazennec, Gwendal; Canaple, Laurence; Saugy, Damien; Wahli, Walter

    2000-01-01

    The nuclear peroxisome proliferator-activated receptors (PPARs) α, β and γ activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. The activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas the activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase dependent induction of PPARs but also their ligand-dependent induction, suggesting that the ligands may also mobilize the PKA pathway to lead to maximal transcriptional induction by PPARs. Moreover, comparing PPARα KO with PPARα wild-type mice, we show that the expression of the ACO gene can be regulated by PKA-activated PPARα in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity and we propose a model associating this pathway in the control of fatty acid β-oxidation under conditions of fasting, stress and exercise. PMID:11117527

  8. Interactions between imidazoline I2 receptor ligands and acetaminophen in adult male rats: antinociception and schedule-controlled responding

    PubMed Central

    Siemian, Justin N.; Li, Jiuzhou; Zhang, Yanan; Li, Jun-Xu

    2015-01-01

    Rationale Recent evidence suggests that imidazoline I2 receptor ligands are suitable for combination therapy with opioids. Quantitative analysis of I2 receptor ligands combined with non-opioid drugs is necessary for justification of alternative pain therapies. Objective This study systematically examined the anti-hyperalgesic and response rate-suppressing effects of selective I2 receptor ligands (2-BFI and phenyzoline) alone and in combination with acetaminophen. Methods Von Frey and Hargreaves tests were used to examine the anti-hyperalgesic effects of drugs in complete Freund’s adjuvant (CFA)-induced inflammatory pain in rats. Food-reinforced schedule-controlled responding was used to assess the rate-suppressing effects of study drugs. Dose-addition and isobolographic analyses were used to assess drug-drug interactions for all assays. Results 2-BFI (3.2–17.8 mg/kg, i.p.), phenyzoline (17.8–100 mg/kg, i.p.), and acetaminophen (56–178 mg/kg, i.p.) all dose-dependently produced significant antinociceptive effects. When studied as combinations, 2-BFI and acetaminophen produced infra-additive to additive interactions while phenyzoline and acetaminophen produced additive to supra-additive interactions. The same drug combinations suppressed response rate in a supra-additive manner. Conclusions Quantitative analysis of the anti-hyperalgesic and response rate-suppressing effects suggests that I2 receptor ligands are not well suited to combination therapy with acetaminophen. PMID:26613734

  9. Hypocretin Receptor Expression in Canine and Murine Narcolepsy Models and in Hypocretin-Ligand Deficient Human Narcolepsy

    PubMed Central

    Mishima, Kazuo; Fujiki, Nobuhiro; Yoshida, Yasushi; Sakurai, Takeshi; Honda, Makoto; Mignot, Emmanuel; Nishino, Seiji

    2008-01-01

    Study Objective: To determine whether hypocretin receptor gene (hcrtR1 and hcrtR2) expression is affected after long-term hypocretin ligand loss in humans and animal models of narcolepsy. Design: Animal and human study. We measured hcrtR1 and hcrtR2 expression in the frontal cortex and pons using the RT-PCR method in murine models (8-week-old and 27-week-old orexin/ataxin-3 transgenic (TG) hypocretin cell ablated mice and wild-type mice from the same litter, 10 mice for each group), in canine models (8 genetically narcoleptic Dobermans with null mutations in the hcrtR2, 9 control Dobermans, 3 sporadic ligand-deficient narcoleptics, and 4 small breed controls), and in humans (5 narcolepsy-cataplexy patients with hypocretin deficiency (average age 77.0 years) and 5 control subjects (72.6 years). Measurement and Results: 27-week-old (but not 8-week-old) TG mice showed significant decreases in hcrtR1 expression, suggesting the influence of the long-term ligand loss on the receptor expression. Both sporadic narcoleptic dogs and human narcolepsy-cataplexy subjects showed a significant decrease in hcrtR1 expression, while declines in hcrtR2 expression were not significant in these cases. HcrtR2-mutated narcoleptic Dobermans (with normal ligand production) showed no alteration in hcrtR1 expression. Conclusions: Moderate declines in hcrtR expressions, possibly due to long-term postnatal loss of ligand production, were observed in hypocretin-ligand deficient narcoleptic subjects. These declines are not likely to be progressive and complete. The relative preservation of hcrtR2 expression also suggests that hypocretin based therapies are likely to be a viable therapeutic options in human narcolepsy-cataplexy. Citation: Mishima K; Fujiki N; Yoshida Y; Sakurai T; Honda M; Mignot E; Nishino S. Hypocretin receptor expression in canine and murine narcolepsy models and in hypocretin-ligand deficient human narcolepsy. SLEEP 2008;31(8):1119-1126. PMID:18714784

  10. Differential expression of ligands for NKG2D and DNAM-1 receptors by epithelial ovarian cancer-derived exosomes and its influence on NK cell cytotoxicity.

    PubMed

    Labani-Motlagh, Alireza; Israelsson, Pernilla; Ottander, Ulrika; Lundin, Eva; Nagaev, Ivan; Nagaeva, Olga; Dehlin, Eva; Baranov, Vladimir; Mincheva-Nilsson, Lucia

    2016-04-01

    Cancers constitutively produce and secrete into the blood and other biofluids 30-150 nm-sized endosomal vehicles called exosomes. Cancer-derived exosomes exhibit powerful influence on a variety of biological mechanisms to the benefit of the tumors that produce them. We studied the immunosuppressive ability of epithelial ovarian cancer (EOC) exosomes on two cytotoxic pathways of importance for anticancer immunity-the NKG2D receptor-ligand pathway and the DNAM-1-PVR/nectin-2 pathway. Using exosomes, isolated from EOC tumor explant and EOC cell-line culture supernatants, and ascitic fluid from EOC patients, we studied the expression of NKG2D and DNAM-1 ligands on EOC exosomes and their ability to downregulate the cognate receptors. Our results show that EOC exosomes differentially and constitutively express NKG2D ligands from both MICA/B and ULBP families on their surface, while DNAM-1 ligands are more seldom expressed and not associated with the exosomal membrane surface. Consequently, the NKG2D ligand-bearing EOC exosomes significantly downregulated the NKG2D receptor expression on peripheral blood mononuclear cells (PBMC) while the DNAM-1 receptor was unaffected. The downregulation of NKG2D receptor expression was coupled to inhibition of NKG2D receptor-ligand-mediated degranulation and cytotoxicity measured in vitro with OVCAR-3 and K562 cells as targets. The EOC exosomes acted as a decoy impairing the NKG2D mediated cytotoxicity while the DNAM-1 receptor-ligand system remained unchanged. Taken together, our results support and explain the mechanism behind the recently reported finding that in EOC, NK-cell recognition and killing of tumor cells was mainly dependent on DNAM-1 signaling while the contribution of the NKG2D receptor-ligand pathway was complementary and uncertain.

  11. Impacts of cannabinoid receptor ligands on nicotine- and chronic mild stress-induced cognitive and depression-like effects in mice.

    PubMed

    Pekala, Karolina; Michalak, Agnieszka; Kruk-Slomka, Marta; Budzynska, Barbara; Biala, Grazyna

    2018-07-16

    Taking into account the rather frequent concomitance of nicotine abuse and stress, we aimed to research memory- and depression-related effects of nicotine administration in combination with chronic mild unpredictable stress (CMUS) in mice and an involvement of the endocannabinoid system through CB1 and CB2 receptors. Mice were submitted to the CMUS for 4 weeks. Effects on depression-like behaviors and cognition, exerted by a combined administration of CB1, i.e., Oleamide (2.5, 5.0 mg/kg), AM 251 (0.1, 0.25 mg/kg) and CB2, i.e., JWH 133 (0.5, 2.0 mg/kg), AM 630 (0.25, 2.0 mg/kg) receptor ligands and nicotine (0.05, 0.1, 0.2 and 0.5 mg/kg), were then studied in stressed and unstressed mice by the forced swimming test and the passive avoidance paradigm, respectively. The results revealed that the CMUS-exposed mice exhibited depression-like behaviors and memory disturbances, while both effects were alleviated by nicotine. CB1 receptor ligands decreased antidepressive and cognitive (the latter for CB1 receptor antagonist only) effects of subchronic nicotine administration in stressed mice. CB1 and CB2 receptor antagonists exerted themselves some procognitive effects in those mice. Regarding the unstressed mice, CB1 and CB2 receptor ligands reversed the antidepressive effects of subchronic nicotine administration, while nicotine, in an ineffective dose, co-administered with CB2 receptor ligands, improved cognition. We confirmed the role of the two main subtypes of cannabinoid receptors, termed CB1 and CB2, on stress- and nicotine-related behavioral changes in mice. Our study has contributed to the understanding of the mechanisms involved in stress- and nicotine-induced disorders, such as anhedonia and memory disturbances. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Quartz crystal microbalance (QCM) with immobilized protein receptors: comparison of response to ligand binding for direct protein immobilization and protein attachment via disulfide linker.

    PubMed

    Baltus, Ruth E; Carmon, Kendra S; Luck, Linda A

    2007-03-27

    Results from an investigation of the frequency response resulting from ligand binding for a genetically engineered hormone-binding domain of the alpha-estrogen receptor immobilized to a piezoelectric quartz crystal are reported. Two different approaches were used to attach a genetically altered receptor to the gold electrode on the quartz surface: (1) the mutant receptor containing a single solvent-exposed cysteine was directly attached to the crystal via a sulfur to gold covalent bond, forming a self-assembled protein monolayer, and (2) the N-terminal histidine-tagged end was utilized to attach the receptor via a 3,3-dithiobis[N-(5-amino-5-carboxypentyl)propionamide-N',N'-diacetic acid] linker complexed with nickel. Previous studies have shown that these engineered constructs bind 17beta-estradiol and are fully functional. Exposure of the receptor directly attached to the piezoelectric crystal to the known ligand 17beta-estradiol resulted in a measurable frequency response, consistent with a change in conformation of the receptor with ligand binding. However, no response was observed when the receptor immobilized via the linker was exposed to the same ligand. The presence of the linker between the quartz surface and the protein receptor does not allow the crystal to sense the conformational change in the receptor that occurs with ligand binding. These results illustrate that the immobilization strategy used to bind the receptor to the sensor platform is key to eliciting an appropriate response from this biosensor. This study has important implications for the development of QCM-based sensors using protein receptors.

  13. Studies on the Role of The Ah Receptor (AhR) on the Etiology of Breast Cancer: A Novel Idea of Identifying this Receptor as a New Therapeutic Target

    DTIC Science & Technology

    2010-09-01

    found that the most potent phytochemical suppressors of cell proliferation of P20E cells were curcumin (10 µM approximately 80 to 90% suppression...effectiveness of a number of phytochemicals from edible plants known to block AhR in attenuating the expression of high rates of cell proliferation...selected number of those phytochemicals , by xenografting those AhR overexpressing human breast cancer cells into athymic nude mice, and by treating

  14. Pivotal Advance: Interconversion between pure chemotactic ligands and chemoattractant/secretagogue ligands of neutrophil C5a receptor by a single amino acid substitution.

    PubMed

    Jia, Nan; Semba, Umeko; Nishiura, Hiroshi; Kuniyasu, Akihiko; Nsiama, Tienabe K; Nishino, Norikazu; Yamamoto, Tetsuro

    2010-06-01

    Skp derived from Escherichia coli attracts leukocytes as a pure chemotactic ligand of the C5a receptor. We identified the submolecular region of Skp that binds and activates the C5a receptor to be -Gln103-Asp104-Arg105- using synthetic peptide fragments and site-directed mutants of Skp. As the C5a amino acid residue equivalent to Gln103 of Skp is Leu72, we prepared a Gln103Leu-Skp mutant as a recombinant protein. With this mutation, Skp gained secretagogue functions including induction of the respiratory burst and granule release reactions and leukotriene generation, in addition to the chemoattraction displayed by C5a. However, when we substituted Leu72 with Gln in C5a, the L72Q-C5a mutant largely lost its secretagogue function. These functional conversions were reproduced using synthetic peptides mimicking the receptor-binding/-activating regions of the recombinant proteins. Receptor-binding assays using the mimicking peptides demonstrated only a small difference between the Leu72-C5a and Gln72-C5a peptides. Consistently, L72Q-C5a apparently antagonized C5a secretagogue function. These results indicate that the difference between a chemotactic response and a combined chemotactic/secretory response can be attributed not to the nature of the receptor but to guidance by the ligand, at least in the case of C5a receptor-mediated leukocyte responses.

  15. Regulation of phagocytosis by TAM receptors and their ligands

    PubMed Central

    Lu, Qingxian; Li, Qiutang; Lu, Qingjun

    2010-01-01

    The TAM family of receptors is preferentially expressed by professional and non-professional phagocytes, including macrophages, dendritic cells and natural killer cells in the immune system, osteoclasts in bone, Sertoli cells in testis, and retinal pigmental epithelium cells in the retina. Mutations in the Mertk single gene or in different combinations of the double or triple gene mutations in the same cell cause complete or partial impairment in phagocytosis of their preys; and as a result, either the normal apoptotic cells cannot be efficiently removed or the tissue neighbor cells die by apoptosis. This scenario of TAM regulation represents a widely adapted model system used by phagocytes in all different tissues. The present review will summarize current known functional roles of TAM receptors and their ligands, Gas 6 and protein S, in the regulation of phagocytosis. PMID:21057587

  16. Aryl Hydrocarbon Receptor Plays Protective Roles against High Fat Diet (HFD)-induced Hepatic Steatosis and the Subsequent Lipotoxicity via Direct Transcriptional Regulation of Socs3 Gene Expression*

    PubMed Central

    Wada, Taira; Sunaga, Hiroshi; Miyata, Kazuki; Shirasaki, Haruno; Uchiyama, Yuki; Shimba, Shigeki

    2016-01-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor regulating the expression of genes involved in xenobiotic response. Recent studies have suggested that AhR plays essential roles not only in xenobiotic detoxification but also energy metabolism. Thus, in this study, we studied the roles of AhR in lipid metabolism. Under high fat diet (HFD) challenge, liver-specific AhR knock-out (AhR LKO) mice exhibited severe steatosis, inflammation, and injury in the liver. Gene expression analysis and biochemical study revealed that de novo lipogenesis activity was significantly increased in AhR LKO mice. In contrast, induction of suppressor of cytokine signal 3 (Socs3) expression by HFD was attenuated in the livers of AhR LKO mice. Rescue of the Socs3 gene in the liver of AhR LKO mice cancelled the HFD-induced hepatic lipotoxicities. Promoter analysis established Socs3 as novel transcriptional target of AhR. These results indicated that AhR plays a protective role against HFD-induced hepatic steatosis and the subsequent lipotoxicity effects, such as inflammation, and that the mechanism of protection involves the direct transcriptional regulation of Socs3 expression by AhR. PMID:26865635

  17. Improving virtual screening of G protein-coupled receptors via ligand-directed modeling

    PubMed Central

    Simms, John; Christopoulos, Arthur; Wootten, Denise

    2017-01-01

    G protein-coupled receptors (GPCRs) play crucial roles in cell physiology and pathophysiology. There is increasing interest in using structural information for virtual screening (VS) of libraries and for structure-based drug design to identify novel agonist or antagonist leads. However, the sparse availability of experimentally determined GPCR/ligand complex structures with diverse ligands impedes the application of structure-based drug design (SBDD) programs directed to identifying new molecules with a select pharmacology. In this study, we apply ligand-directed modeling (LDM) to available GPCR X-ray structures to improve VS performance and selectivity towards molecules of specific pharmacological profile. The described method refines a GPCR binding pocket conformation using a single known ligand for that GPCR. The LDM method is a computationally efficient, iterative workflow consisting of protein sampling and ligand docking. We developed an extensive benchmark comparing LDM-refined binding pockets to GPCR X-ray crystal structures across seven different GPCRs bound to a range of ligands of different chemotypes and pharmacological profiles. LDM-refined models showed improvement in VS performance over origin X-ray crystal structures in 21 out of 24 cases. In all cases, the LDM-refined models had superior performance in enriching for the chemotype of the refinement ligand. This likely contributes to the LDM success in all cases of inhibitor-bound to agonist-bound binding pocket refinement, a key task for GPCR SBDD programs. Indeed, agonist ligands are required for a plethora of GPCRs for therapeutic intervention, however GPCR X-ray structures are mostly restricted to their inactive inhibitor-bound state. PMID:29131821

  18. Molecular identification and partial sequence analysis of an aryl hydrocarbon receptor from beluga (Delphinapterus leucas)

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

    Jensen, B.A.; Hahn, M.E.

    1995-12-31

    The aryl hydrocarbon receptor (AhR) mediates the effects of many common and potentially toxic organic hydrocarbons, including some polychlorinated biphenyls and dioxins. Since small cetaceans often inhabit industrially polluted coastal waters, comparison of the molecular structure and function of this protein in cetaeans with other marine and mammalian species is important for evaluating the sensitivity of cetaceans to these pollutants. An AhR protein has been identified in beluga liver by photoaffinity labeling. In the present study, the authors sought to clone and sequence an AhR cDNA from beluga as a prelude to studying its structure and function, using reverse-transcription polymerasemore » chain reaction (RT-PCR) and degenerate primers, a 515 base pair fragment was amplified, cloned and sequenced, revealing homology to the PAS domain (ligand binding and dimerization region) of AhRs from terrestrial mammals. This portion of the putative beluga AhR has 82% amino acid and 81% nucleotide sequence identity to the mouse AhR, and 63% amino acid and 64% nucleotide sequence identity to an AhR from the marine fish Fundulus heteroclitus. A beluga cDNA library was synthesized and is currently being screened with the PCR-generated fragment to obtain the complete coding sequence. This is the first molecular evidence of AhR presence in cetaceans.« less

  19. Identification of Critical Residues Involved in Ligand Binding and G Protein Signaling in Human Somatostatin Receptor Subtype 2

    PubMed Central

    Parry, Jesse J.; Chen, Ronald; Andrews, Rebecca; Lears, Kimberly A.

    2012-01-01

    G protein signaling through human somatostatin receptor subtype 2 (SSTR2) is well known, but the amino acids involved in stimulation of intracellular responses upon ligand binding have not been characterized. We constructed a series of point mutants in SSTR2 at amino acid positions 89, 139, and 140 in attempts to disrupt G protein signaling upon ligand binding. The aspartic acid changes at position 89 to either Ala, Leu, or Arg generated mutant receptors with varying expression profiles and a complete inability to bind somatostatin-14 (SST). Mutations to Asp 139 and Arg 140 also led to varying expression profiles with some mutants maintaining their affinity for SST. Mutation of Arg 140 to Ala resulted in a mutated receptor that had a Bmax and dissociation constant (Kd) similar to wild-type receptor but was still coupled to the G protein as determined in both a cAMP assay and a calcium-release assay. In contrast, mutation of Asp 139 to Asn resulted in a mutated receptor with Bmax and Kd values that were similar to wild type but was uncoupled from G protein-mediated cAMP signaling, but not calcium release. Thus, we identified mutations in SSTR2 that result in either receptor expression levels that are similar to wild type but is completely ablated for ligand binding or a receptor that maintains affinity for SST and is uncoupled from G protein-mediated cAMP signaling. PMID:22495673

  20. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression

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

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong

    2014-10-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 andmore » CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO

  1. Lysosomal membrane permeabilization is an early event in Sigma-2 receptor ligand mediated cell death in pancreatic cancer.

    PubMed

    Hornick, John R; Vangveravong, Suwanna; Spitzer, Dirk; Abate, Carmen; Berardi, Francesco; Goedegebuure, Peter; Mach, Robert H; Hawkins, William G

    2012-05-02

    Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD

  2. INSIGHTS FROM AHR AND ARNT GENE KNOCKOUT STUDIES REGARDING RESPONSES TO TCDD AND REGULATION OF NORMAL EMBRYONIC DEVELOPMENT

    EPA Science Inventory

    The aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator (ARNT) are members of the Per-ARNT-Sim (PAS) family of proteins. The AhR binds members of the chemical family that includes dioxins, furans and coplanar polychlorinated biphenyls (PCBs). A ligand-AhR-ARNT comp...

  3. The AhR and NF-κB/Rel Proteins Mediate the Inhibitory Effect of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on the 3′ Immunoglobulin Heavy Chain Regulatory Region

    PubMed Central

    Salisbury, Richard L.; Sulentic, Courtney E. W.

    2015-01-01

    Transcriptional regulation of the murine immunoglobulin (Ig) heavy chain gene (Igh) involves several regulatory elements including the 3′Igh regulatory region (3′IghRR), which is composed of at least 4 enhancers (hs3A, hs1.2, hs3B, and hs4). The hs1.2 and hs4 enhancers exhibit the greatest transcriptional activity and contain binding sites for several transcription factors including nuclear factor kappaB/Rel (NF-κB/Rel) proteins and the aryl hydrocarbon receptor (AhR). Interestingly, the environmental immunosuppressant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which potently inhibits antibody secretion, also profoundly inhibits 3′IghRR and hs1.2 enhancer activation induced by the B-lymphocyte activator lipopolysaccharide (LPS), but enhances LPS-induced activation of the hs4 enhancer. Within the hs1.2 and hs4 enhancers, the AhR binding site is in close proximity or overlaps an NF-κB/Rel binding site suggesting a potential reciprocal modulation of the 3′IghRR by AhR and NF-κB/Rel. The objective of the current study was to evaluate the role of NF-κB/Rel and the AhR on the 3′IghRR and its enhancers using the AhR ligand TCDD, the AhR antagonist CH223191, and toll-like receptor agonists LPS, Resiquimod (R848), or cytosine-phosphate-guanine-oligodeoxynucleotides (CpG). Utilizing the CH12.LX B-lymphocyte cell line and variants expressing either a 3′IghRR-regulated transgene reporter or an inducible IκBα (inhibitor kappa B-alpha protein) superrepressor (IκBαAA), we demonstrate an AhR- and NF-κB/Rel-dependent modulation of 3′IghRR and hs4 activity. Additionally, in mouse splenocytes or CH12.LX cells, binding within the hs1.2 and hs4 enhancer of the AhR and the NF-κB/Rel proteins RelA and RelB was differentially altered by the cotreatment of LPS and TCDD. These results suggest that the AhR and NF-κB/Rel protein binding profile within the 3′IghRR mediates the inhibitory effects of TCDD on Ig expression and therefore antibody levels. PMID:26377645

  4. Generation of signaling specificity in Arabidopsis by spatially restricted buffering of ligand-receptor interactions.

    PubMed

    Abrash, Emily B; Davies, Kelli A; Bergmann, Dominique C

    2011-08-01

    Core signaling pathways function in multiple programs during multicellular development. The mechanisms that compartmentalize pathway function or confer process specificity, however, remain largely unknown. In Arabidopsis thaliana, ERECTA (ER) family receptors have major roles in many growth and cell fate decisions. The ER family acts with receptor TOO MANY MOUTHS (TMM) and several ligands of the EPIDERMAL PATTERNING FACTOR LIKE (EPFL) family, which play distinct yet overlapping roles in patterning of epidermal stomata. Here, our examination of EPFL genes EPFL6/CHALLAH (CHAL), EPFL5/CHALLAH-LIKE1, and EPFL4/CHALLAH-LIKE2 (CLL2) reveals that this family may mediate additional ER-dependent processes. chal cll2 mutants display growth phenotypes characteristic of er mutants, and genetic interactions are consistent with CHAL family molecules acting as ER family ligands. We propose that different classes of EPFL genes regulate different aspects of ER family function and introduce a TMM-based discriminatory mechanism that permits simultaneous, yet compartmentalized and distinct, function of the ER family receptors in growth and epidermal patterning.

  5. Functional expression of Squalus acanthias melanocortin-5 receptor in CHO cells: ligand selectivity and interaction with MRAP.

    PubMed

    Reinick, Christina L; Liang, Liang; Angleson, Josepha K; Dores, Robert M

    2012-04-05

    The melanocortin-5 receptor (MC(5)) of the dogfish Squalus acanthias (SacMC(5) receptor) can be functionally expressed in CHO cells in the absence of the co-expression of an exogenous MRAP cDNA. Both human ACTH(1-24) and dogfish ACTH(1-25) were much better stimulators of the SacMC(5) receptor than any of the mammalian or dogfish MSH ligands that were tested. The order of ligand selectivity for the dogfish melanocortins was ACTH(1-25)>αMSH>γ-MSH=δ-MSH>β-MSH. Unlike mammalian MC(5) receptors, the functional expression of the SacMC(5) receptor was not negatively impacted when the receptor was co-expressed with a cartilaginous fish (Callorhinchus milii) MRAP2 cDNA. However, co-expression with either mouse mMRAP1 or zebrafish zfMRAP1 increased the sensitivity of SacMC(5) receptor for hACTH(1-24) by at least one order of magnitude. Hence, SacMC(5) receptor has the potential to interact with MRAP1 orthologs and in this regard behaved more like a melanocortin MC(2) receptor ortholog than a melanocortin MC(5) receptor ortholog. These observations are discussed in light of the evolution of the melanocortin receptor gene family in cartilaginous fish, and the physiological implications of these observations are considered. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. An in vitro and in vivo investigation of bivalent ligands that display preferential binding and functional activity for different melanocortin receptor homodimers

    PubMed Central

    Lensing, Cody J.; Freeman, Katie T.; Schnell, Sathya M.; Adank, Danielle N.; Speth, Robert C.; Haskell-Luevano, Carrie

    2017-01-01

    Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 (CJL-1-140) possessed a functional profile that was unique from its monovalent counterparts providing evidence of the discrete effects of bivalent ligands. Lead compound 7 (CJL-1-87) significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects. PMID:26959173

  7. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

    PubMed

    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  8. Peroxisome proliferator-activated receptor α ligands and modulators from dietary compounds: Types, screening methods and functions.

    PubMed

    Yang, Haixia; Xiao, Lei; Wang, Nanping

    2017-04-01

    Peroxisome proliferator-activated receptor α (PPARα) plays a key role in lipid metabolism and glucose homeostasis and a crucial role in the prevention and treatment of metabolic diseases. Natural dietary compounds, including nutrients and phytochemicals, are PPARα ligands or modulators. High-throughput screening assays have been developed to screen for PPARα ligands and modulators in our diet. In the present review, we discuss recent advances in our knowledge of PPARα, including its structure, function, and ligand and modulator screening assays, and summarize the different types of dietary PPARα ligands and modulators. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  9. Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction.

    PubMed

    Eble, Johannes A

    2018-02-15

    The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of α2β1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

  10. Effects of imidazoline I2 receptor ligands on acute nociception in rats.

    PubMed

    Sampson, Cristal; Zhang, Yanan; Del Bello, Fabio; Li, Jun-Xu

    2012-01-25

    This study examined the antinociceptive effects of seven imidazoline I2 receptor ligands in a rat warm water tail withdrawal procedure (46 and 50 °C). Agmatine, 2-BFI, phenyzoline, and diphenyzoline produced a significant antinociceptive activity at 46 °C. BU224, S22687, and idazoxan had no effect at 46 °C up to doses that altered the locomotor activity. None of the drugs showed antinociceptive activity at 50 °C. It is suggested that I2 receptor agonists have antinociceptive activity for acute phasic pain under weak noxious stimulus, and the effects are efficacy-dependent. These data explain the findings that I2 receptor agonists enhance the antinociceptive effects of opioids and support developing higher-efficacy I2 receptor agonists for the treatment of pain. © 2012 Wolters Kluwer Health | Lippincott Williams & Wilkins.

  11. Induction of AhR-Mediated Gene Transcription by Coffee

    PubMed Central

    Ishikawa, Toshio; Takahashi, Satoshi; Morita, Koji; Okinaga, Hiroko; Teramoto, Tamio

    2014-01-01

    Background Aryl hydrocarbon receptor (AhR) is classically known to be activated by xenobiotics such as dioxins and polycyclic aromatic hydrocarbons (PAHs). Although it has been reported that PAHs are contained in roasted coffee beans, in general coffee beverages are not considered to be AhR activators. We tested whether exposure to coffee would activate AhR in cultured cells. Methods HepG2 cells stably expressing an AhR-responsive reporter gene were treated with coffee samples. Also, expression of CYP1A1, an endogenous AhR-responsive gene, was quantitated by RT-PCR and Western blotting in HepG2, Caco-2, and MCF-7 cells, after treatment with coffee. In order to obtain sensitive and reproducible results, all the experiments were performed with the cells placed in either phosphate-buffered saline (PBS) or pure serum, instead of routinely-used culture medium, whose intrinsic AhR-stimulating activity turned out to be so strong as to interfere with the analyses. Results All the coffee samples tested robustly stimulated AhR-mediated transcription in the reporter gene assays. Of note, to what extent coffee and other AhR agonists activated AhR was different, depending on whether the experiments were done in PBS or serum. CYP1A1 mRNA was induced by coffee, in HepG2, Caco-2, and MCF-7 cells placed in either PBS or serum. CYP1A1 protein expression, which was not detected in these cells incubated in PBS, was also increased by coffee in cells placed in serum. Conclusions By using culture medium-free experimental settings, we have shown that coffee is a strong AhR activator. Our observation may help elucidate as-yet-unrecognized effects of coffee on human health. PMID:25007155

  12. Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

    PubMed

    Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

    2015-01-01

    Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

  13. Patents for Toll-like receptor ligands as radiation countermeasures for acute radiation syndrome.

    PubMed

    Singh, Vijay K; Pollard, Harvey B

    2015-01-01

    Acute radiation exposure induces apoptosis of tissues in the hematopoietic, digestive, cutaneous, cardiovascular and nervous systems; extensive apoptosis of these tissues ultimately leads to acute radiation syndrome. A novel strategy for developing radiation countermeasures has been to imitate the genetic mechanisms acquired by radiation-resistant tumors. Two mechanisms that underlie this ability of tumor cells are the p53 and NF-κB pathways. The loss of p53 function results in the inactivation of pro-apoptotic control mechanisms, while constitutive activation of NF-κB results in the up-regulation of anti-apoptotic genes. Various Toll-like receptor ligands are capable of up regulating the NF-κB pathway, which increases radio-resistance and reduces radiation-induced apoptosis in various tissues. Several Toll-like receptor ligands have been patented and are currently under development as radiation countermeasures for acute radiation syndrome. Ongoing studies suggest that a few of these attractive agents are progressing well along the US FDA approval pathway to become radiation countermeasures.

  14. Patents for Toll-like receptor ligands as radiation countermeasures for acute radiation syndrome

    PubMed Central

    Singh, Vijay K; Pollard, Harvey B

    2015-01-01

    Acute radiation exposure induces apoptosis of tissues in the hematopoietic, digestive, cutaneous, cardiovascular and nervous systems; extensive apoptosis of these tissues ultimately leads to acute radiation syndrome. A novel strategy for developing radiation countermeasures has been to imitate the genetic mechanisms acquired by radiation-resistant tumors. Two mechanisms that underlie this ability of tumor cells are the p53 and NF-κB pathways. The loss of p53 function results in the inactivation of pro-apoptotic control mechanisms, while constitutive activation of NF-κB results in the up-regulation of anti-apoptotic genes. Various Toll-like receptor ligands are capable of up regulating the NF-κB pathway, which increases radio-resistance and reduces radiation-induced apoptosis in various tissues. Several Toll-like receptor ligands have been patented and are currently under development as radiation countermeasures for acute radiation syndrome. Ongoing studies suggest that a few of these attractive agents are progressing well along the US FDA approval pathway to become radiation countermeasures. PMID:26135043

  15. Cancer exosomes and NKG2D receptor-ligand interactions: impairing NKG2D-mediated cytotoxicity and anti-tumour immune surveillance.

    PubMed

    Mincheva-Nilsson, Lucia; Baranov, Vladimir

    2014-10-01

    Human cancers constitutively produce and release endosome-derived nanometer-sized vesicles called exosomes that carry biologically active proteins, messenger and micro RNAs and serve as vehicles of intercellular communication. The tumour exosomes are present in the blood, urine and various malignant effusions such as peritoneal and pleural fluid of cancer patients and can modulate immune cells and responses thus deranging the immune system of cancer patients and giving advantage to the cancer to establish and spread itself. Here, the role of exosomes in the NKG2D receptor-ligand system's interactions is discussed. The activating NK cell receptor NKG2D and its multiple ligands, the MHC class I-related chain (MIC) A/B and the retinoic acid transcript-1/UL-16 binding proteins (RAET1/ULBP) 1-6 comprise a powerful stress-inducible danger detector system that targets infected, inflamed and malignantly transformed cells and plays a decisive role in anti-tumour immune surveillance. Mounting evidence reveals that the MIC- and RAET1/ULBP ligand family members are enriched in the endosomal compartment of various tumour cells and expressed and released into the intercellular space and bodily fluids on exosomes thus preserving their entire molecule, three-dimensional protein structure and biologic activity. The NKG2D ligand-expressing exosomes serve as decoys with a powerful ability to down regulate the cognate receptor and impair the cytotoxic function of NK-, NKT-, gamma/delta- and cytotoxic T cells. This review summarizes recent findings concerning the role of NKG2D receptor-ligand system in cancer with emphasis on regulation of NKG2D ligand expression and the immunosuppressive role of exosomally expressed NKG2D ligands. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    PubMed Central

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; Mcdougal, Owen M.

    2017-01-01

    Computational molecular docking is a fast and effective in silico method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The DockoMatic tutorial described herein provides a framework by which instructors can guide students through a drug screening exercise. Using receptor models derived from readily available protein crystal structures, docking programs have the ability to predict ligand binding properties, such as preferential binding orientations and binding affinities. The use of computational studies can significantly enhance complimentary wet chemical experimentation by providing insight into the important molecular interactions within the system of interest, as well as guide the design of new candidate ligands based on observed binding motifs and energetics. In this laboratory tutorial, the graphical user interface, DockoMatic, facilitates docking job submissions to the docking engine, AutoDock 4.2. The purpose of this exercise is to successfully dock a 17-amino acid peptide, α-conotoxin TxIA, to the acetylcholine binding protein from Aplysia californica-AChBP to determine the most stable binding configuration. Each student will then propose two specific amino acid substitutions of α-conotoxin TxIA to enhance peptide binding affinity, create the mutant in DockoMatic, and perform docking calculations to compare their results with the class. Students will also compare intermolecular forces, binding energy, and geometric orientation of their prepared analog to their initial α-conotoxin TxIA docking results. PMID:26537635

  17. A python-based docking program utilizing a receptor bound ligand shape: PythDock.

    PubMed

    Chung, Jae Yoon; Cho, Seung Joo; Hah, Jung-Mi

    2011-09-01

    PythDock is a heuristic docking program that uses Python programming language with a simple scoring function and a population based search engine. The scoring function considers electrostatic and dispersion/repulsion terms. The search engine utilizes a particle swarm optimization algorithm. A grid potential map is generated using the shape information of a bound ligand within the active site. Therefore, the searching area is more relevant to the ligand binding. To evaluate the docking performance of PythDock, two well-known docking programs (AutoDock and DOCK) were also used with the same data. The accuracy of docked results were measured by the difference of the ligand structure between x-ray structure, and docked pose, i.e., average root mean squared deviation values of the bound ligand were compared for fourteen protein-ligand complexes. Since the number of ligands' rotational flexibility is an important factor affecting the accuracy of a docking, the data set was chosen to have various degrees of flexibility. Although PythDock has a scoring function simpler than those of other programs (AutoDock and DOCK), our results showed that PythDock predicted more accurate poses than both AutoDock4.2 and DOCK6.2. This indicates that PythDock could be a useful tool to study ligand-receptor interactions and could also be beneficial in structure based drug design.

  18. Residues W320 and Y328 within the binding site of the μ-opioid receptor influence opiate ligand bias.

    PubMed

    Hothersall, J Daniel; Torella, Rubben; Humphreys, Sian; Hooley, Monique; Brown, Alastair; McMurray, Gordon; Nickolls, Sarah A

    2017-05-15

    The development of G protein-biased agonists for the μ-opioid receptor (MOR) offers a clear drug discovery rationale for improved analgesia and reduced side-effects of opiate pharmacotherapy. However, our understanding of the molecular mechanisms governing ligand bias is limited, which hinders our ability to rationally design biased compounds. We have investigated the role of MOR binding site residues W320 and Y328 in controlling bias, by receptor mutagenesis. The pharmacology of a panel of ligands in a cAMP and a β-arrestin2 assay were compared between the wildtype and mutated receptors, with bias factors calculated by operational analysis using ΔΔlog(τ/K A ) values. [ 3 H]diprenorphine competition binding was used to estimate affinity changes. Introducing the mutations W320A and Y328F caused changes in pathway bias, with different patterns of change between ligands. For example, DAMGO increased relative β-arrestin2 activity at the W320A mutant, whilst its β-arrestin2 response was completely lost at Y328F. In contrast, endomorphin-1 gained activity with Y328F but lost activity at W320A, in both pathways. For endomorphin-2 there was a directional shift from cAMP bias at the wildtype towards more β-arrestin2 bias at W320A. We also observe clear uncoupling between mutation-driven changes in function and binding affinity. These findings suggest that the mutations influenced the balance of pathway activation in a ligand-specific manner, thus identifying residues in the MOR binding pocket that govern ligand bias. This increases our understanding of how ligand/receptor binding interactions can be translated into agonist-specific pathway activation. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Tissue distribution of aryl hydrocarbon receptor in the intestine: Implication of putative roles in tumor suppression

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

    Ikuta, Togo, E-mail: togo@cancer-c.pref.saitama.jp; Kurosumi, Masafumi, E-mail: mkurosumi@cancer-c.pref.saitama.jp; Yatsuoka, Toshimasa, E-mail: yatsuoka-gi@umin.ac.jp

    Intestinal homeostasis is maintained by complex interactions between intestinal microorganisms and the gut immune system. Dysregulation of gut immunity may lead to inflammatory disorders and tumorigenesis. We previously have shown the tumor suppressive effects of aryl hydrocarbon receptor (AhR) in intestinal carcinogenesis. In the present study, we investigated AhR distribution in the mouse and human intestine by histochemical analysis. In the normal intestine, AhR was mainly localized in the stroma containing immune cells in the lamina propria and lymphoid follicles. On the other hand, in the tumor tissue from human colon cancer and that developed in Apc{sup Min/+}mice, AhR expressionmore » was elevated. AhR immunostaining was found in both stromal and tumor cells. Although AhR was localized in the cytoplasm of tumor cells in most cases, nuclear AhR was also observed in some. AhR knockdown using siRNA resulted in significant promotion of cell growth in colon cancer cell lines. Furthermore, AhR activation by AhR ligands supplemented in culture medium suppressed cell growth. Our study results suggest that tumor suppressive roles of AhR are estimated in two distinct ways: in normal tissue, AhR is associated with tumor prevention by regulating gut immunity, whereas in tumor cells, it is involved in growth suppression. - Highlights: • In the normal intestine, AhR was mainly localized in stroma containing immune cells. • In the tumor tissue, AhR expression was found in both stromal and tumor cells. • AhR knockdown promoted cell growth in colon cancer cell lines.« less

  20. A Lipid Pathway for Ligand Binding Is Necessary for a Cannabinoid G Protein-coupled Receptor*

    PubMed Central

    Hurst, Dow P.; Grossfield, Alan; Lynch, Diane L.; Feller, Scott; Romo, Tod D.; Gawrisch, Klaus; Pitman, Michael C.; Reggio, Patricia H.

    2010-01-01

    Recent isothiocyanate covalent labeling studies have suggested that a classical cannabinoid, (−)-7′-isothiocyanato-11-hydroxy-1′,1′dimethylheptyl-hexahydrocannabinol (AM841), enters the cannabinoid CB2 receptor via the lipid bilayer (Pei, Y., Mercier, R. W., Anday, J. K., Thakur, G. A., Zvonok, A. M., Hurst, D., Reggio, P. H., Janero, D. R., and Makriyannis, A. (2008) Chem. Biol. 15, 1207–1219). However, the sequence of steps involved in such a lipid pathway entry has not yet been elucidated. Here, we test the hypothesis that the endogenous cannabinoid sn-2-arachidonoylglycerol (2-AG) attains access to the CB2 receptor via the lipid bilayer. To this end, we have employed microsecond time scale all-atom molecular dynamics (MD) simulations of the interaction of 2-AG with CB2 via a palmitoyl-oleoyl-phosphatidylcholine lipid bilayer. Results suggest the following: 1) 2-AG first partitions out of bulk lipid at the transmembrane α-helix (TMH) 6/7 interface; 2) 2-AG then enters the CB2 receptor binding pocket by passing between TMH6 and TMH7; 3) the entrance of the 2-AG headgroup into the CB2 binding pocket is sufficient to trigger breaking of the intracellular TMH3/6 ionic lock and the movement of the TMH6 intracellular end away from TMH3; and 4) subsequent to protonation at D3.49/D6.30, further 2-AG entry into the ligand binding pocket results in both a W6.48 toggle switch change and a large influx of water. To our knowledge, this is the first demonstration via unbiased molecular dynamics that a ligand can access the binding pocket of a class A G protein-coupled receptor via the lipid bilayer and the first demonstration via molecular dynamics of G protein-coupled receptor activation triggered by a ligand binding event. PMID:20220143

  1. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor.

    PubMed

    Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

    2016-07-25

    The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

  2. Advances in methods to characterize ligand-induced ionic lock and rotamer toggle molecular switch in G protein-coupled receptors.

    PubMed

    Xie, Xiang-Qun; Chowdhury, Ananda

    2013-01-01

    Structural biology of GPCRs has made significant progress upon recently developed technologies for GPCRs expression/purification and elucidation of GPCRs crystal structures. The crystal structures provide a snapshot of the receptor structural disposition of GPCRs itself or with cocrystallized ligands, and the results are congruent with biophysical and computer modeling studies reported about GPCRs conformational and dynamics flexibility, regulated activation, and the various stabilizing interactions, such as "molecular switches." The molecular switches generally constitute the most conserved domains within a particular GPCR superfamily. Often agonist-induced receptor activation proceeds by the disruption of majority of these interactions, while antagonist and inverse agonist act as blockers and structural stabilizers, respectively. Several elegant studies, particularly for the β2AR, have demonstrated the relationship between ligand structure, receptor conformational changes, and corresponding pharmacological outcomes. Thus, it is of great importance to understand GPCRs activation related to cell signaling pathways. Herein, we summarize the steps to produce functional GPCRs, generate suitably fluorescent labeled GPCRs and the procedure to use that to understand if ligand-induced activation can proceed by activation of the GPCRs via ionic lock switch and/or rotamer toggle switch mechanisms. Such understanding of ligand structure and mechanism of receptor activation will provide great insight toward uncovering newer pathways of GPCR activation and aid in structure-based drug design. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Normal mast cell numbers in the tissues of AhR-deficient mice.

    PubMed

    Pilz, Caroline; Feyerabend, Thorsten; Sonner, Jana; Redaelli, Chiara; Peter, Katharina; Kunze, Anja; Haas, Katharina; Esser, Charlotte; Schäkel, Knut; Wick, Wolfgang; Rodewald, Hans-Reimer; Lanz, Tobias V; Platten, Michael

    2016-01-01

    The transcription factor aryl hydrocarbon receptor (AhR) acts as an immunomodulatory molecule in several immune cell lineages. Recently, it has been implicated in development and maintenance of immune cells in barrier tissues such as skin and mucosa. To investigate its role on mast cell development and maintenance in skin, peritoneal exudate cells (PECs) and lymph nodes, we studied in depth their phenotype in AhR-deficient mice. Our findings do not provide any evidence for a suspected role of the AhR in mast cell homeostasis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Steroid receptors and their ligands: Effects on male gamete functions

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

    Aquila, Saveria; De Amicis, Francesca, E-mail: francesca.deamicis@unical.it

    In recent years a new picture of human sperm biology is emerging. It is now widely recognized that sperm contain nuclear encoded mRNA, mitochondrial encoded RNA and different transcription factors including steroid receptors, while in the past sperm were considered incapable of transcription and translation. One of the main targets of steroid hormones and their receptors is reproductive function. Expression studies on Progesterone Receptor, estrogen receptor, androgen receptor and their specific ligands, demonstrate the presence of these systems in mature spermatozoa as surface but also as nuclear conventional receptors, suggesting that both systemic and local steroid hormones, through sperm receptors,more » may influence male reproduction. However, the relationship between the signaling events modulated by steroid hormones and sperm fertilization potential as well as the possible involvement of the specific receptors are still controversial issues. The main line of this review highlights the current research in human sperm biology examining new molecular systems of response to the hormones as well as specific regulatory pathways controlling sperm cell fate and biological functions. Most significant studies regarding the identification of steroid receptors are reported and the mechanistic insights relative to signaling pathways, together with the change in sperm metabolism energy influenced by steroid hormones are discussed.The reviewed evidences suggest important effects of Progesterone, Estrogen and Testosterone and their receptors on spermatozoa and implicate the involvement of both systemic and local steroid action in the regulation of male fertility potential. - Highlights: • One of the main targets of steroid hormones and their receptors is reproductive function. • Pg/PR co-work to stimulate enzymatic activities to sustain a capacitation process. • E2/ERs regulate sperm motility, capacitation and acrosome reaction and act as survival factors.

  5. Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

    NASA Astrophysics Data System (ADS)

    Zhang, Xianjun; Zhao, Fei; Wu, Yiran; Yang, Jun; Han, Gye Won; Zhao, Suwen; Ishchenko, Andrii; Ye, Lintao; Lin, Xi; Ding, Kang; Dharmarajan, Venkatasubramanian; Griffin, Patrick R.; Gati, Cornelius; Nelson, Garrett; Hunter, Mark S.; Hanson, Michael A.; Cherezov, Vadim; Stevens, Raymond C.; Tan, Wenfu; Tao, Houchao; Xu, Fei

    2017-05-01

    The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

  6. Expression pattern of the type 1 sigma receptor in the brain and identity of critical anionic amino acid residues in the ligand-binding domain of the receptor.

    PubMed

    Seth, P; Ganapathy, M E; Conway, S J; Bridges, C D; Smith, S B; Casellas, P; Ganapathy, V

    2001-07-25

    The type 1 sigma receptor (sigmaR1) has been shown to participate in a variety of functions in the central nervous system. To identify the specific regions of the brain that are involved in sigmaR1 function, we analyzed the expression pattern of the receptor mRNA in the mouse brain by in situ hybridization. SigmaR1 mRNA was detectable primarily in the cerebral cortex, hippocampus, and Purkinje cells of cerebellum. To identify the critical anionic amino acid residues in the ligand-binding domain of sigmaR1, we employed two different approaches: chemical modification of anionic amino acid residues and site-directed mutagenesis. Chemical modification of anionic amino acids in sigmaR1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide reduced the ligand-binding activity markedly. Since it is known that a splice variant of this receptor which lacks exon 3 does not have the ability to bind sigma ligands, the ligand-binding domain with its critical anionic amino acid residues is likely to be present in or around the region coded by exon 3. Therefore, each of the anionic amino acids in this region was mutated individually and the influence of each mutation on ligand binding was assessed. These studies have identified two anionic amino acids, D126 and E172, that are obligatory for ligand binding. Even though the ligand-binding function was abolished by these two mutations, the expression of these mutants was normal at the protein level. These results show that sigmaR1 is expressed at high levels in specific areas of the brain that are involved in memory, emotion and motor functions. The results also provide important information on the chemical nature of the ligand-binding site of sigmaR1 that may be of use in the design of sigmaR1-specific ligands with potential for modulation of sigmaR1-related brain functions.

  7. Phosphatidylserine Is the Signal for TAM Receptors and Their Ligands.

    PubMed

    Lemke, Greg

    2017-09-01

    Nature repeatedly repurposes, in that molecules that serve as metabolites, energy depots, or polymer subunits are at the same time used to deliver signals within and between cells. The preeminent example of this repurposing is ATP, which functions as a building block for nucleic acids, an energy source for enzymatic reactions, a phosphate donor to regulate intracellular signaling, and a neurotransmitter to control the activity of neurons. A series of recent studies now consolidates the view that phosphatidylserine (PtdSer), a common phospholipid constituent of membrane bilayers, is similarly repurposed for use as a signal between cells and that the ligands and receptors of the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases (RTKs) are prominent transducers of this signal. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. An Efficient Metadynamics-Based Protocol To Model the Binding Affinity and the Transition State Ensemble of G-Protein-Coupled Receptor Ligands.

    PubMed

    Saleh, Noureldin; Ibrahim, Passainte; Saladino, Giorgio; Gervasio, Francesco Luigi; Clark, Timothy

    2017-05-22

    A generally applicable metadynamics scheme for predicting the free energy profile of ligand binding to G-protein-coupled receptors (GPCRs) is described. A common and effective collective variable (CV) has been defined using the ideally placed and highly conserved Trp6.48 as a reference point for ligand-GPCR distance measurement and the common orientation of GPCRs in the cell membrane. Using this single CV together with well-tempered multiple-walker metadynamics with a funnel-like boundary allows an efficient exploration of the entire ligand binding path from the extracellular medium to the orthosteric binding site, including vestibule and intermediate sites. The protocol can be used with X-ray structures or high-quality homology models (based on a high-quality template and after thorough refinement) for the receptor and is universally applicable to agonists, antagonists, and partial and reverse agonists. The root-mean-square error (RMSE) in predicted binding free energies for 12 diverse ligands in five receptors (a total of 23 data points) is surprisingly small (less than 1 kcal mol -1 ). The RMSEs for simulations that use receptor X-ray structures and homology models are very similar.

  9. Structure-Activity Relationships of Constrained Phenylethylamine Ligands for the Serotonin 5-HT2 Receptors

    PubMed Central

    Isberg, Vignir; Paine, James; Leth-Petersen, Sebastian; Kristensen, Jesper L.; Gloriam, David E.

    2013-01-01

    Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine class. Conformationally constrained phenethylamine analogs have demonstrated that for optimal activity the free lone pair electrons of the 2-oxygen must be oriented syn and the 5-oxygen lone pairs anti relative to the ethylamine moiety. Also the ethyl linker has been constrained providing information about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9–11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure showed that the 1,2-heterocyclized compounds can be accommodated in the binding site. Conformational analysis showed that 11 can only bind in a higher-energy conformation, which would explain its absent or low affinity. The amine and 2-oxygen interactions with D3.32 and S3.36, respectively, can form but shift the placement of the core scaffold. The constraints in 9–11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9–11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure-activity relationships of constrained phenethylamines and contributes towards the development of 5-HT2 receptor subtype-selective ligands. PMID:24244317

  10. Structure-activity relationships of constrained phenylethylamine ligands for the serotonin 5-HT2 receptors.

    PubMed

    Isberg, Vignir; Paine, James; Leth-Petersen, Sebastian; Kristensen, Jesper L; Gloriam, David E

    2013-01-01

    Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine class. Conformationally constrained phenethylamine analogs have demonstrated that for optimal activity the free lone pair electrons of the 2-oxygen must be oriented syn and the 5-oxygen lone pairs anti relative to the ethylamine moiety. Also the ethyl linker has been constrained providing information about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9-11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure showed that the 1,2-heterocyclized compounds can be accommodated in the binding site. Conformational analysis showed that 11 can only bind in a higher-energy conformation, which would explain its absent or low affinity. The amine and 2-oxygen interactions with D3.32 and S3.36, respectively, can form but shift the placement of the core scaffold. The constraints in 9-11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9-11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure-activity relationships of constrained phenethylamines and contributes towards the development of 5-HT2 receptor subtype-selective ligands.

  11. International Union of Basic and Clinical Pharmacology. LXXXVIII. G Protein-Coupled Receptor List: Recommendations for New Pairings with Cognate Ligands

    PubMed Central

    Alexander, Stephen P. H.; Sharman, Joanna L.; Pawson, Adam J.; Benson, Helen E.; Monaghan, Amy E.; Liew, Wen Chiy; Mpamhanga, Chidochangu P.; Bonner, Tom I.; Neubig, Richard R.; Pin, Jean Philippe; Spedding, Michael; Harmar, Anthony J.

    2013-01-01

    In 2005, the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) published a catalog of all of the human gene sequences known or predicted to encode G protein-coupled receptors (GPCRs), excluding sensory receptors. This review updates the list of orphan GPCRs and describes the criteria used by NC-IUPHAR to recommend the pairing of an orphan receptor with its cognate ligand(s). The following recommendations are made for new receptor names based on 11 pairings for class A GPCRs: hydroxycarboxylic acid receptors [HCA1 (GPR81) with lactate, HCA2 (GPR109A) with 3-hydroxybutyric acid, HCA3 (GPR109B) with 3-hydroxyoctanoic acid]; lysophosphatidic acid receptors [LPA4 (GPR23), LPA5 (GPR92), LPA6 (P2Y5)]; free fatty acid receptors [FFA4 (GPR120) with omega-3 fatty acids]; chemerin receptor (CMKLR1; ChemR23) with chemerin; CXCR7 (CMKOR1) with chemokines CXCL12 (SDF-1) and CXCL11 (ITAC); succinate receptor (SUCNR1) with succinate; and oxoglutarate receptor [OXGR1 with 2-oxoglutarate]. Pairings are highlighted for an additional 30 receptors in class A where further input is needed from the scientific community to validate these findings. Fifty-seven human class A receptors (excluding pseudogenes) are still considered orphans; information has been provided where there is a significant phenotype in genetically modified animals. In class B, six pairings have been reported by a single publication, with 28 (excluding pseudogenes) still classified as orphans. Seven orphan receptors remain in class C, with one pairing described by a single paper. The objective is to stimulate research into confirming pairings of orphan receptors where there is currently limited information and to identify cognate ligands for the remaining GPCRs. Further information can be found on the IUPHAR Database website (http://www.iuphar-db.org). PMID:23686350

  12. Characterization of B61, the ligand for the Eck receptor protein-tyrosine kinase.

    PubMed

    Shao, H; Pandey, A; O'Shea, K S; Seldin, M; Dixit, V M

    1995-03-10

    B61 was originally described as a novel secreted tumor necrosis factor-alpha-inducible gene product in endothelial cells (Holzman, L. B., Marks, R. M., and Dixit, V. M. (1990) Mol. Cell. Biol. 10, 5830-5838). It was recently discovered that soluble recombinant B61 could serve as a ligand for the Eck receptor protein-tyrosine kinase, a member of the Eph/Eck subfamily of receptor protein-tyrosine kinases (Bartley, T.D., Hunt, R. W., Welcher, A. A., Boyle, W. J., Parker, V. P., Lindberg, R. A., Lu, H. S., Colombero, A. M., Elliott, R. L., Guthrie, R. A., Holst, P. L., Skrine, J. D., Toso, R. J., Zhang, M., Fernandez, E., Trail, G., Yarnum, B., Yarden, Y., Hunter, T., and Fox, G. M. (1994) Nature 368, 558-560). We now show that B61 can also exist as a cell surface glycosylphosphatidyl-inositol-linked protein that is capable of activating the Eck receptor protein-tyrosine kinase, the first such report of a receptor protein-tyrosine kinase ligand that is glycosylphosphatidylinositol-linked. In addition, the expression patterns of B61 and Eck during mouse ontogeny were determined by in situ hybridization. Both were found to be highly expressed in the developing lung and gut, while Eck was preferentially expressed in the thymus. Finally, the gene for B61 was localized to a specific position on mouse chromosome 3 by interspecific back-cross analysis.

  13. Alternative dimerization interfaces in the glucocorticoid receptorligand binding domain.

    PubMed

    Bianchetti, Laurent; Wassmer, Bianca; Defosset, Audrey; Smertina, Anna; Tiberti, Marion L; Stote, Roland H; Dejaegere, Annick

    2018-04-30

    Nuclear hormone receptors (NRs) constitute a large family of multi-domain ligand-activated transcription factors. Dimerization is essential for their regulation, and both DNA binding domain (DBD) and ligand binding domain (LBD) are implicated in dimerization. Intriguingly, the glucocorticoid receptor-α (GRα) presents a DBD dimeric architecture similar to that of the homologous estrogen receptor-α (ERα), but an atypical dimeric architecture for the LBD. The physiological relevance of the proposed GRα LBD dimer is a subject of debate. We analyzed all GRα LBD homodimers observed in crystals using an energetic analysis based on the PISA and on the MM/PBSA methods and a sequence conservation analysis, using the ERα LBD dimer as a reference point. Several dimeric assemblies were observed for GRα LBD. The assembly generally taken to be physiologically relevant showed weak binding free energy and no significant residue conservation at the contact interface, while an alternative homodimer mediated by both helix 9 and C-terminal residues showed significant binding free energy and residue conservation. However, none of the GRα LBD assemblies found in crystals are as stable or conserved as the canonical ERα LBD dimer. GRα C-terminal sequence (F-domain) forms a steric obstacle to the canonical dimer assembly in all available structures. Our analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures. This work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research. Copyright © 2018. Published by Elsevier B.V.

  14. Using novel descriptor accounting for ligand-receptor interactions to define and visually explore biologically relevant chemical space.

    PubMed

    Rabal, Obdulia; Oyarzabal, Julen

    2012-05-25

    The definition and pragmatic implementation of biologically relevant chemical space is critical in addressing navigation strategies in the overlapping regions where chemistry and therapeutically relevant targets reside and, therefore, also key to performing an efficient drug discovery project. Here, we describe the development and implementation of a simple and robust method for representing biologically relevant chemical space as a general reference according to current knowledge, independently of any reference space, and analyzing chemical structures accordingly. Underlying our method is the generation of a novel descriptor (LiRIf) that converts structural information into a one-dimensional string accounting for the plausible ligand-receptor interactions as well as for topological information. Capitalizing on ligand-receptor interactions as a descriptor enables the clustering, profiling, and comparison of libraries of compounds from a chemical biology and medicinal chemistry perspective. In addition, as a case study, R-groups analysis is performed to identify the most populated ligand-receptor interactions according to different target families (GPCR, kinases, etc.), as well as to evaluate the coverage of biologically relevant chemical space by structures annotated in different databases (ChEMBL, Glida, etc.).

  15. Different regulation of aryl hydrocarbon receptor-regulated genes in response to dioxin in undifferentiated and neuronally differentiated human neuroblastoma SH-SY5Y cells.

    PubMed

    Imran, Saima; Ferretti, Patrizia; Vrzal, Radim

    2015-01-01

    Some environmental pollutants derived from industrial processes have been suggested to be responsible for neurological impairment in children, especially in heavily polluted areas. Since these compounds are usually activators of aryl hydrocarbon receptor (AhR), it would be important to better understand the molecular pathways downstream of AhR leading to neural deficits. To this purpose, appropriate in vitro human neural model is much needed. Here we have investigated whether undifferentiated and neuronally differentiated human neuroblastoma cells, SH-SY5Y cells, can provide a suitable model for monitoring AhR activity induced by environmental pollutants, focusing on 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD), a known activator of AhR. Further characterization of differentiated SH-SY5Y showed an increase in AhRR (aryl hydrocarbon receptor repressor), no change in ARNT1 (AhR nuclear translocator 1), and a decrease in ARNT2 expression with differentiation; in contrast, AhR was undetectable in both undifferentiated and differentiated cells. Nonetheless, treatment of parental as well as differentiated SH-SY5Y cells with TCDD resulted in the induction of AhR-regulated genes, CYP1A1 and CYP1B1; AhRR expression was also affected, but to a much smaller extent. These results indicate that undifferentiated SH-SY5Y are less sensitive to TCDD than neuronally differentiated ones, suggesting a higher resistance of the undifferentiated tumor cells to toxic insults. They also suggest that TCDD in these cells may not act via direct activation of AhR that is undetectable in SH-SY5Y as well as in differentiated neurons. Hence, these cells do not provide an appropriate model for studying ligand-mediated activation of AhR.

  16. Lysosomal Membrane Permeabilization is an Early Event in Sigma-2 Receptor Ligand Mediated Cell Death in Pancreatic Cancer

    PubMed Central

    2012-01-01

    Background Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. Results Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. Conclusions Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death

  17. Probing ligand recognition of the opioid pan antagonist AT-076 at nociceptin, kappa, mu, and delta opioid receptors through structure-activity relationships.

    PubMed

    Journigan, V Blair; Polgar, Willma E; Tuan, Edward W; Lu, James; Daga, Pankaj R; Zaveri, Nurulain T

    2017-10-16

    Few opioid ligands binding to the three classic opioid receptor subtypes, mu, kappa and delta, have high affinity at the fourth opioid receptor, the nociceptin/orphanin FQ receptor (NOP). We recently reported the discovery of AT-076 (1), (R)-7-hydroxy-N-((S)-1-(4-(3-hydroxyphenyl)piperidin-1-yl)-3-methylbutan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide, a pan antagonist with nanomolar affinity for all four subtypes. Since AT-076 binds with high affinity at all four subtypes, we conducted a structure-activity relationship (SAR) study to probe ligand recognition features important for pan opioid receptor activity, using chemical modifications of key pharmacophoric groups. SAR analysis of the resulting analogs suggests that for the NOP receptor, the entire AT-076 scaffold is crucial for high binding affinity, but the binding mode is likely different from that of NOP antagonists C-24 and SB-612111 bound in the NOP crystal structure. On the other hand, modifications of the 3-hydroxyphenyl pharmacophore, but not the 7-hydroxy Tic pharmacophore, are better tolerated at kappa and mu receptors and yield very high affinity multifunctional (e.g. 12) or highly selective (e.g. 16) kappa ligands. With the availability of the opioid receptor crystal structures, our SAR analysis of the common chemotype of AT-076 suggests rational approaches to modulate binding selectivity, enabling the design of multifunctional or selective opioid ligands from such scaffolds.

  18. Estrogen receptor ligands: a patent review update.

    PubMed

    Paterni, Ilaria; Bertini, Simone; Granchi, Carlotta; Macchia, Marco; Minutolo, Filippo

    2013-10-01

    The role of estrogens is mostly mediated by two nuclear receptors (ERα and ERβ) and a membrane-associated G-protein (GPR30 or GPER), and it is not limited to reproduction, but it extends to the skeletal, cardiovascular and central nervous systems. Various pathologies such as cancer, inflammatory, neurodegenerative and metabolic diseases are often associated with dysfunctions of the estrogenic system. Therapeutic interventions by agents that affect the estrogenic signaling pathway might be useful in the treatment of many dissimilar diseases. The massive chemodiversity of ER ligands, limited to patented small molecules, is herein reviewed. The reported compounds are classified on the basis of their chemical structures. Non-steroidal derivatives, which mostly consist of diphenolic compounds, are further segregated into chemical classes based on their central scaffold. Estrogens have been used for almost a century and their earlier applications have concerned interventions in the female reproductive functions, as well as the treatment of some estrogen-dependent cancers and osteoporosis. Since the discovery of ERβ in 1996, the patent literature has started to pay a progressively increasing attention to this newer receptor subtype, which holds promise as a target for new indications, most of which still need to be clinically validated.

  19. Selective binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonic acid to peroxisome proliferator-activated receptor gamma allows ligand identification and characterization.

    PubMed

    Zorrilla, Silvia; Garzón, Beatriz; Pérez-Sala, Dolores

    2010-04-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily involved in insulin sensitization, atherosclerosis, inflammation, and carcinogenesis. PPARgamma transcriptional activity is modulated by specific ligands that promote conformational changes allowing interaction with coactivators. Here we show that the fluorophore 1-anilinonaphthalene-8-sulfonic acid (ANS) binds to PPARgamma-LBD (ligand binding domain), displaying negligible interaction with other nuclear receptors such as PPARalpha and retinoid X receptor alpha (RXRalpha). ANS binding is competed by PPARgamma agonists such as rosiglitazone, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), and 9,10-dihydro-15-deoxy-Delta(12,14)-prostaglandin J(2) (CAY10410). Moreover, the affinity of PPARgamma for these ligands, determined through ANS competition titrations, is within the range of that reported previously, thereby suggesting that ANS competition could be useful in the screening and characterization of novel PPARgamma agonists. In contrast, gel-based competition assays showed limited performance with noncovalently bound ligands. We applied the ANS binding assay to characterize a biotinylated analog of 15d-PGJ(2) that does not activate PPAR in cells. We found that although this compound bound to PPARgamma with low affinity, it failed to promote PPARgamma interaction with a fluorescent SRC-1 peptide, indicating a lack of receptor activation. Therefore, combined approaches using ANS and fluorescent coactivator peptides to monitor PPARgamma binding and interactions may provide valuable strategies to fully understand the role of PPARgamma ligands. Copyright 2009 Elsevier Inc. All rights reserved.

  20. Rapid, portable detection of endocrine disrupting chemicals through ligand-nuclear hormone receptor interactions.

    PubMed

    Hunt, J Porter; Schinn, Song-Min; Jones, Matthew D; Bundy, Bradley C

    2017-12-04

    Endocrine disrupting chemicals (EDC) are structurally diverse compounds that can interact with nuclear hormone receptors, posing significant risk to human and ecological health. Unfortunately, many conventional biosensors have been too structure-specific, labor-intensive or laboratory-oriented to detect broad ranges of EDC effectively. Recently, several technological advances are providing more rapid, portable, and affordable detection of endocrine-disrupting activity through ligand-nuclear hormone receptor interactions. Here, we overview these recent advances applied to EDC biosensors - including cell lyophilization, cell immobilization, cell-free systems, smartphone-based signal detection, and improved competitive binding assays.

  1. The complex role of NOTCH receptors and their ligands in the development of hepatoblastoma, cholangiocarcinoma and hepatocellular carcinoma.

    PubMed

    Gil-García, Borja; Baladrón, Victoriano

    2016-02-01

    The NOTCH signalling pathway is one of the key molecular pathways of embryonic development and adult tissues homeostasis in mammals. Mammals have four NOTCH receptors and various ligands that modulate their activity. Many cell disorders, whose genesis involves the NOTCH signalling pathway, have been discovered, including cancer. The mechanisms by which these receptors and their ligands affect liver cell transformation are not yet well understood, and they seem to behave as both oncogenes and tumour-suppressor proteins. In this review, we discuss the published data regarding the role of these proteins in the development of hepatoblastoma, cholangiocarcinoma and hepatocellular carcinoma malignancies. The alteration of the NOTCH signalling pathway may be one of the main drivers of hepatic neoplastic growth. However, this signalling pathway might also modulate the development of specific liver tumour features. The complexity of the function of NOTCH receptors and their ligands may be due to their interactions with many other cell signalling pathways. Furthermore, the different levels of expression and activation of these receptors could be a reason for their distinct and sometimes contradictory effects. © 2015 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

  2. A Search for CD36 Ligands from Flavor Volatiles in Foods with an Aldehyde Moiety: Identification of Saturated Aliphatic Aldehydes with 9-16 Carbon Atoms as Potential Ligands of the Receptor.

    PubMed

    Tsuzuki, Satoshi; Amitsuka, Takahiko; Okahashi, Tatsuya; Kimoto, Yusaku; Inoue, Kazuo

    2017-08-09

    Volatile compounds with an aldehyde moiety such as (Z)-9-octadecenal are potential ligands for cluster of differentiation 36 (CD36), a transmembrane receptor that has recently been shown to play a role in mammalian olfaction. In this study, by performing an assay using a peptide mimic of human CD36, we aimed to discover additional ligands for the receptor from volatiles containing a single aldehyde group commonly found in human foods. Straight-chain, saturated aliphatic aldehydes with 9-16 carbons exhibited CD36 ligand activities, albeit to varying degrees. Notably, the activities of tridecanal and tetradecanal were higher than that of oleic acid, the most potent ligand among the fatty acids tested. Among the aldehydes other than aliphatic aldehydes, only phenylacetaldehyde showed a weak activity. These findings make a contribution to our knowledge of recognition mechanisms for flavor volatiles in foods with an aldehyde group.

  3. MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells

    PubMed Central

    2012-01-01

    Introduction The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Methods Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. Results MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Conclusions Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at

  4. MIBE acts as antagonist ligand of both estrogen receptor α and GPER in breast cancer cells.

    PubMed

    Lappano, Rosamaria; Santolla, Maria Francesca; Pupo, Marco; Sinicropi, Maria Stefania; Caruso, Anna; Rosano, Camillo; Maggiolini, Marcello

    2012-01-17

    The multiple biological responses to estrogens are mainly mediated by the classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors. ERα exerts a main role in the development of breast cancer; therefore, the ER antagonist tamoxifen has been widely used although its effectiveness is limited by de novo and acquired resistance. Recently, GPR30/GPER, a member of the seven-transmembrane G protein-coupled receptor family, has been implicated in mediating the effects of estrogens in various normal and cancer cells. In particular, GPER triggered gene expression and proliferative responses induced by estrogens and even ER antagonists in hormone-sensitive tumor cells. Likewise, additional ER ligands showed the ability to bind to GPER eliciting promiscuous and, in some cases, opposite actions through the two receptors. We synthesized a novel compound (ethyl 3-[5-(2-ethoxycarbonyl-1-methylvinyloxy)-1-methyl-1H-indol-3-yl]but-2-enoate), referred to as MIBE, and investigated its properties elicited through ERα and GPER in breast cancer cells. Molecular modeling, binding experiments and functional assays were performed in order to evaluate the biological action exerted by MIBE through ERα and GPER in MCF7 and SkBr3 breast cancer cells. MIBE displayed the ability to act as an antagonist ligand for ERα and GPER as it elicited inhibitory effects on gene transcription and growth effects by binding to both receptors in breast cancer cells. Moreover, GPER was required for epidermal growth factor receptor (EGFR) and ERK activation by EGF as ascertained by using MIBE and performing gene silencing experiments. Our findings provide novel insights on the functional cross-talk between GPER and EGFR signaling. Furthermore, the exclusive antagonistic activity exerted by MIBE on ERα and GPER could represent an innovative pharmacological approach targeting breast carcinomas which express one or both receptors at the beginning and/or during tumor

  5. Glucocorticoid receptor ligand binding in monocytic cells using a microplate assay.

    PubMed

    Jansen, J; Uitdehaag, B; Koper, J W; van Den Berg, T K

    1999-01-01

    Glucocorticoids have profound effects on macrophage function and are widely used as anti-inflammatory drugs. Glucocorticoids receptor (GR) ligand binding capacity is a major determinant of cellular glucocorticoid sensitivity. The number and affinity of GR can be measured in a whole cell binding assay using (3)H-dexamethasone. Here, we describe a rapid and simple microplate assay for GR measurement using the human promonocytic cell line THP-1. Copyright 2000 S. Karger AG, Basel.

  6. Aryl‐hydrocarbon receptor activity modulates prolactin expression in the pituitary

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

    Moran, Tyler B.; Brannick, Katherine E.; Raetzman, Lori T., E-mail: raetzman@life.illinois.edu

    2012-11-15

    Pituitary tumors account for 15% of intracranial neoplasms, however the extent to which environmental toxicants contribute to the proliferation and hormone expression of pituitary cells is unknown. Aryl-hydrocarbon receptor (AhR) interacting protein (AIP) loss of function mutations cause somatotrope and lactotrope adenomas in humans. AIP sequesters AhR and inhibits its transcriptional function. Because of the link between AIP and pituitary tumors, we hypothesize that exposure to dioxins, potent exogenous ligands for AhR that are persistent in the environment, may predispose to pituitary dysfunction through activation of AhR. In the present study, we examined the effect of AhR activation on proliferationmore » and endogenous pituitary hormone expression in the GH3 rat somatolactotrope tumor cell line and the effect of loss of AhR action in knockout mice. GH3 cells respond to nM doses of the reversible AhR agonist β-naphthoflavone with a robust induction of Cyp1a1. Although mRNA levels of the anti-proliferative signaling cytokine TGFbeta1 are suppressed upon β-naphthoflavone treatment, we did not observe an alteration in cell proliferation. AhR activation with β-naphthoflavone suppresses Ahr expression and impairs expression of prolactin (PRL), but not growth hormone (GH) mRNA in GH3 cells. In mice, loss of Ahr similarly leads to a reduction in Prl mRNA at P3, while Gh is unaffected. Additionally, there is a significant reduction in pituitary hormones Lhb and Fshb in the absence of Ahr. Overall, these results demonstrate that AhR is important for pituitary hormone expression and suggest that environmental dioxins can exert endocrine disrupting effects at the pituitary. -- Highlights: ► AhR signaling suppresses Prl mRNA expression. ► AhR signaling does not influence pituitary proliferation in culture. ► AhR is necessary for Prl, Lhb and Fshb expression at postnatal day 3.« less

  7. Computational exploration of a protein receptor binding space with student proposed peptide ligands.

    PubMed

    King, Matthew D; Phillips, Paul; Turner, Matthew W; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M

    2016-01-01

    Computational molecular docking is a fast and effective in silico method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The DockoMatic tutorial described herein provides a framework by which instructors can guide students through a drug screening exercise. Using receptor models derived from readily available protein crystal structures, docking programs have the ability to predict ligand binding properties, such as preferential binding orientations and binding affinities. The use of computational studies can significantly enhance complimentary wet chemical experimentation by providing insight into the important molecular interactions within the system of interest, as well as guide the design of new candidate ligands based on observed binding motifs and energetics. In this laboratory tutorial, the graphical user interface, DockoMatic, facilitates docking job submissions to the docking engine, AutoDock 4.2. The purpose of this exercise is to successfully dock a 17-amino acid peptide, α-conotoxin TxIA, to the acetylcholine binding protein from Aplysia californica-AChBP to determine the most stable binding configuration. Each student will then propose two specific amino acid substitutions of α-conotoxin TxIA to enhance peptide binding affinity, create the mutant in DockoMatic, and perform docking calculations to compare their results with the class. Students will also compare intermolecular forces, binding energy, and geometric orientation of their prepared analog to their initial α-conotoxin TxIA docking results. © 2015 The International Union of Biochemistry and Molecular Biology.

  8. Plant polyphenols differentially modulate inflammatory responses of human keratinocytes by interfering with activation of transcription factors NF{kappa}B and AhR and EGFR-ERK pathway

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

    Potapovich, Alla I.; Biology Department, Belarus State University, Skorina Prosp. 10, Minsk 220050; Lulli, Daniela

    Molecular mechanisms underlying modulation of inflammatory responses in primary human keratinocytes by plant polyphenols (PPs), namely the glycosylated phenylpropanoid verbascoside, the stilbenoid resveratrol and its glycoside polydatin, and the flavonoid quercetin and its glycoside rutin were evaluated. As non-lethal stimuli, the prototypic ligand for epidermal growth factor receptor (EGFR) transforming growth factor alpha (TGFalpha), the combination of tumor necrosis factor (TNFalpha) and interferon (IFNgamma) (T/I), UVA + UVB irradiation, and bacterial lipopolysaccharide (LPS) were used. We demonstrated differential modulation of inflammatory responses in keratinocytes at signal transduction, gene transcription, and protein synthesis levels as a function of PP chemical structure,more » the pro-inflammatory trigger used, and PP interaction with intracellular detoxifying systems. The PPs remarkably inhibited constitutive, LPS- and T/I-induced but not TGFalpha-induced ERK phosphorylation. They also suppressed NFkappaB activation by LPS and T/I. Verbascoside and quercetin invariably impaired EGFR phosphorylation and UV-associated aryl hydrocarbon receptor (AhR)-mediated signaling, while rutin, polydatin and resveratrol did not affect EGFR phosphorylation and further activated AhR machinery in UV-exposed keratinocytes. In general, PPs down-regulated gene expression of pro-inflammatory cytokines/enzymes, except significant up-regulation of IL-8 observed under stimulation with TGFalpha. Both spontaneous and T/I-induced release of IL-8 and IP-10 was suppressed, although 50 {mu}M resveratrol and polydatin up-regulated IL-8. At this concentration, resveratrol activated both gene expression and de novo synthesis of IL-8 and AhR-mediated mechanisms were involved. We conclude that PPs differentially modulate the inflammatory response of human keratinocytes through distinct signal transduction pathways, including AhR and EGFR. - Graphical abstract: Display Omitted

  9. Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

    DOE PAGES

    Zhang, Xianjun; Zhao, Fei; Wu, Yiran; ...

    2017-05-17

    Here, the Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combiningmore » the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.« less

  10. Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

    PubMed

    Antoine, Thomas; Ott, David; Ebell, Katharina; Hansen, Kerrin; Henry, Luc; Becker, Frank; Hannus, Stefan

    2016-06-01

    G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory.

    PubMed

    Weikl, Thomas R; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

    2016-09-02

    The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant [Formula: see text] and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between [Formula: see text] and the binding constant [Formula: see text] of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D).

  12. Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory

    PubMed Central

    Weikl, Thomas R.; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

    2016-01-01

    ABSTRACT The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant K2D and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between K2D and the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D). PMID:27294442

  13. Divergence between human and murine peroxisome proliferator-activated receptor alpha ligand specificities[S

    PubMed Central

    Oswal, Dhawal P.; Balanarasimha, Madhumitha; Loyer, Jeannette K.; Bedi, Shimpi; Soman, Frances L.; Rider, S. Dean; Hostetler, Heather A.

    2013-01-01

    Peroxisome proliferator-activated receptor α (PPARα) belongs to the family of ligand-dependent nuclear transcription factors that regulate energy metabolism. Although there exists remarkable overlap in the activities of PPARα across species, studies utilizing exogenous PPARα ligands suggest species differences in binding, activation, and physiological effects. While unsaturated long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) function as ligands for recombinant mouse PPARα (mPPARα), no such studies have been conducted with full-length human PPARα (hPPARα). The objective of the current study was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for hPPARα or whether there exist species differences for ligand specificity and affinity. Both hPPARα and mPPARα bound with high affinity to LCFA-CoA; however, differences were noted in LCFA affinities. A fluorescent LCFA analog was bound strongly only by mPPARα, and naturally occurring saturated LCFA was bound more strongly by hPPARα than mPPARα. Similarly, unsaturated LCFA induced transactivation of both hPPARα and mPPARα, whereas saturated LCFA induced transactivation only in hPPARα-expressing cells. These data identified LCFA and LCFA-CoA as endogenous ligands of hPPARα, demonstrated species differences in binding specificity and activity, and may help delineate the role of PPARα as a nutrient sensor in metabolic regulation. PMID:23797899

  14. Kinetic and Thermodynamic Characterization of Dihydrotestosterone-Induced Conformational Perturbations in Androgen Receptor Ligand-Binding Domain

    PubMed Central

    Jasuja, Ravi; Ulloor, Jagadish; Yengo, Christopher M.; Choong, Karen; Istomin, Andrei Y.; Livesay, Dennis R.; Jacobs, Donald J.; Swerdloff, Ronald S.; Mikšovská, Jaroslava; Larsen, Randy W.; Bhasin, Shalender

    2009-01-01

    Ligand-induced conformational perturbations in androgen receptor (AR) are important in coactivator recruitment and transactivation. However, molecular rearrangements in AR ligand-binding domain (AR-LBD) associated with agonist binding and their kinetic and thermodynamic parameters are poorly understood. We used steady-state second-derivative absorption and emission spectroscopy, pressure and temperature perturbations, and 4,4′-bis-anilinonaphthalene 8-sulfonate (bis-ANS) partitioning to determine the kinetics and thermodynamics of the conformational changes in AR-LBD after dihydrotestosterone (DHT) binding. In presence of DHT, the second-derivative absorption spectrum showed a red shift and a change in peak-to-peak distance. Emission intensity increased upon DHT binding, and center of spectral mass was blue shifted, denoting conformational changes resulting in more hydrophobic environment for tyrosines and tryptophans within a more compact DHT-bound receptor. In pressure perturbation calorimetry, DHT-induced energetic stabilization increased the Gibbs free energy of unfolding to 8.4 ± 1.3 kcal/mol from 3.5 ± 1.6 kcal/mol. Bis-ANS partitioning studies revealed that upon DHT binding, AR-LBD underwent biphasic rearrangement with a high activation energy (13.4 kcal/mol). An initial, molten globule-like burst phase (k ∼30 sec−1) with greater solvent accessibility was followed by rearrangement (k ∼0.01 sec−1), leading to a more compact conformation than apo-AR-LBD. Molecular simulations demonstrated unique sensitivity of tyrosine and tryptophan residues during pressure unfolding with rearrangement of residues in the coactivator recruitment surfaces distant from the ligand-binding pocket. In conclusion, DHT binding leads to energetic stabilization of AR-LBD domain and substantial rearrangement of residues distant from the ligand-binding pocket. DHT binding to AR-LBD involves biphasic receptor rearrangement including population of a molten globule

  15. Effects of serotonin (5-HT)1B receptor ligands on amphetamine-seeking behavior in rats.

    PubMed

    Miszkiel, Joanna; Przegaliński, Edmund

    2013-01-01

    Numerous studies have indicated that serotonin (5-HT)1B receptor ligands affect the behavioral effects of psychostimulants (cocaine, amphetamine), including the reinforcing activities of these drugs. To substantiate a role for those receptors in incentive motivation for amphetamine, we used the extinction/reinstatement model to examine the effects of the 5-HT1B receptor ligands on the reinstatement of extinguished amphetamine-seeking behavior. Rats trained to self-administer amphetamine (0.06 mg/kg/infusion) subsequently underwent the extinction procedure. These rats were then tested for the amphetamine-primed or amphetamine-associated cue-induced reinstatement of extinguished amphetamine-seeking behavior. The 5-HT1B receptor antagonist SB 216641 (5-7.5 mg/kg) attenuated the amphetamine (1.5 mg/kg)- and the amphetamine-associated cue combined with the threshold dose of amphetamine (0.5 mg/kg)-induced reinstatement of amphetamine-seeking behavior. The 5-HT1B receptor agonist CP 94253 (1.25-5 mg/kg) also inhibited the amphetamine-seeking behavior induced by amphetamine (1.5 mg/kg) but not by the cue combined with the threshold dose of amphetamine. The inhibitory effect of CP94253 on amphetamine-seeking behavior remained unaffected by the 5-HT1B receptor antagonist. Our results indicate that tonic activation of 5-HT1B receptors is involved in amphetamine- and cue-induced reinstatement of amphetamine-seeking behavior and that the inhibitory effects of 5-HT1B receptor antagonists on these phenomena are directly related to the motivational aspects of amphetamine abuse. The inhibitory effect of CP 94253 on amphetamine-seeking behavior seems to be unrelated to 5-HT1B receptor activation and may result from a general reduction of motivation.

  16. Baicalein induces G1 arrest in oral cancer cells by enhancing the degradation of cyclin D1 and activating AhR to decrease Rb phosphorylation

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

    Cheng, Ya-Hsin, E-mail: yhcheng@mail.cmu.edu.tw; Li, Lih-Ann; Lin, Pinpin

    Baicalein is a flavonoid, known to have anti-inflammatory and anti-cancer effects. As an aryl hydrocarbon receptor (AhR) ligand, baicalein at high concentrations blocks AhR-mediated dioxin toxicity. Because AhR had been reported to play a role in regulating the cell cycle, we suspected that the anti-cancer effect of baicalein is associated with AhR. This study investigated the molecular mechanism involved in the anti-cancer effect of baicalein in oral cancer cells HSC-3, including whether such effect would be AhR-mediated. Results revealed that baicalein inhibited cell proliferation and increased AhR activity in a dose-dependent manner. Cell cycle was arrested at the G1 phasemore » and the expression of CDK4, cyclin D1, and phosphorylated retinoblastoma (pRb) was decreased. When the AhR was suppressed by siRNA, the reduction of pRb was partially reversed, accompanied by a decrease of cell population at G1 phase and an increase at S phase, while the reduction of cyclin D1 and CDK4 did not change. This finding suggests that the baicalein activation of AhR is indeed associated with the reduction of pRb, but is independent of the reduction of cyclin D1 and CDK4. When cells were pre-treated with LiCl, the inhibitor of GSK-3β, the decrease of cyclin D1 was blocked and the reduction of pRb was recovered. The data indicates that in HSC-3 the reduction of pRb is both mediated by baicalein through activation of AhR and facilitation of cyclin D1 degradation, which causes cell cycle arrest at the G1 phase, and results in the inhibition of cell proliferation. -- Highlights: ► Baicalein causes the G1 phase arrest by decreasing Rb phosphorylation. ► Baicalein modulates AhR-mediated cell proliferation. ► Both AhR activation and cyclin D1 degradation results in hypophosphorylation of Rb. ► Baicalein facilitates cyclin D1 degradation by signalling the GSK-3β pathway.« less

  17. The effects of administration of ligands for Toll-like receptor 4 and 21 against Marek’s disease in chickens

    USDA-ARS?s Scientific Manuscript database

    Ligands for Toll-like receptors (TLRs) are known to stimulate immune responses, leading to protection against bacterial and viral pathogens. Here, we aimed to examine the effects of various TLR ligands on the development of Marek’s disease in chickens. Specific-pathogen free chickens were treated wi...

  18. Gene expression profiling of the Notch-AhR-IL22 axis at homeostasis and in response to tissue injury.

    PubMed

    Weidenbusch, Marc; Rodler, Severin; Song, Shangqing; Romoli, Simone; Marschner, Julian A; Kraft, Franziska; Holderied, Alexander; Kumar, Santosh; Mulay, Shrikant R; Honarpisheh, Mohsen; Kumar Devarapu, Satish; Lech, Maciej; Anders, Hans-Joachim

    2017-12-22

    Notch and interleukin-22 (IL-22) signaling are known to regulate tissue homeostasis and respond to injury in humans and mice, and the induction of endogenous aryl hydrocarbon receptor (Ahr) ligands through Notch links the two pathways in a hierarchical fashion. However in adults, the species-, organ- and injury-specific gene expression of the Notch-AhR-IL22 axis components is unknown. We therefore performed gene expression profiling of DLL1, DLL3, DLL4, DLK1, DLK2, JAG1, JAG2, Notch1, Notch2, Notch3, Notch4, ADAM17/TNF-α ADAM metalloprotease converting enzyme (TACE), PSEN1, basigin (BSG)/CD147, RBP-J, HES1, HES5, HEY1, HEYL, AHR, ARNT, ARNT2, CYP1A1, CYP24A1, IL-22, IL22RA1, IL22RA2, IL10RB, and STAT3 under homeostatic conditions in ten mature murine and human organs. Additionally, the expression of these genes was assessed in murine models of acute sterile inflammation and progressive fibrosis. We show that there are organ-specific gene expression profiles of the Notch-AhR-IL22 axis in humans and mice. Although there is an overall interspecies congruency, specific differences between human and murine expression signatures do exist. In murine tissues with AHR/ARNT expression CYP1A1 and IL-22 were correlated with HES5 and HEYL expression, while in human tissues no such correlation was found. Notch and AhR signaling are involved in renal inflammation and fibrosis with specific gene expression changes in each model. Despite the presence of all Notch pathway molecules in the kidney and a model-specific induction of Notch ligands, IL-22 was only up-regulated in acute inflammation, but rapidly down-regulated during regeneration. This implies that for targeting injury responses, e.g. via IL-22, species-specific differences, injury type and time points have to be considered. © 2017 The Author(s).

  19. The pregnane X receptor regulates gene expression in a ligand- and promoter-selective fashion.

    PubMed

    Masuyama, Hisashi; Suwaki, Naoko; Tateishi, Yoko; Nakatsukasa, Hideki; Segawa, Tomonori; Hiramatsu, Yuji

    2005-05-01

    Recent studies have revealed that pregnane X receptor (PXR) can function as a master regulator to control the expression of phase I and phase II drug-metabolizing enzymes, as well as members of the drug transporter family, including multiple drug resistance (MDR) 1, which has a major role in multidrug resistance. Previously, we have demonstrated that steroid/xenobiotics metabolism by tumor tissue through the PXR-cytochrome P-450 3A (CYP3A) pathway might play an important role in endometrial cancer. In this study, we examined which endocrine-disrupting chemicals (EDCs) and anticancer agents might be ligands for PXR and whether these chemicals enhanced PXR-mediated transcription through two different PXR-responsive elements (PXREs), CYP3A4 and MDR1, in endometrial cancer cell lines. Some steroids/EDCs strongly activated PXR-mediated transcription through the CYP3A4-responsive element compared with the MDR1-responsive element, whereas these steroids/EDCs also enhanced the CYP3A4 expression compared with the MDR1 expression. In contrast, the anticancer agents, cisplatin and paclitaxel, strongly activated PXR-mediated transcription through the MDR1-responsive element compared with the CYP3A4-responsive element, whereas these drugs also enhanced the MDR1 expression compared with the CYP3A4 expression. We also analyzed how these ligands regulated PXR-mediated transcription through two different PXREs. In the presence of PXR ligands, there was no difference in the DNA binding affinity of the PXR/retinoid X receptor heterodimer to each PXRE, but there were different interactions of the coactivator to each PXR/PXRE complex. These data suggested that PXR ligands enhanced PXR-mediated transcription in a ligand- and promoter-dependent fashion, which in turn differentially regulated the expression of individual PXR targets, especially CYP3A4 and MDR1.

  20. Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5-HT2C Receptor Underlying the Pharmacology of Distinct Ligands.

    PubMed

    Liu, Yue; Canal, Clinton E; Cordova-Sintjago, Tania C; Zhu, Wanying; Booth, Raymond G

    2017-01-18

    While exploring the structure-activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT 2C receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT 2C receptors. In HEK293 cells expressing human 5-HT 2C-INI receptors, for example, (-)-trans-3'-Br-PAT and (-)-trans-3'-Cl-PAT are agonists regarding Gα q -inositol phosphate signaling, whereas (-)-trans-3'-CF 3 -PAT is an inverse agonist. To investigate the ligand-receptor interactions that govern this change in function, we performed site-directed mutagenesis of 14 amino acids of the 5-HT 2C receptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that S3.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (K i ) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC 50 functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (-)-trans-3'-CF 3 -PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (-)-trans-3'-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (K D ) of the antagonist radioligand [ 3 H]mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT 2C ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT 2C receptor residues important for pharmacology of one ligand are not necessarily important for another ligand.

  1. Visualization of ligand-induced transmembrane signaling in the full-length human insulin receptor

    PubMed Central

    2018-01-01

    Insulin receptor (IR) signaling plays a critical role in the regulation of metabolism and growth in multicellular organisms. IRs are unique among receptor tyrosine kinases in that they exist exclusively as covalent (αβ)2 homodimers at the cell surface. Transmembrane signaling by the IR can therefore not be based on ligand-induced dimerization as such but must involve structural changes within the existing receptor dimer. In this study, using glycosylated full-length human IR reconstituted into lipid nanodiscs, we show by single-particle electron microscopy that insulin binding to the dimeric receptor converts its ectodomain from an inverted U-shaped conformation to a T-shaped conformation. This structural rearrangement of the ectodomain propagates to the transmembrane domains, which are well separated in the inactive conformation but come close together upon insulin binding, facilitating autophosphorylation of the cytoplasmic kinase domains. PMID:29453311

  2. Differential hydrogen/deuterium exchange mass spectrometry analysis of protein–ligand interactions

    PubMed Central

    Chalmers, Michael J; Busby, Scott A; Pascal, Bruce D; West, Graham M; Griffin, Patrick R

    2011-01-01

    Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptorligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule–receptor interactions, this technique has also been applied to study protein–protein complexes, such as mapping antibody–antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein–ligand interactions has had an impact on biology and drug discovery. PMID:21329427

  3. The role of TAM family receptors and ligands in the nervous system: From development to pathobiology.

    PubMed

    Shafit-Zagardo, Bridget; Gruber, Ross C; DuBois, Juwen C

    2018-03-04

    Tyro3, Axl, and Mertk, referred to as the TAM family of receptor tyrosine kinases, are instrumental in maintaining cell survival and homeostasis in mammals. TAM receptors interact with multiple signaling molecules to regulate cell migration, survival, phagocytosis and clearance of metabolic products and cell debris called efferocytosis. The TAMs also function as rheostats to reduce the expression of proinflammatory molecules and prevent autoimmunity. All three TAM receptors are activated in a concentration-dependent manner by the vitamin K-dependent growth arrest-specific protein 6 (Gas6). Gas6 and the TAMs are abundantly expressed in the nervous system. Gas6, secreted by neurons and endothelial cells, is the sole ligand for Axl. ProteinS1 (ProS1), another vitamin K-dependent protein functions mainly as an anti-coagulant, and independent of this function can activate Tyro3 and Mertk, but not Axl. This review will focus on the role of the TAM receptors and their ligands in the nervous system. We highlight studies that explore the function of TAM signaling in myelination, the visual cortex, neural cancers, and multiple sclerosis (MS) using Gas6 -/- and TAM mutant mice models. Copyright © 2018. Published by Elsevier Inc.

  4. The orthosteric GABAA receptor ligand Thio-4-PIOL displays distinctly different functional properties at synaptic and extrasynaptic receptors

    PubMed Central

    Hoestgaard-Jensen, K; O'Connor, R M; Dalby, N O; Simonsen, C; Finger, B C; Golubeva, A; Hammer, H; Bergmann, M L; Kristiansen, U; Krogsgaard-Larsen, P; Bräuner-Osborne, H; Ebert, B; Frølund, B; Cryan, J F; Jensen, A A

    2013-01-01

    BACKGROUND AND PURPOSE Explorations into the heterogeneous population of native GABA type A receptors (GABAARs) and the physiological functions governed by the multiple GABAAR subtypes have for decades been hampered by the lack of subtype-selective ligands. EXPERIMENTAL APPROACH The functional properties of the orthosteric GABAA receptor ligand 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) have been investigated in vitro, ex vivo and in vivo. KEY RESULTS Thio-4-PIOL displayed substantial partial agonist activity at the human extrasynaptic GABAAR subtypes expressed in Xenopus oocytes, eliciting maximal responses of up to ∼30% of that of GABA at α5β3γ2S, α4β3δ and α6β3δ and somewhat lower efficacies at the corresponding α5β2γ2S, α4β2δ and α6β2δ subtypes (maximal responses of 4–12%). In contrast, it was an extremely low efficacious agonist at the α1β3γ2S, α1β2γ2S, α2β2γ2S, α2β3γ2S, α3β2γ2S and α3β3γ2S GABAARs (maximal responses of 0–4%). In concordance with its agonism at extrasynaptic GABAARs and its de facto antagonism at the synaptic receptors, Thio-4-PIOL elicited robust tonic currents in electrophysiological recordings on slices from rat CA1 hippocampus and ventrobasal thalamus and antagonized phasic currents in hippocampal neurons. Finally, the observed effects of Thio-4-PIOL in rat tests of anxiety, locomotion, nociception and spatial memory were overall in good agreement with its in vitro and ex vivo properties. CONCLUSION AND IMPLICATIONS The diverse signalling characteristics of Thio-4-PIOL at GABAARs represent one of the few examples of a functionally subtype-selective orthosteric GABAAR ligand reported to date. We propose that Thio-4-PIOL could be a useful pharmacological tool in future studies exploring the physiological roles of native synaptic and extrasynaptic GABAARs. PMID:23957253

  5. Role of hydrogen bonding in ligand interaction with the N-methyl-D-aspartate receptor ion channel

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

    Leeson, P.D.; Carling, R.W.; James, K.

    1990-05-01

    Displacement of (3H)MK-801 (dizocilpine, 1) binding to rat brain membranes has been used to evaluate the affinities of novel dibenzocycloalkenimines related to 1 for the ion channel binding site (also known as the phencyclidine or PCP receptor) on the N-methyl-D-aspartate (NMDA) subtype of excitory amino acid receptor. In common with many other agents having actions in the central nervous system, these compounds contain a hydrophobic aromatic moiety and a basic nitrogen atom. The conformational rigidity of these ligands provides a unique opportunity to evaluate the importance of specific geometrical properties that influence active-site recognition, in particular the role of themore » nitrogen atom in hydrogen-bonding interactions. The relative affinities (IC50s) of hydrocarbon-substituted analogues of 1 and ring homologated cyclooctenimines illustrate the importance of size-limited hydrophobic binding of both aryl rings and of the quaternary C-5 methyl group. Analysis of the binding of a series of the 10 available structurally rigid dibenzoazabicyclo(x.y.z)alkanes, by using molecular modeling techniques, uncovered a highly significant correlation between affinity and a proposed ligand-active site hydrogen bonding vector (r = 0.950, p less than 0.001). These results are used to generate a pharmacophore of the MK-801 recognition site/PCP receptor, which accounts for the binding of all of the known ligands.« less

  6. Considerations for potency equivalent calculations in the Ah receptor-based CALUX bioassay: normalization of superinduction results for improved sample potency estimation.

    PubMed

    Baston, David S; Denison, Michael S

    2011-02-15

    The chemically activated luciferase expression (CALUX) system is a mechanistically based recombinant luciferase reporter gene cell bioassay used in combination with chemical extraction and clean-up methods for the detection and relative quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like halogenated aromatic hydrocarbons in a wide variety of sample matrices. While sample extracts containing complex mixtures of chemicals can produce a variety of distinct concentration-dependent luciferase induction responses in CALUX cells, these effects are produced through a common mechanism of action (i.e. the Ah receptor (AhR)) allowing normalization of results and sample potency determination. Here we describe the diversity in CALUX response to PCDD/Fs from sediment and soil extracts and not only report the occurrence of superinduction of the CALUX bioassay, but we describe a mechanistically based approach for normalization of superinduction data that results in a more accurate estimation of the relative potency of such sample extracts. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Considerations for potency equivalent calculations in the Ah receptor-based CALUX bioassay: Normalization of superinduction results for improved sample potency estimation

    PubMed Central

    Baston, David S.; Denison, Michael S.

    2011-01-01

    The chemically activated luciferase expression (CALUX) system is a mechanistically based recombinant luciferase reporter gene cell bioassay used in combination with chemical extraction and clean-up methods for the detection and relative quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like halogenated aromatic hydrocarbons in a wide variety of sample matrices. While sample extracts containing complex mixtures of chemicals can produce a variety of distinct concentration-dependent luciferase induction responses in CALUX cells, these effects are produced through a common mechanism of action (i.e. the Ah receptor (AhR)) allowing normalization of results and sample potency determination. Here we describe the diversity in CALUX response to PCDD/Fs from sediment and soil extracts and not only report the occurrence of superinduction of the CALUX bioassay, but we describe a mechanistically based approach for normalization of superinduction data that results in a more accurate estimation of the relative potency of such sample extracts. PMID:21238730

  8. Fish genomes provide novel insights into the evolution of vertebrate secretin receptors and their ligand.

    PubMed

    Cardoso, João C R; Félix, Rute C; Trindade, Marlene; Power, Deborah M

    2014-12-01

    The secretin receptor (SCTR) is a member of Class 2 subfamily B1 GPCRs and part of the PAC1/VPAC receptor subfamily. This receptor has long been known in mammals but has only recently been identified in other vertebrates including teleosts, from which it was previously considered to be absent. The ligand for SCTR in mammals is secretin (SCT), an important gastrointestinal peptide, which in teleosts has not yet been isolated, or the gene identified. This study revises the evolutionary model previously proposed for the secretin-GPCRs in metazoan by analysing in detail the fishes, the most successful of the extant vertebrates. All the Actinopterygii genomes analysed and the Chondrichthyes and Sarcopterygii fish possess a SCTR gene that shares conserved sequence, structure and synteny with the tetrapod homologue. Phylogenetic clustering and gene environment comparisons revealed that fish and tetrapod SCTR shared a common origin and diverged early from the PAC1/VPAC subfamily group. In teleosts SCTR duplicated as a result of the fish specific whole genome duplication but in all the teleost genomes analysed, with the exception of tilapia (Oreochromis niloticus), one of the duplicates was lost. The function of SCTR in teleosts is unknown but quantitative PCR revealed that in both sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) transcript abundance is high in the gastrointestinal tract suggesting it may intervene in similar processes to those in mammals. In contrast, no gene encoding the ligand SCT was identified in the ray-finned fishes (Actinopterygii) although it was present in the coelacanth (lobe finned fish, Sarcopterygii) and in the elephant shark (holocephalian). The genes in linkage with SCT in tetrapods and coelacanth were also identified in ray-finned fishes supporting the idea that it was lost from their genome. At present SCTR remains an orphan receptor in ray-finned fishes and it will be of interest in the future to establish why SCT was

  9. Identification of Amino Acid Substitutions Supporting Antigenic Change of Influenza A(H1N1)pdm09 Viruses

    PubMed Central

    Koel, Björn F.; Mögling, Ramona; Chutinimitkul, Salin; Fraaij, Pieter L.; Burke, David F.; van der Vliet, Stefan; de Wit, Emmie; Bestebroer, Theo M.; Rimmelzwaan, Guus F.; Osterhaus, Albert D. M. E.; Smith, Derek J.; Fouchier, Ron A. M.

    2015-01-01

    ABSTRACT The majority of currently circulating influenza A(H1N1) viruses are antigenically similar to the virus that caused the 2009 influenza pandemic. However, antigenic variants are expected to emerge as population immunity increases. Amino acid substitutions in the hemagglutinin protein can result in escape from neutralizing antibodies, affect viral fitness, and change receptor preference. In this study, we constructed mutants with substitutions in the hemagglutinin of A/Netherlands/602/09 in an attenuated backbone to explore amino acid changes that may contribute to emergence of antigenic variants in the human population. Our analysis revealed that single substitutions affecting the loop that consists of amino acid positions 151 to 159 located adjacent to the receptor binding site caused escape from ferret and human antibodies elicited after primary A(H1N1)pdm09 virus infection. The majority of these substitutions resulted in similar or increased replication efficiency in vitro compared to that of the virus carrying the wild-type hemagglutinin and did not result in a change of receptor preference. However, none of the substitutions was sufficient for escape from the antibodies in sera from individuals that experienced both seasonal and pandemic A(H1N1) virus infections. These results suggest that antibodies directed against epitopes on seasonal A(H1N1) viruses contribute to neutralization of A(H1N1)pdm09 antigenic variants, thereby limiting the number of possible substitutions that could lead to escape from population immunity. IMPORTANCE Influenza A viruses can cause significant morbidity and mortality in humans. Amino acid substitutions in the hemagglutinin protein can result in escape from antibody-mediated neutralization. This allows the virus to reinfect individuals that have acquired immunity to previously circulating strains through infection or vaccination. To date, the vast majority of A(H1N1)pdm09 strains remain antigenically similar to the virus

  10. Development of an image analysis screen for estrogen receptor alpha (ERα) ligands through measurement of nuclear translocation dynamics.

    PubMed

    Dull, Angie; Goncharova, Ekaterina; Hager, Gordon; McMahon, James B

    2010-11-01

    We have developed a robust high-content assay to screen for novel estrogen receptor alpha (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen utilizes a green fluorescent protein tagged-glucocorticoid/estrogen receptor (GFP-GRER) chimera which consisted of the N-terminus of the glucocorticoid receptor fused to the human ER ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands, and translocated to the nucleus in response to stimulation with ERα agonists or antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. The assay was validated with known ERα agonists and antagonists, and the Library of Pharmacologically Active Compounds (LOPAC 1280). Additionally, screening of crude natural product extracts demonstrated the robustness of the assay, and the ability to quantitate the effects of toxicity on nuclear translocation dynamics. The GFP-GRER nuclear translocation assay was very robust, with z' values >0.7, CVs <5%, and has been validated with known ER ligands, and inclusion of cytotoxicity filters will facilitate screening of natural product extracts. This assay has been developed for future primary screening of synthetic, pure natural products, and natural product extracts libraries available at the National Cancer Institute at Frederick. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. Single-molecule force spectroscopy study of interactions between angiotensin II type 1 receptor and different biased ligands in living cells.

    PubMed

    Li, Wenhui; Xu, Jiachao; Kou, Xiaolong; Zhao, Rong; Zhou, Wei; Fang, Xiaohong

    2018-05-01

    Angiotensin II type 1 receptor (AT1R), a typical G protein-coupled receptor, plays a key role in regulating many cardiovascular functions. Different ligands can bind with AT1R to selectively activate either G protein (Gq) or β-arrestin (β-arr) pathway, or both pathways, but the molecular mechanism is not clear yet. In this work, we used, for the first time, atomic force microscopy-based single molecule force spectroscopy (SMFS) to study the interactions of AT1R with three types of ligands, balanced ligand, Gq-biased ligand, and β-arr-biased ligand, in living cells. The results revealed their difference in binding force and binding stability. The complex of the Gq-biased ligand-AT1R overcame two energy barriers with an intermediate state during dissociation, whereas that of β-arr-biased ligand-AT1R complex overcame one energy barrier. This indicated that AT1R had different ligand-binding conformational substates and underwent different structural changes to activate downstream signaling pathways with variable agonist efficacies. Quantitative analysis of AT1R-ligand binding in living cells at the single-molecule level offers a new tool to study the molecular mechanism of AT1R biased activation. Graphical Abstract Single-molecule force measurement on the living cell expressing AT1R-eGFP with a ligand modified AFM tip (left), the dynamic force spectra of β-arrestin biased ligands-AT1R (middle), and Gq-biased ligands-AT1R (right). The complexes of β-arr-biased ligand-AT1R overcame one energy barrier, with one linear region in the spectra, whereas the Gq-biased ligand-AT1R complexes overcame two energy barriers with two linear regions.

  12. Biased ligand quantification in drug discovery: from theory to high throughput screening to identify new biased μ opioid receptor agonists

    PubMed Central

    Winpenny, David; Clark, Mellissa

    2016-01-01

    Background and Purpose Biased GPCR ligands are able to engage with their target receptor in a manner that preferentially activates distinct downstream signalling and offers potential for next generation therapeutics. However, accurate quantification of ligand bias in vitro is complex, and current best practice is not amenable for testing large numbers of compound. We have therefore sought to apply ligand bias theory to an industrial scale screening campaign for the identification of new biased μ receptor agonists. Experimental Approach μ receptor assays with appropriate dynamic range were developed for both Gαi‐dependent signalling and β‐arrestin2 recruitment. Δlog(Emax/EC50) analysis was validated as an alternative for the operational model of agonism in calculating pathway bias towards Gαi‐dependent signalling. The analysis was applied to a high throughput screen to characterize the prevalence and nature of pathway bias among a diverse set of compounds with μ receptor agonist activity. Key Results A high throughput screening campaign yielded 440 hits with greater than 10‐fold bias relative to DAMGO. To validate these results, we quantified pathway bias of a subset of hits using the operational model of agonism. The high degree of correlation across these biased hits confirmed that Δlog(Emax/EC50) was a suitable method for identifying genuine biased ligands within a large collection of diverse compounds. Conclusions and Implications This work demonstrates that using Δlog(Emax/EC50), drug discovery can apply the concept of biased ligand quantification on a large scale and accelerate the deliberate discovery of novel therapeutics acting via this complex pharmacology. PMID:26791140

  13. The prelude on novel receptor and ligand targets involved in the treatment of diabetes mellitus.

    PubMed

    Jonnalagadda, Venu Gopal; Ram Raju, Allam Venkata Sita; Pittala, Srinivas; Shaik, Afsar; Selkar, Nilakash Annaji

    2014-01-01

    Metabolic disorders are a group of disorders, due to the disruption of the normal metabolic process at a cellular level. Diabetes Mellitus and Tyrosinaemia are the majorly reported metabolic disorders. Among them, Diabetes Mellitus is a one of the leading metabolic syndrome, affecting 5 to 7 % of the population worldwide and mainly characterised by elevated levels of glucose and is associated with two types of physiological event disturbances such as impaired insulin secretion and insulin resistance. Up to now, various treatment strategies are like insulin, alphaglucosidase inhibitors, biguanides, incretins were being followed. Concurrently, various novel therapeutic strategies are required to advance the therapy of Diabetes mellitus. For the last few decades, there has been an extensive research in understanding the metabolic pathways involved in Diabetes Mellitus at the cellular level and having the profound knowledge on cell-growth, cell-cycle, and apoptosis at a molecular level provides new targets for the treatment of Diabetes Mellitus. Receptor signalling has been involved in these mechanisms, to translate the information coming from outside. To understand the various receptors involved in these pathways, we must have a sound knowledge on receptors and ligands involved in it. This review mainly summarises the receptors and ligands which are involved the Diabetes Mellitus. Finally, researchers have to develop the alternative chemical moieties that retain their affinity to receptors and efficacy. Diabetes Mellitus being a metabolic disorder due to the glucose surfeit, demands the need for regular exercise along with dietary changes.

  14. Ligand-Mediated Activation of an Engineered Gs G Protein-Coupled Receptor in Osteoblasts Increases Trabecular Bone Formation

    PubMed Central

    Hsiao, Edward C.; Millard, Susan M.; Louie, Alyssa; Huang, Yong; Conklin, Bruce R.; Nissenson, Robert A.

    2010-01-01

    Age-dependent changes in skeletal growth play important roles in regulating skeletal expansion and in the course of many diseases affecting bone. How G protein-coupled receptor (GPCR) signaling affects these changes is poorly understood. Previously, we described a mouse model expressing Rs1, an engineered receptor with constitutive Gs activity. Rs1 expression in osteoblasts from gestation induced a dramatic age-dependent increase in trabecular bone with features resembling fibrous dysplasia; however, these changes were greatly minimized if Rs1 expression was delayed until after puberty. To further investigate whether ligand-induced activation of the Gs-GPCR pathway affects bone formation in adult mice, we activated Rs1 in adult mice with the synthetic ligand RS67333 delivered continuously via an osmotic pump or intermittently by daily injections. We found that osteoblasts from adult animals can be stimulated to form large amounts of bone, indicating that adult mice are sensitive to the dramatic bone- forming actions of Gs signaling in osteoblasts. In addition, our results show that intermittent and continuous activation of Rs1 led to structurally similar but quantitatively different degrees of trabecular bone formation. These results indicate that activation of a Gs-coupled receptor in osteoblasts of adult animals by either intermittent or continuous ligand administration can increase trabecular bone formation. In addition, osteoblasts located at the bone epiphyses may be more responsive to Gs signaling than osteoblasts at the bone diaphysis. This model provides a powerful tool for investigating the effects of ligand-activated Gs-GPCR signaling on dynamic bone growth and remodeling. PMID:20150184

  15. The Activating Human NK Cell Receptor KIR2DS2 Recognizes a β2-Microglobulin-Independent Ligand on Cancer Cells.

    PubMed

    Thiruchelvam-Kyle, Lavanya; Hoelsbrekken, Sigurd E; Saether, Per C; Bjørnsen, Elisabeth Gyllensten; Pende, Daniela; Fossum, Sigbjørn; Daws, Michael R; Dissen, Erik

    2017-04-01

    The functions of activating members of the killer cell Ig-like receptor (KIR) family are not fully understood, as the ligands for these receptors are largely unidentified. In this study, we report that KIR2DS2 reporter cells recognize a ligand expressed by cancer cell lines. All cancer targets recognized by KIR2DS2 were also recognized by KIR2DL2 and KIR2DL3 reporters. Trogocytosis of membrane proteins from the cancer targets was observed with responding reporter cells, indicating the formation of KIR2DS2 ligand-specific immunological synapses. HLA-C typing of target cells showed that KIR2DS2 recognition was independent of the HLA C1 or C2 group, whereas targets cells that were only recognized by KIR2DL3 expressed C1 group alleles. Anti-HLA class I Abs blocked KIR2DL3 responses toward C1-expressing targets, but they did not block KIR2DS2 recognition of cancer cells. Small interfering RNA knockdown of β 2 -microglobulin reduced the expression of class I H chain on the cancer targets by >97%, but it did not reduce the KIR2DS2 reporter responses, indicating a β 2 -microglobulin-independent ligand for KIR2DS2. Importantly, KIR2DL3 responses toward some KIR2DS2 ligand-expressing cells were also undiminished after β 2 -microglobulin knockdown, and they were not blocked by anti-HLA class I Abs, suggesting that KIR2DL3, in addition to the traditional HLA-C ligands, can bind to the same β 2 -microglobulin-independent ligand as KIR2DS2. These observations indicate the existence of a novel, presently uncharacterized ligand for the activating NK cell receptor KIR2DS2. Molecular identification of this ligand may lead to improved KIR-HLA mismatching in hematopoietic stem cell transplantation therapy for leukemia and new, more specific NK cell-based cancer therapies. Copyright © 2017 by The American Association of Immunologists, Inc.

  16. AutoSite: an automated approach for pseudo-ligands prediction—from ligand-binding sites identification to predicting key ligand atoms

    PubMed Central

    Ravindranath, Pradeep Anand; Sanner, Michel F.

    2016-01-01

    Motivation: The identification of ligand-binding sites from a protein structure facilitates computational drug design and optimization, and protein function assignment. We introduce AutoSite: an efficient software tool for identifying ligand-binding sites and predicting pseudo ligand corresponding to each binding site identified. Binding sites are reported as clusters of 3D points called fills in which every point is labelled as hydrophobic or as hydrogen bond donor or acceptor. From these fills AutoSite derives feature points: a set of putative positions of hydrophobic-, and hydrogen-bond forming ligand atoms. Results: We show that AutoSite identifies ligand-binding sites with higher accuracy than other leading methods, and produces fills that better matches the ligand shape and properties, than the fills obtained with a software program with similar capabilities, AutoLigand. In addition, we demonstrate that for the Astex Diverse Set, the feature points identify 79% of hydrophobic ligand atoms, and 81% and 62% of the hydrogen acceptor and donor hydrogen ligand atoms interacting with the receptor, and predict 81.2% of water molecules mediating interactions between ligand and receptor. Finally, we illustrate potential uses of the predicted feature points in the context of lead optimization in drug discovery projects. Availability and Implementation: http://adfr.scripps.edu/AutoDockFR/autosite.html Contact: sanner@scripps.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27354702

  17. Detection of Aryl Hydrocarbon Receptor Activation by Some Chemicals in Food Using a Reporter Gene Assay

    PubMed Central

    Amakura, Yoshiaki; Tsutsumi, Tomoaki; Yoshimura, Morio; Nakamura, Masafumi; Handa, Hiroshi; Matsuda, Rieko; Teshima, Reiko; Watanabe, Takahiro

    2016-01-01

    The purpose of this study was to examine whether a simple bioassay used for the detection of dioxins (DXNs) could be applied to detect trace amounts of harmful DXN-like substances in food products. To identify substances with possible DXN-like activity, we assessed the ability of various compounds in the environment to bind the aryl hydrocarbon receptor (AhR) that binds specifically to DXNs. The compounds tested included 19 polycyclic aromatic hydrocarbons (PAHs), 20 PAH derivatives (nitrated, halogenated, and aminated derivatives), 23 pesticides, six amino acids, and eight amino acid metabolites. The AhR binding activities (AhR activity) of these compounds were measured using the chemical activated luciferase gene expression (CALUX) reporter gene assay system. The majority of the PAHs exhibited marked AhR activity that increased in a concentration-dependent manner. Furthermore, there was a positive link between AhR activity and the number of aromatic rings in the PAH derivatives. Conversely, there appeared to be a negative correlation between AhR activity and the number of chlorine residues present on halogenated PAH derivatives. However, there was no correlation between AhR activity and the number and position of substituents among nitrated and aminated derivatives. Among the pesticides tested, the indole-type compounds carbendazim and thiabendazole showed high levels of activity. Similarly, the indole compound tryptamine was the only amino acid metabolite to induce AhR activity. The results are useful in understanding the identification and characterization of AhR ligands in the CALUX assay. PMID:28231110

  18. Ligand selectivity of estrogen receptors by a molecular dynamics study.

    PubMed

    Hu, Guodong; Wang, Jihua

    2014-03-03

    Estrogen receptors α (ERα) and β (ERβ) have different physiological functions and expression levels in different tissues. ERα and ERβ are highly homologous and have only two residue substitutions in the binding pocket. This high similarity at the active site stimulates the interests for discovering subtype selective ligands. In this study, molecular dynamics (MD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) method have been carried out to analyze the basis of selectivity of three ligands (659, 818 and 041). The calculated binding free energies show that all the ligands bind more tightly to ERβ than to ERα. The dominant free energy components of selectivity for 659 are similar to that for 041, but different from that for 818. The decompositions of free energy contributions and structural analysis imply that there are eight residues primarily contributing to the selectivity for 659, five residues for 041, as well as two residues for 818. The structural analysis implies that two residue substitutions in binding packet cause the position of 659 in ERβ-659 complex to shift relative to that in ERα-659 complex and also cause the conformational changes of other residues in the binding pocket. The higher selectivity for 041 is mainly caused by three residues, Ile373 (Met421), His475 (His524) and Leu476 (Leu525). Copyright © 2013. Published by Elsevier Masson SAS.

  19. Evolution of the hemagglutinin expressed by human influenza A(H1N1)pdm09 and A(H3N2) viruses circulating between 2008-2009 and 2013-2014 in Germany.

    PubMed

    Wedde, Marianne; Biere, Barbara; Wolff, Thorsten; Schweiger, Brunhilde

    2015-10-01

    This report describes the evolution of the influenza A(H1N1)pdm09 and A(H3N2) viruses circulating in Germany between 2008-2009 and 2013-2014. The phylogenetic analysis of the hemagglutinin (HA) genes of both subtypes revealed similar evolution of the HA variants that were also seen worldwide with minor exceptions. The analysis showed seven distinct HA clades for A(H1N1)pdm09 and six HA clades for A(H3N2) viruses. Herald strains of both subtypes appeared sporadically since 2008-2009. Regarding A(H1N1)pdm09, herald strains of HA clade 3 and 4 were detected late in the 2009-2010 season. With respect to A(H3N2), we found herald strains of HA clade 3, 4 and 7 between 2009 and 2012. Those herald strains were predominantly seen for minor and not for major HA clades. Generally, amino acid substitutions were most frequently found in the globular domain, including substitutions near the antigenic sites or the receptor binding site. Differences between both influenza A subtypes were seen with respect to the position of the indicated substitutions in the HA. For A(H1N1)pdm09 viruses, we found more substitutions in the stem region than in the antigenic sites. In contrast, in A(H3N2) viruses most changes were identified in the major antigenic sites and five changes of potential glycosylation sites were identified in the head of the HA monomer. Interestingly, we found in seasons with less influenza activity a relatively high increase of substitutions in the head of the HA in both subtypes. This might be explained by the fact that mutations under negative selection are subsequently compensated by secondary mutations to restore important functions e.g. receptor binding properties. A better knowledge of basic evolution strategies of influenza viruses will contribute to the refinement of predictive mathematical models for identifying novel antigenic drift variants. Copyright © 2015 Elsevier GmbH. All rights reserved.

  20. The Aryl Hydrocarbon Receptor: Differential Contribution to T Helper 17 and T Cytotoxic 17 Cell Development

    PubMed Central

    Hayes, Mark D.; Ovcinnikovs, Vitalijs; Smith, Andrew G.; Kimber, Ian; Dearman, Rebecca J.

    2014-01-01

    The aryl hydrocarbon receptor (AhR) has been shown to be required for optimal Thelper (Th) 17 cell activation. Th17 cells provide immunity against extracellular pathogens and are implicated in autoimmune diseases. Herein, the role of the AhR in cytokine production by Th17, and by the analogous population of T cytotoxic (Tc)17 cells, has been examined. Lymph node Tc (CD8+) and Th (CD4+) cells were isolated by negative selection from naive AhR+/− and AhR−/− mice and polarised to Tc1/Th1 or Tc17/Th17 phenotypes with appropriate cytokines. Cell differentiation was assessed as a function of mRNA and protein (ELISA and flow cytometry) expression for interferon (IFN)-γ and for key Th17 cytokines. In AhR+/− mice, Th17 cells displayed an exclusive IL-17 profile, which was markedly inhibited by a selective AhR antagonist to levels observed in AhR knockout mice. Addition of the natural AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) markedly enhanced Th17 cell activity in the heterozygotes. In contrast, Tc17 cells polarised into 3 distinct subsets: producing either IL-17 or IFN-γ alone, or both cytokines. Blocking AhR was also detrimental to Tc17 development, with reduced responses recorded in AhR−/− mice and antagonist-mediated reduction of IL-17 expression in the heterozygotes. However, Tc17 cells were largely refractory to exogenous FICZ, presumably because Tc17 cells express baseline AhR mRNA, but unlike Th17 cells, there is no marked up-regulation during polarisation. Thus, Th17 cell development is more dependent upon AhR activation than is Tc17 cell development, suggesting that endogenous AhR ligands play a much greater role in driving Th17 cell responses. PMID:25203682