Science.gov

Sample records for receptor selectively coupled

  1. Integrating Pharmacophore into Membrane Molecular Dynamics Simulations to Improve Homology Modeling of G Protein-coupled Receptors with Ligand Selectivity: A2A Adenosine Receptor as an Example.

    PubMed

    Zeng, Lingxiao; Guan, Mengxin; Jin, Hongwei; Liu, Zhenming; Zhang, Liangren

    2015-12-01

    Homology modeling has been applied to fill in the gap in experimental G protein-coupled receptors structure determination. However, achievement of G protein-coupled receptors homology models with ligand selectivity remains challenging due to structural diversity of G protein-coupled receptors. In this work, we propose a novel strategy by integrating pharmacophore and membrane molecular dynamics (MD) simulations to improve homology modeling of G protein-coupled receptors with ligand selectivity. To validate this integrated strategy, the A2A adenosine receptor (A2A AR), whose structures in both active and inactive states have been established, has been chosen as an example. We performed blind predictions of the active-state A2A AR structure based on the inactive-state structure and compared the performance of different refinement strategies. The blind prediction model combined with the integrated strategy identified ligand-receptor interactions and conformational changes of key structural elements related to the activation of A2 A AR, including (i) the movements of intracellular ends of TM3 and TM5/TM6; (ii) the opening of ionic lock; (iii) the movements of binding site residues. The integrated strategy of pharmacophore with molecular dynamics simulations can aid in the optimization in the identification of side chain conformations in receptor models. This strategy can be further investigated in homology modeling and expand its applicability to other G protein-coupled receptor modeling, which should aid in the discovery of more effective and selective G protein-coupled receptor ligands.

  2. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    SciTech Connect

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G.

    2015-02-13

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  3. Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

    SciTech Connect

    Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian; Huber, Jochen; Tesmer, John J.G.

    2012-07-11

    G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G{beta}{gamma} complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G{beta}{gamma} structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

  4. Novel chiral-diazepines function as specific, selective receptor agonists with variable coupling and species variability in human, mouse and rat BRS-3 receptor cells.

    PubMed

    Ramos-Álvarez, Irene; Nakamura, Taichi; Mantey, Samuel A; Moreno, Paola; Nuche-Berenguer, Bernardo; Jensen, Robert T

    2016-01-01

    Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein coupled receptor which is classified in the bombesin receptor (BnR) family with which it shares high homology. It is present widely in the central nervous system and peripheral tissues and primarily receptor-knockout studies suggest it is involved in metabolic-glucose-insulin homeostasis, feeding and other CNS behaviors, gastrointestinal motility and cancer growth. However, the role of BRS-3 physiologically or in pathologic disorders has been not well defined because the natural ligand is unknown. Until recently, no selective agonists/antagonists were available; however, recently synthetic high-affinity agonists, chiral-diazepines nonpeptide-analogs (3F, 9D, 9F, 9G) with low CNS penetrance, were described, but are not well-categorized pharmacologically or in different labarotory species. The present study characterizes the affinities, potencies, selectivities of the chiral-diazepine BRS-3 agonists in human and rodents (mice,rat). In human BRS-3 receptors, the relative affinities of the chiral-diazepines was 9G>9D>9F>3F; each was selective for BRS-3. For stimulating PLC activity, in h-BRS-3 each of the four chiral diazepine analogs was fully efficacious and their relative potencies were: 9G (EC50: 9 nM)>9D (EC50: 9.4 nM)>9F (EC50: 39 nM)>3F (EC50: 48 nM). None of the four chiral diazepine analogs activated r,m,h-GRPR/NMBR. The nonpeptide agonists showed marked differences from each other and a peptide agonist in receptor-coupling-stiochiometry and in affinities/potencies in different species. These results demonstrate that chiral diazepine analogs (9G, 9D, 9F, 3F) have high/affinity/potency for the BRS-3 receptor in human and rodent cells, but different coupling-relationships and species differences from a peptide agonist.

  5. Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic {beta}-cell mass

    SciTech Connect

    Cline, Gary W.; Zhao, Xiaojian; Jakowski, Amy B.; Soeller, Walter C.; Treadway, Judith L.

    2011-09-02

    Highlights: {yields} We screened G-protein coupled receptors for imaging pancreatic. {yields} Database mining and immunohistochemistry identified GPCRs enriched in {beta}-cells. {yields} In vitro and in vivo assays were used to determine exocrine vs endocrine specificity. {yields} GPCR candidates for imaging of {beta}-cell mass are Prokineticin-1R, mGluR5, and GLP-1R. -- Abstract: A critical unmet need exists for methods to quantitatively measure endogenous pancreatic {beta}-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet {beta}-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R > GLP-1R > NPY-2R > mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R > VMAT2 {approx} GLP-1R > mGluR5. Favorable islet selectivity and biodistribution

  6. Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

    SciTech Connect

    Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J.G.

    2012-08-31

    Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic {alpha}F-{alpha}G loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase.

  7. Adenylyl cyclase 2 selectively couples to E prostanoid type 2 receptors, whereas adenylyl cyclase 3 is not receptor-regulated in airway smooth muscle.

    PubMed

    Bogard, Amy S; Adris, Piyatilake; Ostrom, Rennolds S

    2012-08-01

    Adenylyl cyclases (ACs) are important regulators of airway smooth muscle function, because β-adrenergic receptor (βAR) agonists stimulate AC activity and cAMP production. We have previously shown in a number of cell types that AC6 selectively couples to βAR and these proteins are coexpressed in lipid rafts. We overexpressed AC2, AC3, and AC6 in mouse bronchial smooth muscle cells (mBSMCs) and human embryonic kidney (HEK)-293 cells by using recombinant adenoviruses and assessed their localization and regulation by various G protein-coupled receptors (GPCRs). AC3 and AC6 were expressed primarily in caveolin-rich fractions, whereas AC2 expression was excluded from these domains. AC6 expression enhanced cAMP production in response to isoproterenol but did not increase responses to butaprost, reflecting the colocalization of AC6 with β(2)AR but not E prostanoid type 2 receptor (EP(2)R) in lipid raft fractions. AC2 expression enhanced butaprost-stimulated cAMP production but had no effect on the β(2)AR-mediated response. AC3 did not couple to any GPCR tested. Forskolin-induced arborization of mBSMCs was assessed as a functional readout of cAMP signaling. Arborization was enhanced by overexpression of AC6 and AC3, but AC2 had no effect. GPCR-stimulated arborization mirrored the selective coupling observed for cAMP production. With the addition of the phosphodiesterase 4 (PDE4) inhibitor rolipram AC2 accelerated forskolin-stimulated arborization. Thus, AC2 selectively couples to EP(2)R, but signals from this complex are limited by PDE4 activity. AC3 does not seem to couple to GPCR in either mBSMCs or HEK-293 cells, so it probably exists in a distinct signaling domain in these cells.

  8. Select Neuropeptides and their G-Protein Coupled Receptors in Caenorhabditis Elegans and Drosophila Melanogaster

    PubMed Central

    Bendena, William G.; Campbell, Jason; Zara, Lian; Tobe, Stephen S.; Chin-Sang, Ian D.

    2012-01-01

    The G-protein coupled receptor (GPCR) family is comprised of seven transmembrane domain proteins and play important roles in nerve transmission, locomotion, proliferation and development, sensory perception, metabolism, and neuromodulation. GPCR research has been targeted by drug developers as a consequence of the wide variety of critical physiological functions regulated by this protein family. Neuropeptide GPCRs are the least characterized of the GPCR family as genetic systems to characterize their functions have lagged behind GPCR gene discovery. Drosophila melanogaster and Caenorhabditis elegans are genetic model organisms that have proved useful in characterizing neuropeptide GPCRs. The strength of a genetic approach leads to an appreciation of the behavioral plasticity that can result from subtle alterations in GPCRs or regulatory proteins in the pathways that GPCRs control. Many of these invertebrate neuropeptides, GPCRs, and signaling pathway components serve as models for mammalian counterparts as they have conserved sequences and function. This review provides an overview of the methods to match neuropeptides to their cognate receptor and a state of the art account of neuropeptide GPCRs that have been characterized in D. melanogaster and C. elegans and the behaviors that have been uncovered through genetic manipulation. PMID:22908006

  9. Differential helical orientations among related G protein-coupled receptors provide a novel mechanism for selectivity. Studies with salvinorin A and the kappa-opioid receptor.

    PubMed

    Vortherms, Timothy A; Mosier, Philip D; Westkaemper, Richard B; Roth, Bryan L

    2007-02-02

    Salvinorin A, the active component of the hallucinogenic sage Salvia divinorum, is an apparently selective and highly potent kappa-opioid receptor (KOR) agonist. Salvinorin A is unique among ligands for peptidergic G protein-coupled receptors in being nonnitrogenous and lipid-like in character. To examine the molecular basis for the subtype-selective binding of salvinorin A, we utilized an integrated approach using chimeric opioid receptors, site-directed mutagenesis, the substituted cysteine accessibility method, and molecular modeling and dynamics studies. We discovered that helix 2 is required for salvinorin A binding to KOR and that two residues (Val-108(2.53) and Val-118(2.63)) confer subtype selectivity. Intriguingly, molecular modeling studies predicted that these loci exhibit an indirect effect on salvinorin A binding, presumably through rotation of helix 2. Significantly, and in agreement with our in silico predictions, substituted cysteine accessibility method analysis of helix 2 comparing KOR and the delta-opioid receptor, which has negligible affinity for salvinorin A, revealed that residues known to be important for salvinorin A binding exhibit a differential pattern of water accessibility. These findings imply that differences in the helical orientation of helix 2 are critical for the selectivity of salvinorin A binding to KOR and provide a structurally novel basis for ligand selectivity.

  10. Analysis of Drug Design for a Selection of G Protein-Coupled Neuro- Receptors Using Neural Network Techniques.

    PubMed

    Agerskov, Claus; Mortensen, Rasmus M; Bohr, Henrik G

    2015-01-01

    A study is presented on how well possible drug-molecules can be predicted with respect to their function and binding to a selection of neuro-receptors by the use of artificial neural networks. The ligands investigated in this study are chosen to be corresponding to the G protein-coupled receptors µ-opioid, serotonin 2B (5-HT2B) and metabotropic glutamate D5. They are selected due to the availability of pharmacological drug-molecule binding data for these receptors. Feedback and deep belief artificial neural network architectures (NNs) were chosen to perform the task of aiding drugdesign. This is done by training on structural features, selected using a "minimum redundancy, maximum relevance"-test, and testing for successful prediction of categorized binding strength. An extensive comparison of the neural network performances was made in order to select the optimal architecture. Deep belief networks, trained with greedy learning algorithms, showed superior performance in prediction over the simple feedback NNs. The best networks obtained scores of more than 90 % accuracy in predicting the degree of binding drug molecules to the mentioned receptors and with a maximal Matthew`s coefficient of 0.925. The performance of 8 category networks (8 output classes for binding strength) obtained a prediction accuracy of above 60 %. After training the networks, tests were done on how well the systems could be used as an aid in designing candidate drug molecules. Specifically, it was shown how a selection of chemical characteristics could give the lowest observed IC50 values, meaning largest bio-effect pr. nM substance, around 0.03-0.06 nM. These ligand characteristics could be total number of atoms, their types etc. In conclusion, deep belief networks trained on drug-molecule structures were demonstrated as powerful computational tools, able to aid in drug-design in a fast and cheap fashion, compared to conventional pharmacological techniques.

  11. Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors.

    PubMed

    Guo, Shuohan; Zhang, Xiaohan; Zheng, Mei; Zhang, Xiaowei; Min, Chengchun; Wang, Zengtao; Cheon, Seung Hoon; Oak, Min-Ho; Nah, Seung-Yeol; Kim, Kyeong-Man

    2015-10-01

    Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the β2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

  12. Commercially available antibodies directed against α-adrenergic receptor subtypes and other G protein-coupled receptors with acceptable selectivity in flow cytometry experiments.

    PubMed

    Tripathi, Abhishek; Gaponenko, Vadim; Majetschak, Matthias

    2016-02-01

    Several previous reports suggested that many commercially available antibodies directed against G protein-coupled receptors (GPCR) lack sufficient selectivity. Accordingly, it has been proposed that receptor antibodies should be validated by at least one of several criteria, such as testing tissues or cells after knockout or silencing of the corresponding gene. Here, we tested whether 12 commercially available antibodies directed against α-adrenergic receptor (AR) subtypes (α1A/B/D, α2A/B/C), atypical chemokine receptor 3 (ACKR3), and vasopressin receptor 1A (AVPR1A) suffice these criteria. We detected in flow cytometry experiments with human vascular smooth muscle cells that the fluorescence signals from each of these antibodies were reduced by 46 ± 10 %-91 ± 2 % in cells treated with commercially available small interfering RNA (siRNA) specific for each receptor, as compared with cells that were incubated with non-targeting siRNA. The tested antibodies included anti-ACKR3 (R&D Systems, mab42273), for which specificity has previously been demonstrated. Staining with this antibody resulted in 72 ± 5 % reduction of the fluorescence signal after ACKR3 siRNA treatment. Furthermore, staining with anti-α1A-AR (Santa Cruz, sc1477) and anti-ACKR3 (Abcam, ab38089), which have previously been reported to be non-specific, resulted in 70 ± 19 % and 80 ± 4 % loss of the fluorescence signal after α1A-AR and ACKR3 siRNA treatment, respectively. Our findings demonstrate that the tested antibodies show reasonable selectivity for their receptor target under our experimental conditions. Furthermore, our observations suggest that the selectivity of GPCR antibodies depends on the method for which the antibody is employed, the species from which cells/tissues are obtained, and on the type of specimens (cell, tissue/cell homogenate, or section) tested.

  13. A novel experimental strategy to assess the metabolic effects of selective activation of a G(q)-coupled receptor in hepatocytes in vivo.

    PubMed

    Li, Jian Hua; Jain, Shalini; McMillin, Sara M; Cui, Yinghong; Gautam, Dinesh; Sakamoto, Wataru; Lu, Huiyan; Jou, William; McGuinness, Owen P; Gavrilova, Oksana; Wess, Jürgen

    2013-10-01

    Increased hepatic glucose production is a key pathophysiological feature of type 2 diabetes. Like all other cell types, hepatocytes express many G protein-coupled receptors (GPCRs) that are linked to different functional classes of heterotrimeric G proteins. The important physiological functions mediated by G(s)-coupled hepatic glucagon receptors are well-documented. In contrast, little is known about the in vivo physiological roles of hepatocyte GPCRs that are linked to G proteins of the G(q) family. To address this issue, we established a transgenic mouse line (Hep-Rq mice) that expressed a G(q)-linked designer receptor (Rq) in a hepatocyte-selective fashion. Importantly, Rq could no longer bind endogenous ligands but could be selectively activated by a synthetic drug, clozapine-N-oxide. Clozapine-N-oxide treatment of Hep-Rq mice enabled us to determine the metabolic consequences caused by selective activation of a G(q)-coupled GPCR in hepatocytes in vivo. We found that acute Rq activation in vivo led to pronounced increases in blood glucose levels, resulting from increased rates of glycogen breakdown and gluconeogenesis. We also demonstrated that the expression of the V(1b) vasopressin receptor, a G(q)-coupled receptor expressed by hepatocytes, was drastically increased in livers of ob/ob mice, a mouse model of diabetes. Strikingly, treatment of ob/ob mice with a selective V(1b) receptor antagonist led to reduced glucose excursions in a pyruvate challenge test. Taken together, these findings underscore the importance of G(q)-coupled receptors in regulating hepatic glucose fluxes and suggest novel receptor targets for the treatment of type 2 diabetes.

  14. Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C: Pharmacological and biochemical properties

    SciTech Connect

    Buck, M.A.; Fraser, C.M. )

    1990-12-14

    The pharmacological and biochemical properties of rat m1 and m3 muscarinic acetylcholine receptors (mAChR) stably transfected into Chinese hamster ovary-K1 (CHO) cells were characterized with ligand binding, affinity labeling and biochemical assays. Both mAChR subtypes display saturable, high affinity binding of (3H)-quinuclidinyl benzilate (QNB) and a rank order of antagonist potency of QNB greater than atropine greater than pirenzepine greater than AF-DX 116. Carbachol displacement of (3H)-QNB binding to the m3 mAChR revealed an approximate 17-fold higher affinity than observed with the m1 mAChR. (3H)-propylbenzilylcholine mustard (PrBCM) labeling of mAChR revealed that m1 and m3 mAChR migrated on SDS-polyacrylamide gels with apparent molecular masses of 80,000 and 94,000 daltons, respectively, consistent with the known differences in their molecular sizes. Both m1 and m3 mAChR elicited dose-dependent increases in the hydrolysis of phosphoinositides; however, the maximal increase in total inositol phosphates elicited with the m1 mAChR was approximately 2-fold greater than that observed in cells expressing similar densities of m3 mAChR. Agonist activation of the m1 mAChR also elicited increases in basal and forskolin-stimulated cAMP, whereas the m3 mAChR had no effect on intracellular cAMP levels. These data suggest that although m1 and m3 mAChR display a considerable degree of structural homology, they exhibit distinct pharmacological and biochemical properties.

  15. Selective Glucocorticoid Receptor modulators.

    PubMed

    De Bosscher, Karolien

    2010-05-31

    The ancient two-faced Roman god Janus is often used as a metaphor to describe the characteristics of the Glucocorticoid Receptor (NR3C1), which exhibits both a beneficial side, that serves to halt inflammation, and a detrimental side responsible for undesirable effects. However, recent developments suggest that the Glucocorticoid Receptor has many more faces with the potential to express a range of different functionalities, depending on factors that include the tissue type, ligand type, receptor variants, cofactor surroundings and target gene promoters. This behavior of the receptor has made the development of safer ligands, that trigger the expression program of only a desirable subset of genes, a real challenge. Thus more knowledge-based fundamental research is needed to ensure the design and development of selective Glucocorticoid Receptor modulators capable of reaching the clinic. Recent advances in the characterization of novel selective Glucocorticoid Receptor modulators, specifically in the context of anti-inflammatory strategies, will be described in this review.

  16. Selective impairment of corticotropin-releasing factor1 (CRF1) receptor-mediated function using CRF coupled to saporin.

    PubMed

    Maciejewski-Lenoir, D; Heinrichs, S C; Liu, X J; Ling, N; Tucker, A; Xie, Q; Lappi, D A; Grigoriadis, D E

    2000-02-01

    CRF is the main component in the brain neuropeptide effector system responsible for the behavioral, endocrine, and physiological activation that accompanies stress activation. Reduced CRF system activation plays a role in the etiology of a variety of psychiatric and metabolic disease states. We have developed a novel protein conjugate that joins native rat/human CRF to a ribosome-inactivating protein, saporin (CRF-SAP), for the purpose of targeted inactivation of CRF receptor-expressing cells. Cytotoxicity measurements revealed that CRF-SAP (1-100 nM) produced concentration-dependent and progressive cell death over time in CRF1 receptor-transfected L cells, but at similar concentrations had no effect on CRF2alpha receptor-transfected cells. The CRF-SAP-induced toxicity in CRF1-transfected cells was prevented by coincubation with the competitive CRF1/CRF2 receptor peptide antagonist, [D-Phe12]CRF-(12-41), or the selective nonpeptide CRF1 receptor antagonist, NBI 27914. Finally, in cultured rat pituitary cells that express native CRF1 receptors, CRF-SAP suppressed CRF-induced (1 nM) ACTH release. GnRH (1-10 nM) stimulated LH release was also assessed in the same pituitary cultures. Although there was a slight decrease in LH release from these cultures, this decrease was observed with CRF-SAP or SAP alone, suggesting that the response was nonspecific. Taken together, these results suggest the utility of CRF-SAP as a specific and subtype-selective tool for long term impairment of CRF1 receptor-expressing cells.

  17. Coupling of the thrombin receptor to G12 may account for selective effects of thrombin on gene expression and DNA synthesis in 1321N1 astrocytoma cells.

    PubMed Central

    Post, G R; Collins, L R; Kennedy, E D; Moskowitz, S A; Aragay, A M; Goldstein, D; Brown, J H

    1996-01-01

    In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G protein-coupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2+, diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxin-insensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that G alpha 12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells. Images PMID:8930892

  18. The Selective Estrogen Receptor Modulator Raloxifene Regulates Arginine-Vasopressin Gene Expression in Human Female Neuroblastoma Cells Through G Protein-Coupled Estrogen Receptor and ERK Signaling.

    PubMed

    Grassi, Daniela; Ghorbanpoor, Samar; Acaz-Fonseca, Estefania; Ruiz-Palmero, Isabel; Garcia-Segura, Luis M

    2015-10-01

    The selective estrogen receptor modulator raloxifene reduces blood pressure in hypertensive postmenopausal women. In the present study we have explored whether raloxifene regulates gene expression of arginine vasopressin (AVP), which is involved in the pathogenesis of hypertension. The effect of raloxifene was assessed in human female SH-SY5Y neuroblastoma cells, which have been recently identified as a suitable cellular model to study the estrogenic regulation of AVP. Raloxifene, within a concentration ranging from 10(-10) M to 10(-6) M, decreased the mRNA levels of AVP in SH-SY5Y cells with maximal effect at 10(-7) M. This effect of raloxifene was imitated by an agonist (±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone of G protein-coupled estrogen receptor-1 (GPER) and blocked by an antagonist (3aS*,4R*,9bR*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline of GPER and by GPER silencing. Raloxifene induced a time-dependent increase in the level of phosphorylated ERK1 and ERK2, by a mechanism blocked by the GPER antagonist. The treatment of SH-SY5Y cells with either a MAPK/ERK kinase 1/2-specific inhibitor (1,4-diamino-2, 3-dicyano-1,4-bis(2-aminophenylthio)butadine) or a protein kinase C inhibitor (sotrastaurin) blocked the effects of raloxifene on the phosphorylation of ERK1/2 and the regulation of AVP mRNA levels. These results reveal a mechanism mediating the regulation of AVP expression by raloxifene, involving the activation of GPER, which in turn activates protein kinase C, MAPK/ERK kinase, and ERK. The regulation of AVP by raloxifene and GPER may have implications for the treatment of blood hypertension(.).

  19. Characteristics of muscarinic receptors that selectively couple to inhibition of adenylate cyclase or stimulation of phospholipase C on NG108-15 and 1321N1 cells

    SciTech Connect

    Liang, M.

    1988-01-01

    The purpose of this dissertation was to establish whether different muscarinic receptor proteins selectively couple to different second messenger response system. Although both second messenger response systems are fully functional in both cell lines, activation of muscarinic cholinergic receptors only results in inhibition of adenylate cyclase in NG108-15 neuroblastoma {times} glioma cells and stimulation of phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Muscarinic receptors on both cell types were covalently labeled with ({sup 3}H)Propylbenzilylcholine mustard (({sup 3}H)PBCM) and the mobilities of the ({sup 3}H)PBCM-labelled species of both cells were compared by SDS-PAGE. 1321N1 and NG108-15 cells each primarily expressed a single ({sup 3}H)PBCM-labelled species with an apparent size of approximately 92,000 and 66,000 Da, respectively. ({sup 3}H)PBCM labelling was completely inhibited by 1 {mu}M atropine or by down-regulation of muscarinic receptors by an overnight incubation with carbachol. The apparent size of the ({sup 3}H)PBCM-labelled species of both cell lines was not altered by treatment with a series of protease inhibitors or by treatment with dithiothreitol and iodoacetamide. Another approach for determining differences in the muscarinic receptors of 2 cells lines was to study agonist-induced alteration of muscarinic receptor number. Exposure of both cell types to agonists resulted in rapid loss of muscarinic receptors from cell surface without change of total cellular muscarinic receptors followed by subsequently loss of receptors from cells. Muscarinic receptors on both cell lines were regulated by agonist with similar properties.

  20. Coupling of HIV-1 Antigen to the Selective Autophagy Receptor SQSTM1/p62 Promotes T-Cell-Mediated Immunity

    PubMed Central

    Andersen, Aram Nikolai; Landsverk, Ole Jørgen; Simonsen, Anne; Bogen, Bjarne; Corthay, Alexandre; Øynebråten, Inger

    2016-01-01

    Vaccines aiming to promote T-cell-mediated immune responses have so far showed limited efficacy, and there is a need for novel strategies. Studies indicate that autophagy plays an inherent role in antigen processing and presentation for CD4+ and CD8+ T cells. Here, we report a novel vaccine strategy based on fusion of antigen to the selective autophagy receptor sequestosome 1 (SQSTM1)/p62. We hypothesized that redirection of vaccine antigen from proteasomal degradation into the autophagy pathway would increase the generation of antigen-specific T cells. A hybrid vaccine construct was designed in which the antigen is fused to the C-terminus of p62, a signaling hub, and a receptor that naturally delivers ubiquitinated cargo for autophagic degradation. Fusion of the human immunodeficiency virus-1 antigen Gagp24 to p62 resulted in efficient antigen delivery into the autophagy pathway. Intradermal immunization of mice revealed that, in comparison to Gagp24 delivered alone, fusion to p62 enhanced the number of Gagp24-specific interferon-γ-producing T cells, including CD8+ T cells. The strategy may also have the potential to modulate the antigenic peptide repertoire. Because p62 and autophagy are highly conserved between species, we anticipate this strategy to be a candidate for the development of T-cell-based vaccines in humans. PMID:27242780

  1. Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling.

    PubMed

    Semack, Ansley; Sandhu, Manbir; Malik, Rabia U; Vaidehi, Nagarajan; Sivaramakrishnan, Sivaraj

    2016-08-19

    Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the β2-adrenergic receptor (β2-AR)-Gαs and V1A receptor (V1AR)-Gαq The Gαs and Gαq peptides adopt different orientations in β2-AR and V1AR, respectively. The β2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with β2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions.

  2. Selective coupling of the S1P3 receptor subtype to S1P-mediated RhoA activation and cardioprotection.

    PubMed

    Yung, Bryan S; Brand, Cameron S; Xiang, Sunny Y; Gray, Charles B B; Means, Christopher K; Rosen, Hugh; Chun, Jerold; Purcell, Nicole H; Brown, Joan Heller; Miyamoto, Shigeki

    2017-02-01

    Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P1, S1P2, and S1P3 receptor subtypes to affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to Gq. We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα13 but not Gα12, Gαq, or Gαi. Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P3 in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P3 knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P3 KO mouse heart. CYM-51736, an S1P3-specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P3 receptor- and Gα13-mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P3 receptors could provide therapeutic benefits in ischemic heart disease.

  3. Structural Mapping and Functional Characterization of Zebrafish Class B G-Protein Coupled Receptor (GPCR) with Dual Ligand Selectivity towards GLP-1 and Glucagon

    PubMed Central

    Oren, Deena A.; Wei, Yang; Skrabanek, Luce; Chow, Billy K. C.; Mommsen, Thomas; Mojsov, Svetlana

    2016-01-01

    GLP-1 and glucagon regulate glucose metabolism through a network of metabolic pathways initiated upon binding to their specific receptors that belong to class B G-protein coupled receptors (GPCRs). The therapeutic potential of glucagon is currently being evaluated, while GLP-1 is already used in the treatment of type 2 diabetes and obesity. Development of a second generation of GLP-1 based therapeutics depends on a molecular and structural understanding of the interactions between the GLP-1 receptor (GLP-1R) and its ligand GLP-1. There is considerable sequence conservation between GLP-1 and glucagon and between the hGLP-1R and human glucagon receptor (hGCGR), yet each receptor recognizes only its own specific ligand. Glucagon receptors in fish and frogs also exhibit ligand selectivity only towards glucagon and not GLP-1. Based on competitive binding experiments and assays of increase in intracellular cAMP, we demonstrate here that a GPCR in zebrafish (Danio rerio) exhibits dual ligand selectivity towards GLP-1 and glucagon, a characteristic not found in mammals. Further, many structural features found in hGLP-1R and hGCGR are also found in this zebrafish GPCR (zfGPCR). We show this by mapping of its sequence and structural features onto the hGLP-1R and hGCGR based on their partial and complementary crystal structures. Thus, we propose that zfGPCR represents a dual GLP-1R/GCGR. The main differences between the three receptors are in their stalk regions that connect their N-terminal extracellular domains (NECDs) with their transmembrane domains and the absence of loop 3 in the NECD in zfGLP-1R/GCGR. These observations suggest that the interactions between GLP-1 and glucagon with loop 3 and the stalk regions may induce different conformational changes in hGLP-1R and hGCGR upon ligand binding and activation that lead to selective recognition of their native ligands. PMID:27930690

  4. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  5. Selective Estrogen Receptor Modulators

    PubMed Central

    2016-01-01

    Selective estrogen receptor modulators (SERMs) are now being used as a treatment for breast cancer, osteoporosis and postmenopausal symptoms, as these drugs have features that can act as an estrogen agonist and an antagonist, depending on the target tissue. After tamoxifen, raloxifene, lasofoxifene and bazedoxifene SERMs have been developed and used for treatment. The clinically decisive difference among these drugs (i.e., the key difference) is their endometrial safety. Compared to bisphosphonate drug formulations for osteoporosis, SERMs are to be used primarily in postmenopausal women of younger age and are particularly recommended if there is a family history of invasive breast cancer, as their use greatly reduces the incidence of this type of cancer in women. Among the above mentioned SERMs, raloxifene has been widely used in prevention and treatment of postmenopausal osteoporosis and vertebral compression fractures, and clinical studies are now underway to test the comparative advantages of raloxifene with those of bazedoxifene, a more recently developed SERM. Research on a number of adverse side effects of SERM agents is being performed to determine the long-term safety of this class of compouds for treatment of osteoporosis. PMID:27559463

  6. A cell-based time-resolved fluorescence assay for selection of antibody reagents for G protein-coupled receptor immunohistochemistry.

    PubMed

    Su, Jui-Lan; Fornwald, Jim; Rivers, Philip; Goldsworthy, Susan; Looney, Noeleen A; Hanvey, Jeff; Plumpton, Chris; Parham, Janet; Romanos, Michael; Kost, Thomas A; Kull, Frederick C

    2004-08-01

    A cell-based time-resolved fluorescence (celTRF) immunoassay is described for pre-screening antibodies to G protein-coupled receptor (GPCR) peptides that predicts suitability for immunohistochemistry (IHC). Rat GPCRs were expressed in Saos-2 human osteosarcoma cells via recombinant baculoviruses designed for mammalian cell expression, i.e., the transduced cells were used as a "screening lawn". The lawn was fixed and permeabilized similarly to IHC tissue. The celTRF, a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), employed Eu-labelled goat anti-rabbit IgG. It exhibited a broad dynamic range upon which enzyme-linked immunosorbant assay (ELISA)-positive affinity-purified anti-peptide antibody reagents were examined for specificity and potency. Over 150 anti-peptide reagents to 27 GPCRs were characterized. All celTRF-positive antibodies were found to be suitable for IHC, whereas ELISA alone did not predict IHC utility. Examples are illustrated with five rabbit anti-neuropeptide FF receptor 1 (NPFF1) antibodies, where a strong correlation between celTRF potency and IHC utility was observed in both applications. In contrast, two high anti-peptide ELISA titer but celTRF-negative antibodies failed to recognize the NPFF1 receptor in IHC. The celTRF assay was performed manually and in an automated fashion, in our case, using a Biomek FX station and Sami scheduling software. The celTRF is the first in vitro automated assay that offers confident pre-selection of antibodies for IHC and the versatility to accommodate the rapid screening of large numbers of GPCRs. The celTRF is readily applicable to other protein target classes.

  7. A Miniaturized Screen of a Schistosoma mansoni Serotonergic G Protein-Coupled Receptor Identifies Novel Classes of Parasite-Selective Inhibitors

    PubMed Central

    Chan, John D.; McCorvy, John D.; Acharya, Sreemoyee; Day, Timothy A.; Roth, Bryan L.; Marchant, Jonathan S.

    2016-01-01

    Schistosomiasis is a tropical parasitic disease afflicting ~200 million people worldwide and current therapy depends on a single drug (praziquantel) which exhibits several non-optimal features. These shortcomings underpin the need for next generation anthelmintics, but the process of validating physiologically relevant targets (‘target selection’) and pharmacologically profiling them is challenging. Remarkably, even though over a quarter of current human therapeutics target rhodopsin-like G protein coupled receptors (GPCRs), no library screen of a flatworm GPCR has yet been reported. Here, we have pharmacologically profiled a schistosome serotonergic GPCR (Sm.5HTR) implicated as a downstream modulator of PZQ efficacy, in a miniaturized screening assay compatible with high content screening. This approach employs a split luciferase based biosensor sensitive to cellular cAMP levels that resolves the proximal kinetics of GPCR modulation in intact cells. Data evidence a divergent pharmacological signature between the parasitic serotonergic receptor and the closest human GPCR homolog (Hs.5HTR7), supporting the feasibility of optimizing parasitic selective pharmacophores. New ligands, and chemical series, with potency and selectivity for Sm.5HTR over Hs.5HTR7 are identified in vitro and validated for in vivo efficacy against schistosomules and adult worms. Sm.5HTR also displayed a property resembling irreversible inactivation, a phenomenon discovered at Hs.5HTR7, which enhances the appeal of this abundantly expressed parasite GPCR as a target for anthelmintic ligand design. Overall, these data underscore the feasibility of profiling flatworm GPCRs in a high throughput screening format competent to resolve different classes of GPCR modulators. Further, these data underscore the promise of Sm.5HTR as a chemotherapeutically vulnerable node for development of next generation anthelmintics. PMID:27187180

  8. A combined ligand-based and target-based drug design approach for G-protein coupled receptors: application to salvinorin A, a selective kappa opioid receptor agonist

    NASA Astrophysics Data System (ADS)

    Singh, Nidhi; Chevé, Gwénaël; Ferguson, David M.; McCurdy, Christopher R.

    2006-08-01

    Combined ligand-based and target-based drug design approaches provide a synergistic advantage over either method individually. Therefore, we set out to develop a powerful virtual screening model to identify novel molecular scaffolds as potential leads for the human KOP (hKOP) receptor employing a combined approach. Utilizing a set of recently reported derivatives of salvinorin A, a structurally unique KOP receptor agonist, a pharmacophore model was developed that consisted of two hydrogen bond acceptor and three hydrophobic features. The model was cross-validated by randomizing the data using the CatScramble technique. Further validation was carried out using a test set that performed well in classifying active and inactive molecules correctly. Simultaneously, a bovine rhodopsin based "agonist-bound" hKOP receptor model was also generated. The model provided more accurate information about the putative binding site of salvinorin A based ligands. Several protein structure-checking programs were used to validate the model. In addition, this model was in agreement with the mutation experiments carried out on KOP receptor. The predictive ability of the model was evaluated by docking a set of known KOP receptor agonists into the active site of this model. The docked scores correlated reasonably well with experimental p K i values. It is hypothesized that the integration of these two independently generated models would enable a swift and reliable identification of new lead compounds that could reduce time and cost of hit finding within the drug discovery and development process, particularly in the case of GPCRs.

  9. Multiple mechanisms of GW-9508, a selective G protein-coupled receptor 40 agonist, in the regulation of glucose homeostasis and insulin sensitivity.

    PubMed

    Ou, Horng-Yih; Wu, Hung-Tsung; Hung, Hao-Chang; Yang, Yi-Ching; Wu, Jin-Shang; Chang, Chih-Jen

    2013-03-15

    Activation of G protein-coupled receptor 40 (GPR40) by agonists increases insulin release in isolated islets, whereas it is inconclusive whether GPR40 antagonists decrease blood glucose and increase insulin sensitivity. Although some clinical trials indicated that administration of a GPR40 agonist shows benefits in the regulation of blood glucose homeostasis, the pharmacological mechanisms of this receptor in the improvement of glycemic control remain unclear. Therefore, we used a selective GPR40 agonist, GW-9508, to clarify the role of GPR40 in the regulation of blood glucose. Bolus intraperitoneal injection of GW-9508 in mice showed a slight decrease in blood glucose, with an increase in plasma insulin levels under glucose stimuli. However, long-term treatment with low doses of GW-9508 in high-fat diet-induced (HFD) diabetic mice decreased blood glucose with decreased plasma insulin significantly and improved glucose intolerance and insulin resistance. Using small interfering ribonucleic acid to delete GPR40 in HepG2 cells, we demonstrated that GW-9508 reversed palmitate-induced insulin signaling impairment through a GPR40-dependent pathway. We also found that GW-9508 activates the Akt/GSK-3β pathway to increase glycogen levels in HepG2 cells. Furthermore, administration of GW-9508 decreased the hepatic expression of fetuin-A in HFD mice significantly and regulated high-glucose- or palmitate-induced fetuin-A expression to increase insulin sensitivity through a GPR40/PLC/PKC pathway in HepG2 cells. Taken together, GW-9508 exerts a partial agonist effect to regulate blood glucose through multiple mechanisms. Investigation of chemicals that act on GPR40 might be a new strategy for the treatment of diabetes.

  10. G Protein-Coupled Receptor Biased Agonism

    PubMed Central

    Hodavance, Sima Y.; Gareri, Clarice; Torok, Rachel D.; Rockman, Howard A.

    2016-01-01

    G protein-coupled receptors (GPCR) are the largest family of targets for current therapeutics. The classic model of their activation was binary, where agonist binding induced an active conformation and subsequent downstream signaling. Subsequently, the revised concept of biased agonism emerged, where different ligands at the same GPCR selectively activate one downstream pathway versus another. Advances in understanding the mechanism of biased agonism has led to the development of novel ligands, which have the potential for improved therapeutic and safety profiles. In this review, we summarize the theory and most recent breakthroughs in understanding biased signaling, examine recent laboratory investigations concerning biased ligands across different organ systems, and discuss the promising clinical applications of biased agonism. PMID:26751266

  11. Autophagy selectivity through receptor clustering

    NASA Astrophysics Data System (ADS)

    Rutenberg, Andrew; Brown, Aidan

    Substrate selectivity in autophagy requires an all-or-none cellular response. We focus on peroxisomes, for which autophagy receptor proteins NBR1 and p62 are well characterized. Using computational models, we explore the hypothesis that physical clustering of autophagy receptor proteins on the peroxisome surface provides an appropriate all-or-none response. We find that larger peroxisomes nucleate NBR1 clusters first, and lose them due to competitive coarsening last, resulting in significant size-selectivity. We then consider a secondary hypothesis that p62 inhibits NBR1 cluster formation. We find that p62 inhibition enhances size-selectivity enough that, even if there is no change of the pexophagy rate, the volume of remaining peroxisomes can significantly decrease. We find that enhanced ubiquitin levels suppress size-selectivity, and that this effect is more pronounced for individual peroxisomes. Sufficient ubiquitin allows receptor clusters to form on even the smallest peroxisomes. We conclude that NBR1 cluster formation provides a viable physical mechanism for all-or-none substrate selectivity in pexophagy. We predict that cluster formation is associated with significant size-selectivity. Now at Simon Fraser University.

  12. FTY720 Phosphate Activates Sphingosine-1-Phosphate Receptor 2 and Selectively Couples to Gα12/13/Rho/ROCK to Induce Myofibroblast Contraction.

    PubMed

    Sobel, Katrin; Monnier, Lucile; Menyhart, Katalin; Bolinger, Matthias; Studer, Rolf; Nayler, Oliver; Gatfield, John

    2015-06-01

    FTY720 phosphate (FTY720-P; 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol, monodihydrogen phosphate ester) is a nonselective sphingosine-1-phosphate (S1P) receptor agonist thought to be devoid of activity at the S1P2 receptor subtype. However, we have recently shown that FTY720-P displays significant S1P2 receptor agonist activity in recombinant cells and fibroblasts expressing endogenous S1P2 receptors. To elucidate the S1P2-dependent signaling pathways that were activated by FTY720-P, we employed second messenger assays and impedance-based assays in combination with pharmacological and small interfering RNA-based pathway inhibition in recombinant Chinese hamster ovary (CHO)-S1P2 cells as well as human lung myofibroblasts generated in vitro. In CHO-S1P2 cells, FTY720-P did not modulate cAMP or calcium levels. However, reporter-gene assays, impedance-based assays with a selective Rho-associated kinase (ROCK) inhibitor, Gα12/13 knockdown and activated Rho-pull-down assays demonstrated that FTY720-P potently activated Gα12/13/Rho/ROCK signaling. S1P similarly activated Gα12/13/Rho/ROCK signaling via S1P2 receptors, whereas the two selective S1P1 receptor agonists (Z,Z)-5-(3-chloro-4-[(2R)-2,3-dihydroxy-propoxy]-benzylidene)-2-propylimino-3-o-tolyl-thiazolidin-4-one (ponesimond) and 5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl]1,2,4-oxadiazole (SEW2871) were inactive. In lung myofibroblasts, which mainly expressed the S1P2 receptor subtype, we showed that FTY720-P selectively activated the Gα12/13/Rho/ROCK pathway via the S1P2 receptor. Moreover, the activation of the Gα12/13/Rho/ROCK pathway in myofibroblasts by FTY720-P caused potent myofibroblast contraction similar to that induced by the natural ligand S1P. Thus, complementing second messenger assays with unbiased label-free assays or phenotypic assays in native expression systems can uncover activation of additional pathways, such as Gα12/13/Rho/ROCK signaling.

  13. G Protein-Coupled Receptors in Cancer

    PubMed Central

    Bar-Shavit, Rachel; Maoz, Myriam; Kancharla, Arun; Nag, Jeetendra Kumar; Agranovich, Daniel; Grisaru-Granovsky, Sorina; Uziely, Beatrice

    2016-01-01

    Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of “cancer driver” GPCRs. Emerging data on GPCR biology point to functional selectivity and “biased agonism”; hence, there is a diminishing enthusiasm for the concept of “one drug per GPCR target” and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics. PMID:27529230

  14. G Protein-Coupled Receptors in Cancer.

    PubMed

    Bar-Shavit, Rachel; Maoz, Myriam; Kancharla, Arun; Nag, Jeetendra Kumar; Agranovich, Daniel; Grisaru-Granovsky, Sorina; Uziely, Beatrice

    2016-08-12

    Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of "cancer driver" GPCRs. Emerging data on GPCR biology point to functional selectivity and "biased agonism"; hence, there is a diminishing enthusiasm for the concept of "one drug per GPCR target" and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics.

  15. The use of receptor-specific antibodies to study G-protein-coupled receptors.

    PubMed

    Gupta, Achla; Devi, Lakshmi A

    2006-07-01

    The identification of G-protein-coupled receptor (GPCR) cDNAs has facilitated a number of studies characterizing the biochemical properties of the receptor protein. Most of these studies have used antibodies directed against the epitope-tagged receptor expressed in heterologous cells, because of the lack of sensitive and selective antibodies capable of recognizing endogenous receptors in their native state. In order to facilitate studies with endogenous receptors, efforts have been made to generate receptor-type selective, sensitive antibodies that are able to recognize endogenous receptors. In this review, we discuss the strategies as well as the details of the techniques used for the generation of monoclonal and polyclonal antibodies with a focus on family A GPCRs.

  16. G Protein–Coupled Receptor Heteromers

    PubMed Central

    Gomes, Ivone; Ayoub, Mohammed Akli; Fujita, Wakako; Jaeger, Werner C.; Pfleger, Kevin D.G.; Devi, Lakshmi A.

    2016-01-01

    G protein–coupled receptors (GPCRs) compose one of the largest families of membrane proteins involved in intracellular signaling. They are involved in numerous physiological and pathological processes and are prime candidates for drug development. Over the past decade, an increasing number of studies have reported heteromerization between GPCRs. Many investigations in heterologous systems have provided important indications of potential novel pharmacology; however, the physiological relevance of these findings has yet to be established with endogenous receptors in native tissues. In this review, we focus on family A GPCRs and describe the techniques and criteria to assess their heteromerization. We conclude that advances in approaches to study receptor complex functionality in heterologous systems, coupled with techniques that enable specific examination of native receptor heteromers in vivo, are likely to establish GPCR heteromers as novel therapeutic targets. PMID:26514203

  17. G protein-coupled receptor mutations and human genetic disease.

    PubMed

    Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

    2014-01-01

    Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

  18. [G-protein-coupled receptors targeting: the allosteric approach].

    PubMed

    Sebag, Julien A; Pantel, Jacques

    2012-10-01

    G-protein-coupled receptors (GPCR) are a major family of drug targets. Essentially all drugs targeting these receptors on the market compete with the endogenous ligand (agonists or antagonists) for binding the receptor. Recently, non-competitive compounds binding to distinct sites from the cognate ligand were documented in various classes of these receptors. These compounds, called allosteric modulators, generally endowed of a better selectivity are able to modulate specifically the endogenous signaling of the receptor. To better understand the promising potential of this class of GPCRs targeting compounds, this review highlights the properties of allosteric modulators, the strategies used to identify them and the challenges associated with the development of these compounds.

  19. G protein-coupled receptors as promising cancer targets.

    PubMed

    Liu, Ying; An, Su; Ward, Richard; Yang, Yang; Guo, Xiao-Xi; Li, Wei; Xu, Tian-Rui

    2016-07-01

    G protein-coupled receptors (GPCRs) regulate an array of fundamental biological processes, such as growth, metabolism and homeostasis. Specifically, GPCRs are involved in cancer initiation and progression. However, compared with the involvement of the epidermal growth factor receptor in cancer, that of GPCRs have been largely ignored. Recent findings have implicated many GPCRs in tumorigenesis, tumor progression, invasion and metastasis. Moreover, GPCRs contribute to the establishment and maintenance of a microenvironment which is permissive for tumor formation and growth, including effects upon surrounding blood vessels, signaling molecules and the extracellular matrix. Thus, GPCRs are considered to be among the most useful drug targets against many solid cancers. Development of selective ligands targeting GPCRs may provide novel and effective treatment strategies against cancer and some anticancer compounds are now in clinical trials. Here, we focus on tumor related GPCRs, such as G protein-coupled receptor 30, the lysophosphatidic acid receptor, angiotensin receptors 1 and 2, the sphingosine 1-phosphate receptors and gastrin releasing peptide receptor. We also summarize their tissue distributions, activation and roles in tumorigenesis and discuss the potential use of GPCR agonists and antagonists in cancer therapy.

  20. Oligomeric forms of G protein-coupled receptors (GPCRs)

    PubMed Central

    Palczewski, Krzysztof

    2010-01-01

    Oligomerization is a general characteristic of cell membrane receptors that is shared by G protein-coupled receptors (GPCRs) together with their G protein partners. Recent studies of these complexes, both in vivo and in purified reconstituted forms, unequivocally support this contention for GPCRs, perhaps with only rare exceptions. As evidence has evolved from experimental cell lines to more relevant in vivo studies and from indirect biophysical approaches to well defined isolated complexes of dimeric receptors alone and complexed with G proteins, there is an expectation that the structural basis of oligomerization and the functional consequences for membrane signaling will be elucidated. Oligomerization of cell membrane receptors is fully supported by both thermodynamic calculations and the selectivity and duration of signaling required to reach targets located in various cellular compartments. PMID:20538466

  1. Modification on ursodeoxycholic acid (UDCA) scaffold. discovery of bile acid derivatives as selective agonists of cell-surface G-protein coupled bile acid receptor 1 (GP-BAR1).

    PubMed

    Sepe, Valentina; Renga, Barbara; Festa, Carmen; D'Amore, Claudio; Masullo, Dario; Cipriani, Sabrina; Di Leva, Francesco Saverio; Monti, Maria Chiara; Novellino, Ettore; Limongelli, Vittorio; Zampella, Angela; Fiorucci, Stefano

    2014-09-25

    Bile acids are signaling molecules interacting with the nuclear receptor FXR and the G-protein coupled receptor 1 (GP-BAR1/TGR5). GP-BAR1 is a promising pharmacological target for the treatment of steatohepatitis, type 2 diabetes, and obesity. Endogenous bile acids and currently available semisynthetic bile acids are poorly selective toward GP-BAR1 and FXR. Thus, in the present study we have investigated around the structure of UDCA, a clinically used bile acid devoid of FXR agonist activity, to develop a large family of side chain modified 3α,7β-dihydroxyl cholanoids that selectively activate GP-BAR1. In vivo and in vitro pharmacological evaluation demonstrated that administration of compound 16 selectively increases the expression of pro-glucagon 1, a GP-BAR1 target, in the small intestine, while it had no effect on FXR target genes in the liver. Further, compound 16 results in a significant reshaping of bile acid pool in a rodent model of cholestasis. These data demonstrate that UDCA is a useful scaffold to generate novel and selective steroidal ligands for GP-BAR1.

  2. Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

    PubMed Central

    Zhang, Xiaohan; Kim, Kyeong-Man

    2017-01-01

    Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with β-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis. PMID:28035080

  3. G-protein-coupled receptors and melanoma.

    PubMed

    Lee, Hwa Jin; Wall, Brian; Chen, Suzie

    2008-08-01

    G-protein-coupled receptors (GPCR) are the largest family of receptors with over 500 members. Evaluation of GPCR gene expression in primary human tumors identified over-expression of GPCR in several tumor types. Analysis of cancer samples in different disease stages also suggests that some GPCR may be involved in early tumor progression and others may play a critical role in tumor invasion and metastasis. Currently, >50% of drug targets to various human diseases are based on GPCR. In this review, the relationships between several GPCR and melanoma development and/or progression will be discussed. Finally, the possibility of using one or more of these GPCR as therapeutic targets in melanoma will be summarized.

  4. G-protein-coupled receptors and cancer.

    PubMed

    Dorsam, Robert T; Gutkind, J Silvio

    2007-02-01

    G-protein-coupled receptors (GPCRs), the largest family of cell-surface molecules involved in signal transmission, have recently emerged as crucial players in tumour growth and metastasis. Malignant cells often hijack the normal physiological functions of GPCRs to survive, proliferate autonomously, evade the immune system, increase their blood supply, invade their surrounding tissues and disseminate to other organs. This Review will address our current understanding of the many roles of GPCRs and their signalling circuitry in tumour progression and metastasis. We will also discuss how interfering with GPCRs might provide unique opportunities for cancer prevention and treatment.

  5. Quantifying agonist activity at G protein-coupled receptors.

    PubMed

    Ehlert, Frederick J; Suga, Hinako; Griffin, Michael T

    2011-12-26

    When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors. Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (K(b)) is much greater than that for the inactive state (K(a)). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (K(obs)), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε) (Figure 2). Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the K(obs) and relative efficacy of an agonist. In this report, we show how to modify this analysis to estimate the agonist K(b) value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate K(b) in absolute units of M(-1). Our method of analyzing agonist concentration-response curves consists of global nonlinear regression using the operational model. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of K(obs) and a parameter proportional to efficacy (

  6. Crystallization of G Protein-Coupled Receptors

    PubMed Central

    Salom, David; Padayatti, Pius S.; Palczewski, Krzysztof

    2015-01-01

    Oligomerization is one of several mechanisms that can regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallography and NMR are the only methods able to reveal the details of receptor–receptor interactions at an atomic level, and several GPCR homodimers already have been described from crystal structures. Two clusters of symmetric interfaces have been identified from these structures that concur with biochemical data, one involving helices I, II, and VIII and the other formed mainly by helices V and VI. In this chapter, we describe the protocols used in our laboratory for the crystallization of rhodopsin and the β2-adrenergic receptor (β2-AR). For bovine rhodopsin, we developed a new purification strategy including a (NH4)2SO4-induced phase separation that proved essential to obtain crystals of photoactivated rhodopsin containing parallel dimers. Crystallization of native bovine rhodopsin was achieved by the classic vapor-diffusion technique. For β2-AR, we developed a purification strategy based on previously published protocols employing a lipidic cubic phase to obtain diffracting crystals of a β2-AR/T4-lysozyme chimera bound to the antagonist carazolol. PMID:24143992

  7. Velocity selection in coupled-map lattices

    NASA Astrophysics Data System (ADS)

    Parekh, Nita; Puri, Sanjay

    1993-02-01

    We investigate the phenomenon of velocity selection for traveling wave fronts in a class of coupled-map lattices, derived by discretizations of the Fisher equation [Ann. Eugenics 7, 355 (1937)]. We find that the velocity selection can be understood in terms of a discrete analog of the marginal-stability hypothesis. A perturbative approach also enables us to estimate the selected velocity accurately for small values of the discretization mesh sizes.

  8. G-protein-coupled receptors: past, present and future

    PubMed Central

    Hill, Stephen J

    2006-01-01

    The G-protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Drugs active at these receptors have therapeutic actions across a wide range of human diseases ranging from allergic rhinitis to pain, hypertension and schizophrenia. This review provides a brief historical overview of the properties and signalling characteristics of this important family of receptors. PMID:16402114

  9. Covalent agonists for studying G protein-coupled receptor activation

    PubMed Central

    Weichert, Dietmar; Kruse, Andrew C.; Manglik, Aashish; Hiller, Christine; Zhang, Cheng; Hübner, Harald; Kobilka, Brian K.; Gmeiner, Peter

    2014-01-01

    Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis. PMID:25006259

  10. Model Organisms in G Protein–Coupled Receptor Research

    PubMed Central

    Barr, Maureen M.; Bruchas, Michael R.; Ewer, John; Griffith, Leslie C.; Maiellaro, Isabella; Taghert, Paul H.; White, Benjamin H.

    2015-01-01

    The study of G protein–coupled receptors (GPCRs) has benefited greatly from experimental approaches that interrogate their functions in controlled, artificial environments. Working in vitro, GPCR receptorologists discovered the basic biologic mechanisms by which GPCRs operate, including their eponymous capacity to couple to G proteins; their molecular makeup, including the famed serpentine transmembrane unit; and ultimately, their three-dimensional structure. Although the insights gained from working outside the native environments of GPCRs have allowed for the collection of low-noise data, such approaches cannot directly address a receptor’s native (in vivo) functions. An in vivo approach can complement the rigor of in vitro approaches: as studied in model organisms, it imposes physiologic constraints on receptor action and thus allows investigators to deduce the most salient features of receptor function. Here, we briefly discuss specific examples in which model organisms have successfully contributed to the elucidation of signals controlled through GPCRs and other surface receptor systems. We list recent examples that have served either in the initial discovery of GPCR signaling concepts or in their fuller definition. Furthermore, we selectively highlight experimental advantages, shortcomings, and tools of each model organism. PMID:25979002

  11. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by XMetA, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  12. Structure and Function of Serotonin G protein Coupled Receptors

    PubMed Central

    McCorvy, John D.; Roth, Bryan L.

    2015-01-01

    Serotonin receptors are prevalent throughout the nervous system and the periphery, and remain one of the most lucrative and promising drug discovery targets for disorders ranging from migraine headaches to neuropsychiatric disorders such as schizophrenia and depression. There are 14 distinct serotonin receptors, of which 13 are G protein coupled receptors (GPCRs), which are targets for approximately 40% of the approved medicines. Recent crystallographic and biochemical evidence has provided a converging understanding of the basic structure and functional mechanics of GPCR activation. Currently, two GPCR crystal structures exist for the serotonin family, the 5-HT1B and 5-HT2B receptor, with the antimigraine and valvulopathic drug ergotamine bound. The first serotonin crystal structures not only provide the first evidence of serotonin receptor topography but also provide mechanistic explanations into functional selectivity or biased agonism. This review will detail the findings of these crystal structures from a molecular and mutagenesis perspective for driving rational drug design for novel therapeutics incorporating biased signaling. PMID:25601315

  13. Subtype Differences in Pre-Coupling of Muscarinic Acetylcholine Receptors

    PubMed Central

    Jakubík, Jan; Janíčková, Helena; Randáková, Alena; El-Fakahany, Esam E.; Doležal, Vladimír

    2011-01-01

    Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex “collides” with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to Gq/11, while even-numbered receptors prefer coupling to Gi/o. We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [3H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M1 and M3 receptors also pre-couple with non-preferential Gi/o G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype

  14. Transmission coupling mechanisms: cultural group selection

    PubMed Central

    Boyd, Robert; Richerson, Peter J.

    2010-01-01

    The application of phylogenetic methods to cultural variation raises questions about how cultural adaption works and how it is coupled to cultural transmission. Cultural group selection is of particular interest in this context because it depends on the same kinds of mechanisms that lead to tree-like patterns of cultural variation. Here, we review ideas about cultural group selection relevant to cultural phylogenetics. We discuss why group selection among multiple equilibria is not subject to the usual criticisms directed at group selection, why multiple equilibria are a common phenomena, and why selection among multiple equilibria is not likely to be an important force in genetic evolution. We also discuss three forms of group competition and the processes that cause populations to shift from one equilibrium to another and create a mutation-like process at the group level. PMID:21041204

  15. Selectively targeting estrogen receptors for cancer treatment

    PubMed Central

    Shanle, Erin K.; Xu, Wei

    2010-01-01

    Estrogens regulate growth and development through the action of two distinct estrogen receptors (ERs), ERα and ERβ, which mediate proliferation and differentiation of cells. For decades, ERα mediated estrogen signaling has been therapeutically targeted to treat breast cancer, most notably with the selective estrogen receptor modulator (SERM) tamoxifen. Selectively targeting ERs occurs at two levels: tissue selectivity and receptor subtype selectivity. SERMs have been developed with emphasis on tissue selectivity to target ER signaling for breast cancer treatment. Additionally, new approaches to selectively target the action of ERα going beyond ligand-dependent activity are under current investigation. As evidence of the anti-proliferative role of ERβ accumulates, selectively targeting ERβ is an attractive approach for designing new cancer therapies with the emphasis shifted to designing ligands with subtype selectivity. This review will present the mechanistic and structural features of ERs that determine tissue and subtype selectivity with an emphasis on current approaches to selectively target ERα and ERβ for cancer treatment. PMID:20708050

  16. Generation, use, and validation of receptor-selective antibodies.

    PubMed

    Mackrill, John J

    2004-01-01

    Antibodies have proved invaluable in the study of G-protein-coupled receptors (GPCRs). The utility of these immunoglobulin probes for investigation of protein structures and functions arises from their selectivity as well as their versatility. Antibodies can be used to analyze GPCR size, abundance, distribution, turnover, modification, interaction with other proteins, and functional properties. In this chapter, techniques for the generation and characterization of receptor-selective antibodies are described. Two protocols are given for the generation of antibodies: (1) development of polyclonal antibodies (PAbs) against synthetic peptides corresponding to a specific site within a GPCR and (2) selection of synthetic single-chain fragment variable (scFv) monoclonal antibodies (MAbs) from libraries expressed on the surface of bacteriophage. Immunoblot and enzyme-linked immunosorbent assays for characterization of the selectivity and affinity of such antibodies are described. Finally, methods are given for improvement of the titer and specificity of PAbs.

  17. Production of a bioengineered G-protein coupled receptor of human formyl peptide receptor 3.

    PubMed

    Wang, Xiaoqiang; Zhang, Shuguang

    2011-01-01

    G-protein coupled receptors (GPCRs) participate in a wide range of vital regulations of our physiological actions. They are also of pharmaceutical importance and have become many therapeutic targets for a number of disorders and diseases. Purified GPCR-based approaches including structural study and novel biophysical and biochemical function analyses are increasingly being used in GPCR-directed drug discovery. Before these approaches become routine, however, several hurdles need to be overcome; they include overexpression, solubilization, and purification of large quantities of functional and stable receptors on a regular basis. Here we report milligram production of a human formyl peptide receptor 3 (FPR3). FPR3 comprises a functionally distinct GPCR subfamily that is involved in leukocyte chemotaxis and activation. The bioengineered FPR3 was overexpressed in stable tetracycline-inducible mammalian cell lines (HEK293S). After a systematic detergent screening, fos-choline-14 (FC-14) was selected for subsequent solubilization and purification processes. A two-step purification method, immunoaffinity using anti-rho-tag monoclonal antibody 1D4 and gel filtration, was used to purify the receptors to near homogeneity. Immunofluorescence analysis showed that expressed FPR3 was predominantly displayed on cellular membrane. Secondary structural analysis using circular dichroism showed that the purified FPR3 receptor was correctly folded with >50% α-helix, which is similar to other known GPCR secondary structures. Our method can readily produce milligram quantities of human FPR3, which would facilitate in developing human FPR as therapeutic drug targets.

  18. Antibodies to probe endogenous G protein-coupled receptor heteromer expression, regulation, and function

    PubMed Central

    Gomes, Ivone; Gupta, Achla; Bushlin, Ittai; Devi, Lakshmi A.

    2014-01-01

    Over the last decade an increasing number of studies have focused on the ability of G protein-coupled receptors to form heteromers and explored how receptor heteromerization modulates the binding, signaling and trafficking properties of individual receptors. Most of these studies were carried out in heterologous cells expressing epitope tagged receptors. Very little information is available about the in vivo physiological role of G protein-coupled receptor heteromers due to a lack of tools to detect their presence in endogenous tissue. Recent advances such as the generation of mouse models expressing fluorescently labeled receptors, of TAT based peptides that can disrupt a given heteromer pair, or of heteromer-selective antibodies that recognize the heteromer in endogenous tissue have begun to elucidate the physiological and pathological roles of receptor heteromers. In this review we have focused on heteromer-selective antibodies and describe how a subtractive immunization strategy can be successfully used to generate antibodies that selectively recognize a desired heteromer pair. We also describe the uses of these antibodies to detect the presence of heteromers, to study their properties in endogenous tissues, and to monitor changes in heteromer levels under pathological conditions. Together, these findings suggest that G protein-coupled receptor heteromers represent unique targets for the development of drugs with reduced side-effects. PMID:25520661

  19. Selective Orthosteric Free Fatty Acid Receptor 2 (FFA2) Agonists

    PubMed Central

    Schmidt, Johannes; Smith, Nicola J.; Christiansen, Elisabeth; Tikhonova, Irina G.; Grundmann, Manuel; Hudson, Brian D.; Ward, Richard J.; Drewke, Christel; Milligan, Graeme; Kostenis, Evi; Ulven, Trond

    2011-01-01

    Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [35S]guanosine 5′-(3-O-thio)triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp3-hybridized α-carbons preferentially activate FFA3, whereas ligands with sp2- or sp-hybridized α-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors. PMID:21220428

  20. How Can Mutations Thermostabilize G-Protein-Coupled Receptors?

    PubMed

    Vaidehi, Nagarajan; Grisshammer, Reinhard; Tate, Christopher G

    2016-01-01

    Structures of over 30 different G-protein-coupled receptors (GPCRs) have advanced our understanding of cell signaling and have provided a foundation for structure-guided drug design. This exciting progress has required the development of three complementary methods to facilitate GPCR crystallization, one of which is the thermostabilization of receptors by systematic mutagenesis. However, the reason why a particular mutation, or combination of mutations, stabilizes the receptor is not always evident from a static crystal structure. Molecular dynamics (MD) simulations have been used to identify and estimate the energetic factors that affect thermostability through comparing the dynamics of the thermostabilized receptors with structure-based models of the wild-type receptor. The data indicate that receptors are stabilized through a combination of factors, including an increase in receptor rigidity, a decrease in collective motion, reduced stress at specific residues, and the presence of ordered water molecules. Predicting thermostabilizing mutations computationally represents a major challenge for the field.

  1. [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.

  2. Presynaptic G Protein-Coupled Receptors: Gatekeepers of Addiction?

    PubMed Central

    Johnson, Kari A.; Lovinger, David M.

    2016-01-01

    Drug abuse and addiction cause widespread social and public health problems, and the neurobiology underlying drug actions and drug use and abuse is an area of intensive research. Drugs of abuse alter synaptic transmission, and these actions contribute to acute intoxication as well as the chronic effects of abused substances. Transmission at most mammalian synapses involves neurotransmitter activation of two receptor subtypes, ligand-gated ion channels that mediate fast synaptic responses and G protein-coupled receptors (GPCRs) that have slower neuromodulatory actions. The GPCRs represent a large proportion of neurotransmitter receptors involved in almost all facets of nervous system function. In addition, these receptors are targets for many pharmacotherapeutic agents. Drugs of abuse directly or indirectly affect neuromodulation mediated by GPCRs, with important consequences for intoxication, drug taking and responses to prolonged drug exposure, withdrawal and addiction. Among the GPCRs are several subtypes involved in presynaptic inhibition, most of which are coupled to the Gi/o class of G protein. There is increasing evidence that these presynaptic Gi/o-coupled GPCRs have important roles in the actions of drugs of abuse, as well as behaviors related to these drugs. This topic will be reviewed, with particular emphasis on receptors for three neurotransmitters, Dopamine (DA; D1- and D2-like receptors), Endocannabinoids (eCBs; CB1 receptors) and glutamate (group II metabotropic glutamate (mGlu) receptors). The focus is on recent evidence from laboratory animal models (and some evidence in humans) implicating these receptors in the acute and chronic effects of numerous abused drugs, as well as in the control of drug seeking and taking. The ability of drugs targeting these receptors to modify drug seeking behavior has raised the possibility of using compounds targeting these receptors for addiction pharmacotherapy. This topic is also discussed, with emphasis on

  3. G protein-coupled estrogen receptor protects from atherosclerosis.

    PubMed

    Meyer, Matthias R; Fredette, Natalie C; Howard, Tamara A; Hu, Chelin; Ramesh, Chinnasamy; Daniel, Christoph; Amann, Kerstin; Arterburn, Jeffrey B; Barton, Matthias; Prossnitz, Eric R

    2014-12-23

    Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity.

  4. G Protein-coupled Estrogen Receptor Protects from Atherosclerosis

    PubMed Central

    Meyer, Matthias R.; Fredette, Natalie C.; Howard, Tamara A.; Hu, Chelin; Ramesh, Chinnasamy; Daniel, Christoph; Amann, Kerstin; Arterburn, Jeffrey B.; Barton, Matthias; Prossnitz, Eric R.

    2014-01-01

    Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity. PMID:25532911

  5. Multiple switches in G protein-coupled receptor activation.

    PubMed

    Ahuja, Shivani; Smith, Steven O

    2009-09-01

    The activation mechanism of G protein-coupled receptors has presented a puzzle that finally may be close to solution. These receptors have a relatively simple architecture consisting of seven transmembrane helices that contain just a handful of highly conserved amino acids, yet they respond to light and a range of chemically diverse ligands. Recent NMR structural studies on the active metarhodopsin II intermediate of the visual receptor rhodopsin, along with the recent crystal structure of the apoprotein opsin, have revealed multiple structural elements or 'switches' that must be simultaneously triggered to achieve full activation. The confluence of several required structural changes is an example of "coincidence counting", which is often used by nature to regulate biological processes. In ligand-activated G protein-coupled receptors, the presence of multiple switches may provide an explanation for the differences between full, partial and inverse agonists.

  6. Biased ligands for better cardiovascular drugs: dissecting G-protein-coupled receptor pharmacology.

    PubMed

    DeWire, Scott M; Violin, Jonathan D

    2011-07-08

    Drug discovery efforts targeting G-protein-coupled receptors (GPCR) have been immensely successful in creating new cardiovascular medicines. Currently marketed GPCR drugs are broadly classified as either agonists that activate receptors or antagonists that prevent receptor activation by endogenous stimuli. However, GPCR couple to a multitude of intracellular signaling pathways beyond classical G-protein signals, and these signals can be independently activated by biased ligands to vastly expand the potential for new drugs at these classic targets. By selectively engaging only a subset of a receptor's potential intracellular partners, biased ligands may deliver more precise therapeutic benefit with fewer side effects than current GPCR-targeted drugs. In this review, we discuss the history of biased ligand research, the current understanding of how biased ligands exert their unique pharmacology, and how research into GPCR signaling has uncovered previously unappreciated capabilities of receptor pharmacology. We focus on several receptors to illustrate the approaches taken and discoveries made, and how these are steadily illuminating the intricacies of GPCR pharmacology. Discoveries of biased ligands targeting the angiotensin II type 1 receptor and of separable pharmacology suggesting the potential value of biased ligands targeting the β-adrenergic receptors and nicotinic acid receptor GPR109a highlight the powerful clinical promise of this new category of potential therapeutics.

  7. A monoclonal antibody for G protein-coupled receptor crystallography.

    PubMed

    Day, Peter W; Rasmussen, Søren G F; Parnot, Charles; Fung, Juan José; Masood, Asna; Kobilka, Tong Sun; Yao, Xiao-Jie; Choi, Hee-Jung; Weis, William I; Rohrer, Daniel K; Kobilka, Brian K

    2007-11-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

  8. Photomodulation of G Protein-Coupled Adenosine Receptors by a Novel Light-Switchable Ligand

    PubMed Central

    2015-01-01

    The adenosinergic system operates through G protein-coupled adenosine receptors, which have become promising therapeutic targets for a wide range of pathological conditions. However, the ubiquity of adenosine receptors and the eventual lack of selectivity of adenosine-based drugs have frequently diminished their therapeutic potential. Accordingly, here we aimed to develop a new generation of light-switchable adenosine receptor ligands that change their intrinsic activity upon irradiation, thus allowing the spatiotemporal control of receptor functioning (i.e., receptor activation/inactivation dependent on location and timing). Therefore, we synthesized an orthosteric, photoisomerizable, and nonselective adenosine receptor agonist, nucleoside derivative MRS5543 containing an aryl diazo linkage on the N6 substituent, which in the dark (relaxed isomer) behaved as a full adenosine A3 receptor (A3R) and partial adenosine A2A receptor (A2AR) agonist. Conversely, upon photoisomerization with blue light (460 nm), it remained a full A3R agonist but became an A2AR antagonist. Interestingly, molecular modeling suggested that structural differences encountered within the third extracellular loop of each receptor could modulate the intrinsic, receptor subtype-dependent, activity. Overall, the development of adenosine receptor ligands with photoswitchable activity expands the pharmacological toolbox in support of research and possibly opens new pharmacotherapeutic opportunities. PMID:25248077

  9. G Protein-Coupled Receptors in Anopheles gambiae

    NASA Astrophysics Data System (ADS)

    Hill, Catherine A.; Fox, A. Nicole; Pitts, R. Jason; Kent, Lauren B.; Tan, Perciliz L.; Chrystal, Mathew A.; Cravchik, Anibal; Collins, Frank H.; Robertson, Hugh M.; Zwiebel, Laurence J.

    2002-10-01

    We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.

  10. [Regulation of G protein-coupled receptor kinase activity].

    PubMed

    Haga, T; Haga, K; Kameyama, K; Nakata, H

    1994-09-01

    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  11. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity

    PubMed Central

    Alsamarah, Abdelaziz; LaCuran, Alecander E.; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun

    2015-01-01

    Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550

  12. Biased ligands at G-protein-coupled receptors: promise and progress.

    PubMed

    Violin, Jonathan D; Crombie, Aimee L; Soergel, David G; Lark, Michael W

    2014-07-01

    Drug discovery targeting G protein-coupled receptors (GPCRs) is no longer limited to seeking agonists or antagonists to stimulate or block cellular responses associated with a particular receptor. GPCRs are now known to support a diversity of pharmacological profiles, a concept broadly referred to as functional selectivity. In particular, the concept of ligand bias, whereby a ligand stabilizes subsets of receptor conformations to engender novel pharmacological profiles, has recently gained increasing prominence. This review discusses how biased ligands may deliver safer, better tolerated, and more efficacious drugs, and highlights several biased ligands that are in clinical development. Biased ligands targeting the angiotensin II type 1 receptor and the μ opioid receptor illustrate the translation of the biased ligand concept from basic biology to clinical drug development.

  13. Mate Selection among Married and Cohabiting Couples.

    ERIC Educational Resources Information Center

    Blackwell, Debra L.; Lichter, Daniel T.

    2000-01-01

    Examines comparative patterns of educational and racial assortative mating or homogany among married and cohabiting couples, and evaluates whether women and men trade in socioeconomic status and racial caste prestige. Lists several findings, including married/cohabiting couples are highly homogenous with respect to race and education. Suggests…

  14. Development of selective agonists and antagonists of P2Y receptors

    PubMed Central

    Ivanov, Andrei A.; de Castro, Sonia; Harden, T. Kendall; Ko, Hyojin

    2008-01-01

    Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y1, P2Y2, and P2Y6 receptors and nucleotide antagonists selective for P2Y1 and P2Y12 receptors are now known. Selective nonnucleotide antagonists were reported for P2Y1, P2Y2, P2Y6, P2Y11, P2Y12, and P2Y13 receptors. At the P2Y1 and P2Y12 receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y1 and P2Y6 receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y12 receptor antagonists with antithrombotic activity. Selective agonists for the P2Y4, P2Y11, and P2Y13 receptors and selective antagonists for P2Y4 and P2Y14 receptors have not yet been identified. The P2Y14 receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets. PMID:18600475

  15. Crosstalk between G-protein-coupled receptors and epidermal growth factor receptor in cancer.

    PubMed

    Bhola, Neil E; Grandis, Jennifer R

    2008-01-01

    EGFR and its respective ligands are overexpressed in various tumors and this over-expression correlates with poor prognosis in selected cancers. In addition to direct activation by EGFR autocrine ligands, the large family of G-protein-coupled receptors (GPCRs) has been reported to transactivate EGFR via both ligand-dependent and independent mechanisms. GPCRs can induce the cleavage of membrane-bound EGFR-ligand precursors or directly activate the juxtamembrane tyrosine kinase domain of EGFR. Due to the heterogenous expression of GPCRs in tumors, this form of receptor crosstalk may contribute to the modest clinical responses to EGFR-targeted therapies observed to date. Studies, so far, have indicated that the signaling mechanisms involved in transactivation are specifically influenced by the activated GPCR and the tumor type in question. The progression of colon, lung, breast, head and neck, prostate and ovarian cancers have all been reported to be mediated, at least in part, by GPCR-EGFR crosstalk. Increased understanding of the specific signaling pathways involved in EGFR transactivation by GPCR will facilitate the identification of new biomarkers for molecular targeting strategies.

  16. Forskolin-free cAMP assay for Gi-coupled receptors.

    PubMed

    Gilissen, Julie; Geubelle, Pierre; Dupuis, Nadine; Laschet, Céline; Pirotte, Bernard; Hanson, Julien

    2015-12-01

    G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human diseases. Many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However, agonist ligands for Gi-coupled receptors are difficult to track because inducers such as forskolin (FSK) must be used and are sources of variations and errors. We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or GPR91) which could be an attractive drug target. It has never been validated as such because very few ligands have been described. Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real time, FSK-free detection of an agonist effect. This FSK-free agonist signal was confirmed on other Gi-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false positives when measuring the basal levels. In this report, we validate a cAMP-inducer free method for the detection of Gi-coupled receptors agonists compatible with high-throughput screening. This method will facilitate the study and screening of Gi-coupled receptors for active ligands.

  17. Membrane cholesterol access into a G-protein-coupled receptor

    NASA Astrophysics Data System (ADS)

    Guixà-González, Ramon; Albasanz, José L.; Rodriguez-Espigares, Ismael; Pastor, Manuel; Sanz, Ferran; Martí-Solano, Maria; Manna, Moutusi; Martinez-Seara, Hector; Hildebrand, Peter W.; Martín, Mairena; Selent, Jana

    2017-02-01

    Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

  18. Crystal Structure of a Lipid G Protein-Coupled Receptor

    SciTech Connect

    Hanson, Michael A; Roth, Christopher B; Jo, Euijung; Griffith, Mark T; Scott, Fiona L; Reinhart, Greg; Desale, Hans; Clemons, Bryan; Cahalan, Stuart M; Schuerer, Stephan C; Sanna, M Germana; Han, Gye Won; Kuhn, Peter; Rosen, Hugh; Stevens, Raymond C

    2012-03-01

    The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P1-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P1, resulting in the modulation of immune and stromal cell responses.

  19. Membrane cholesterol access into a G-protein-coupled receptor

    PubMed Central

    Guixà-González, Ramon; Albasanz, José L.; Rodriguez-Espigares, Ismael; Pastor, Manuel; Sanz, Ferran; Martí-Solano, Maria; Manna, Moutusi; Martinez-Seara, Hector; Hildebrand, Peter W.; Martín, Mairena; Selent, Jana

    2017-01-01

    Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs. PMID:28220900

  20. Agonist selectivity in the oxytocin/vasopressin receptor family: new insights and challenges.

    PubMed

    Chini, B; Manning, M

    2007-08-01

    The design and development of selective agonists acting at the OT (oxytocin)/AVP (vasopressin) receptors has been and continues to be a difficult task because of the great similarity among the different receptor subtypes as well as the high degree of chemical similarity between the active ligands. In recent decades, at least a thousand synthetic peptides have been synthesized and examined for their ability to bind to and activate the different OT/AVP receptors; an effort that has led to the identification of several receptor subtype-selective agonists in the rat. However, owing to species differences between rat and human AVP/OT receptors, these peptides do not exhibit the same selectivities in human receptor assays. Furthermore, the discovery of receptor promiscuity, which is the ability of a single receptor subtype to couple to several different G-proteins, has led to the definition of a completely new class of compounds, referred to here as coupling-selective ligands, which may activate, within a single receptor subtype, only a specific signalling pathway. Finally, the accumulating evidence that GPCRs (G-protein-coupled receptors) do not function as monomers, but as dimers/oligomers, opens up the design of another class of specific ligands, bivalent ligands, in which two agonist and/or antagonist moieties are joined by a spacer of the appropriate length to allow the simultaneous binding at the two subunits within the dimer. The pharmacological properties and selectivity profiles of these bivalent ligands, which remain to be investigated, could lead to highly novel research tools and potential therapeutic agents.

  1. Multiple receptors coupled to phospholipase C gate long-term depression in visual cortex.

    PubMed

    Choi, Se-Young; Chang, Jeff; Jiang, Bin; Seol, Geun-Hee; Min, Sun-Seek; Han, Jung-Soo; Shin, Hee-Sup; Gallagher, Michela; Kirkwood, Alfredo

    2005-12-07

    Long-term depression (LTD) in sensory cortices depends on the activation of NMDA receptors. Here, we report that in visual cortical slices, the induction of LTD (but not long-term potentiation) also requires the activation of receptors coupled to the phospholipase C (PLC) pathway. Using immunolesions in combination with agonists and antagonists, we selectively manipulated the activation of alpha1 adrenergic, M1 muscarinic, and mGluR5 glutamatergic receptors. Inactivation of these PLC-coupled receptors prevents the induction of LTD, but only when the three receptors were inactivated together. LTD is fully restored by activating any one of them or by supplying intracellular D-myo-inositol-1,4,5-triphosphate (IP3). LTD was also impaired by intracellular application of PLC or IP3 receptor blockers, and it was absent in mice lacking PLCbeta1, the predominant PLC isoform in the forebrain. We propose that visual cortical LTD requires a minimum of PLC activity that can be supplied independently by at least three neurotransmitter systems. This essential requirement places PLC-linked receptors in a unique position to control the induction of LTD and provides a mechanism for gating visual cortical plasticity via extra-retinal inputs in the intact organism.

  2. The G protein-coupled receptor N-terminus and receptor signalling: N-tering a new era.

    PubMed

    Coleman, James L J; Ngo, Tony; Smith, Nicola J

    2017-05-01

    G protein-coupled receptors (GPCRs) are a vast family of membrane-traversing proteins, essential to the ability of eukaryotic life to detect, and mount an intracellular response to, a diverse range of extracellular stimuli. GPCRs have evolved with archetypal features including an extracellular N-terminus and intracellular C-terminus that flank a transmembrane structure of seven sequential helices joined by intracellular and extracellular loops. These structural domains contribute to the ability of a GPCR to be correctly synthesised and inserted into the cell membrane, to interact with its cognate ligand(s) and to couple with signal-transducing heterotrimeric G proteins, allowing the activated receptor to selectively modulate a number of signalling cascades. Whilst well known for its importance in receptor translation and trafficking, the GPCR N-terminus is underexplored as a participant in receptor signalling. This review aims to discuss and integrate recent advances in knowledge of the vital roles of the GPCR N-terminus in receptor signalling.

  3. Molecular basis of association of receptor activity-modifying protein 3 with the family B G protein-coupled secretin receptor.

    PubMed

    Harikumar, Kaleeckal G; Simms, John; Christopoulos, George; Sexton, Patrick M; Miller, Laurence J

    2009-12-15

    The three receptor activity-modifying proteins (RAMPs) have been recognized as being important for the trafficking and function of a subset of family B G protein-coupled receptors, although the structural basis for this has not been well established. In the current work, we use morphological fluorescence techniques, bioluminescence resonance energy transfer, and bimolecular fluorescence complementation to demonstrate that the secretin receptor associates specifically with RAMP3, but not with RAMP1 or RAMP2. We use truncation constructs, peptide competition experiments, and chimeric secretin-GLP1 receptor constructs to establish that this association is structurally specific, dependent on the intramembranous region of the RAMP and TM6 and TM7 of this receptor. There were no observed changes in secretin-stimulated cAMP, intracellular calcium, ERK1/2 phosphorylation, or receptor internalization in receptor-bearing COS or CHO-K1 cells in the presence or absence of exogenous RAMP transfection, although the secretin receptor trafficks normally to the cell surface in these cells in a RAMP-independent manner, resulting in both free and RAMP-associated receptor on the cell surface. RAMP3 association with this receptor was shown to be capable of rescuing a receptor mutant (G241C) that is normally trapped intracellularly in the biosynthetic machinery. Similarly, secretin receptor expression had functional effects on adrenomedullin activity, with increasing secretin receptor expression competing for RAMP3 association with the calcitonin receptor-like receptor to yield a functional adrenomedullin receptor. These data provide important new insights into the structural basis for RAMP3 interaction with a family B G protein-coupled receptor, potentially providing a highly selective target for drug action. This may be representative of similar interactions between other members of this receptor family and RAMP proteins.

  4. Ryanodine Receptor Allosteric Coupling and the Dynamics of Calcium Sparks

    PubMed Central

    Groff, Jeffrey R.; Smith, Gregory D.

    2008-01-01

    Puffs and sparks are localized intracellular Ca2+ elevations that arise from the cooperative activity of Ca2+-regulated inositol 1,4,5-trisphosphate receptors and ryanodine receptors clustered at Ca2+ release sites on the surface of the endoplasmic reticulum or the sarcoplasmic reticulum. While the synchronous gating of Ca2+-regulated Ca2+ channels can be mediated entirely though the buffered diffusion of intracellular Ca2+, interprotein allosteric interactions also contribute to the dynamics of ryanodine receptor (RyR) gating and Ca2+ sparks. In this article, Markov chain models of Ca2+ release sites are used to investigate how the statistics of Ca2+ spark generation and termination are related to the coupling of RyRs via local [Ca2+] changes and allosteric interactions. Allosteric interactions are included in a manner that promotes the synchronous gating of channels by stabilizing neighboring closed-closed and/or open-open channel pairs. When the strength of Ca2+-mediated channel coupling is systematically varied (e.g., by changing the Ca2+ buffer concentration), simulations that include synchronizing allosteric interactions often exhibit more robust Ca2+ sparks; however, for some Ca2+ coupling strengths the sparks are less robust. We find no evidence that the distribution of spark durations can be used to distinguish between allosteric interactions that stabilize closed channel pairs, open channel pairs, or both in a balanced fashion. On the other hand, the changes in spark duration, interspark interval, and frequency observed when allosteric interactions that stabilize closed channel pairs are gradually removed from simulations are qualitatively different than the changes observed when open or both closed and open channel pairs are stabilized. Thus, our simulations clarify how changes in spark statistics due to pharmacological washout of the accessory proteins mediating allosteric coupling may indicate the type of synchronizing allosteric interactions exhibited

  5. Identifying the receptor subtype selectivity of retinoid X and retinoic acid receptors via quantum mechanics.

    PubMed

    Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

    2017-03-01

    Understanding and identifying the receptor subtype selectivity of a ligand is an important issue in the field of drug discovery. Using a combination of classical molecular mechanics and quantum mechanical calculations, this report assesses the receptor subtype selectivity for the human retinoid X receptor (hRXR) and retinoic acid receptor (hRAR) ligand-binding domains (LBDs) complexed with retinoid ligands. The calculated energies show good correlation with the experimentally reported binding affinities. The technique proposed here is a promising method as it reveals the origin of the receptor subtype selectivity of selective ligands.

  6. The Faith Development of Selected Adult Couples.

    ERIC Educational Resources Information Center

    Gorman, Margaret

    Theories and studies of adult development are largely confined to adult male career development and ignore a moral or faith dimension of adult development. To determine the faith and moral dimension of adult couples, three hypotheses were examined, i.e.,: (1) religion is a significant dimension in their consciousness; (2) the family is integrally…

  7. Disease-Specific Heteromerization of G-Protein-Coupled Receptors That Target Drugs of Abuse

    PubMed Central

    Gomes, Ivone; Fujita, Wakako; Chandrakala, Moraje V.; Devi, Lakshmi A.

    2014-01-01

    Drugs of abuse such as morphine or marijuana exert their effects through the activation of G-protein-coupled receptors (GPCRs), the opioid and cannabinoid receptors, respectively. Moreover, interactions between either of these receptors have been shown to be involved in the rewarding effects of drugs of abuse. Recent advances in the field, using a variety of approaches, have demonstrated that many GPCRs, including opioid, cannabinoid, and dopamine receptors, can form associations between different receptor subtypes or with other GPCRs to form heteromeric complexes. The formation of these complexes, in turn, leads to the modulation of the properties of individual protomers. The development of tools that can selectively disrupt GPCR heteromers as well as monoclonal antibodies that can selectively block signaling by specific heteromer pairs has indicated that heteromers involving opioid, cannabinoid, or dopamine receptors may play a role in various disease states. In this review, we describe evidence for opioid, cannabinoid, and dopamine receptor heteromerization and the potential role of GPCR heteromers in pathophysiological conditions. PMID:23663971

  8. Applications of molecular replacement to G protein-coupled receptors

    SciTech Connect

    Kruse, Andrew C.; Manglik, Aashish; Kobilka, Brian K.; Weis, William I.

    2013-11-01

    The use of molecular replacement in solving the structures of G protein-coupled receptors is discussed, with specific examples being described in detail. G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed.

  9. Cell-free expression of G-protein coupled receptors: new pipelines for challenging targets.

    PubMed

    Rues, Ralf-Bernhardt; Orbán, Erika; Dötsch, Volker; Bernhard, Frank

    2014-12-01

    Based on their eminent importance for medical applications, G-protein coupled receptors are currently amongst the most frequently membrane protein targets analyzed by cell-free expression. The cell-free expression approach removes most bottlenecks known from conventional cell-based protein production pipelines and ensures fast access to a selected receptor target. In addition, receptors can be synthesized in presence of a large variety of artificial solubilization environments comprising detergents, lipids, nanodiscs and other amphiphilic compounds. The currently accumulated data based on a variety of analyzed receptors already opens promising perspectives for applications of cell-free synthesized samples in functional characterization and drug screening. Structural evaluation still suffers from high conformational dynamics causing sample instability and might be addressed in future by molecular engineering or immuno-stabilization approaches.

  10. Strategic Research Institute G-Protein-Coupled Receptors Drug Discovery World Summit.

    PubMed

    Felder, Christian C

    2004-08-01

    The Strategic Research Institute provided a well-organised 2-day summit that offered presentations and posters on new assay technology, structure-based small-molecule discovery and examples of clinical candidates targeted to G-protein-coupled receptor (GPCR) targets. A wide variety of topics were presented providing recent advances in GPCR target selection, bioassay-enabling technology and medicinal chemistry targeted to GPCR-relevant chemical libraries. GPCRs continue to be an attractive platform for drug discovery.

  11. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    SciTech Connect

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K.

    2010-01-14

    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 native 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.

  12. Serial Femtosecond Crystallography of G Protein-Coupled Receptors

    PubMed Central

    Liu, Wei; Wacker, Daniel; Gati, Cornelius; Han, Gye Won; James, Daniel; Wang, Dingjie; Nelson, Garrett; Weierstall, Uwe; Katritch, Vsevolod; Barty, Anton; Zatsepin, Nadia A.; Li, Dianfan; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Koglin, Jason E.; Seibert, M. Marvin; Wang, Chong; Shah, Syed T.A.; Basu, Shibom; Fromme, Raimund; Kupitz, Christopher; Rendek, Kimberley N.; Grotjohann, Ingo; Fromme, Petra; Kirian, Richard A.; Beyerlein, Kenneth R.; White, Thomas A.; Chapman, Henry N.; Caffrey, Martin; Spence, John C.H.; Stevens, Raymond C.; Cherezov, Vadim

    2014-01-01

    X-ray crystallography of G protein-coupled receptors and other membrane proteins is hampered by difficulties associated with growing sufficiently large crystals that withstand radiation damage and yield high-resolution data at synchrotron sources. Here we used an x-ray free-electron laser (XFEL) with individual 50-fs duration x-ray pulses to minimize radiation damage and obtained a high-resolution room temperature structure of a human serotonin receptor using sub-10 µm microcrystals grown in a membrane mimetic matrix known as lipidic cubic phase. Compared to the structure solved by traditional microcrystallography from cryo-cooled crystals of about two orders of magnitude larger volume, the room temperature XFEL structure displays a distinct distribution of thermal motions and conformations of residues that likely more accurately represent the receptor structure and dynamics in a cellular environment. PMID:24357322

  13. Lysophospholipid activation of G protein-coupled receptors.

    PubMed

    Mutoh, Tetsuji; Chun, Jerold

    2008-01-01

    One of the major lipid biology discoveries in last decade was the broad range of physiological activities of lysophospholipids that have been attributed to the actions of lysophospholipid receptors. The most well characterized lysophospholipids are lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). Documented cellular effects of these lipid mediators include growth-factor-like effects on cells, such as proliferation, survival, migration, adhesion, and differentiation. The mechanisms for these actions are attributed to a growing family of 7-transmembrane, G protein-coupled receptors (GPCRs). Their pathophysiological actions include immune modulation, neuropathic pain modulation, platelet aggregation, wound healing, vasopressor activity, and angiogenesis. Here we provide a brief introduction to receptor-mediated lysophospholipid signaling and physiology, and then discuss potential therapeutic roles in human diseases.

  14. G-protein-coupled receptors for free fatty acids: nutritional and therapeutic targets.

    PubMed

    Milligan, Graeme; Ulven, Trond; Murdoch, Hannah; Hudson, Brian D

    2014-06-01

    It is becoming evident that nutrients and metabolic intermediates derived from such nutrients regulate cellular function by activating a number of cell-surface G-protein coupled receptors (GPCRs). Until now, members of the GPCR family have largely been considered as the molecular targets that communicate cellular signals initiated by hormones and neurotransmitters. Recently, based on tissue expression patterns of these receptors and the concept that they may elicit the production of a range of appetite- and hunger-regulating peptides, such nutrient sensing GPCRs are attracting considerable attention due to their potential to modulate satiety, improve glucose homeostasis and supress the production of various pro-inflammatory mediators. Despite the developing interests in these nutrients sensing GPCR both as sensors of nutritional status, and targets for limiting the development of metabolic diseases, major challenges remain to exploit their potential for therapeutic purposes. Mostly, this is due to limited characterisation and validation of these receptors because of paucity of selective and high-potency/affinity pharmacological agents to define the detailed function and regulation of these receptors. However, ongoing clinical trials of agonists of free fatty acid receptor 1 suggest that this receptor and other receptors for free fatty acids may provide a successful strategy for controlling hyperglycaemia and providing novel approaches to treat diabetes. Receptors responsive to free fatty acid have been of particular interest, and some aspects of these are considered herein.

  15. Interaction of G protein coupled receptors and cholesterol.

    PubMed

    Gimpl, Gerald

    2016-09-01

    G protein coupled receptors (GPCRs) form the largest receptor superfamily in eukaryotic cells. Owing to their seven transmembrane helices, large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. Thus, GPCRs are always in proximity to cholesterol. Some of them are functionally dependent on the specific presence of cholesterol. Over the last years, enormous progress on receptor structures has been achieved. While lipophilic ligands other than cholesterol have been shown to bind either inside the helix bundle or at the receptor-lipid interface, the binding site of cholesterol was either a single transmembrane helix or a groove between two or more transmembrane helices. A clear preference for one of the two membrane leaflets has not been observed. Not surprisingly, many hydrophobic residues (primarily leucine and isoleucine) were found to be involved in cholesterol binding. In most cases, the rough β-face of cholesterol contacted the transmembrane helix bundle rather than the surrounding lipid matrix. The polar hydroxy group of cholesterol was localized near the water-membrane interface with potential hydrogen bonding to residues in receptor loop regions. Although a canonical motif, designated as CCM site, was detected as a specific cholesterol binding site in case of the β2AR, this site was not found to be occupied by cholesterol in other GPCRs possessing the same motif. Cholesterol-receptor interactions can increase the compactness of the receptor structure and are able to enhance the conformational stability towards active or inactive receptor states. Overall, all current data suggest a high plasticity of cholesterol interaction sites in GPCRs.

  16. Regulation of G protein-coupled receptor export trafficking

    PubMed Central

    Dong, Chunmin; Filipeanu, Catalin M.; Duvernay, Matthew T.; Wu, Guangyu

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute a superfamily of cell-surface receptors which share a common topology of seven transmembrane domains and modulate a variety of cell functions through coupling to heterotrimeric G proteins by responding to a vast array of stimuli. The magnitude of cellular response elicited by a given signal is dictated by the level of GPCR expression at the plasma membrane, which is the balance of elaborately regulated endocytic and exocytic trafficking. This review will cover recent advances in understanding the molecular mechanism underlying anterograde transport of the newly synthesized GPCRs from the endoplasmic reticulum (ER) through the Golgi to the plasma membrane. We will focus on recently identified motifs involved in GPCR exit from the ER and the Golgi, GPCR folding in the ER and the rescue of misfolded receptors from within, GPCR-interacting proteins that modulate receptor cell-surface targeting, pathways that mediate GPCR traffic, and the functional role of export in controlling GPCR signaling. PMID:17074298

  17. Protease-Activated Receptors and other G-Protein-Coupled Receptors: the Melanoma Connection.

    PubMed

    Rosero, Rebecca A; Villares, Gabriel J; Bar-Eli, Menashe

    2016-01-01

    The vast array of G-protein-coupled receptors (GPCRs) play crucial roles in both physiological and pathological processes, including vision, coagulation, inflammation, autophagy, and cell proliferation. GPCRs also affect processes that augment cell proliferation and metastases in many cancers including melanoma. Melanoma is the deadliest form of skin cancer, yet limited therapeutic modalities are available to patients with metastatic melanoma. Studies have found that both chemokine receptors and protease-activated receptors, both of which are GPCRs, are central to the metastatic melanoma phenotype and may serve as potential targets in novel therapies against melanoma and other cancers.

  18. GABA(B) receptors: altered coupling to G-proteins in rats sensitized to amphetamine.

    PubMed

    Zhang, K; Tarazi, F I; Campbell, A; Baldessarini, R J

    2000-01-01

    Modified dopamine and glutamate neurotransmission in discrete brain regions is implicated in stimulant-induced behavioral sensitization. Release of both neurotransmitters is influenced by GABA(B) metabotropic receptors for the principal inhibitory neurotransmitter GABA. Accordingly, GABA(B) receptors were examined in rats sensitized to amphetamine by measuring receptor density and coupling to G-proteins indicated as [(3)H]baclofen binding and baclofen-mediated [(35)S]GTP gamma S binding. Repeated treatment with (+)-amphetamine (5mg/kg per day, i.p., for five days) sensitized the rats to amphetamine challenge (1mg/kg) at 14 days, but not one day, later. GABA(B) receptor density was not altered at either time. Baclofen-mediated [(35)S]GTP gamma S binding, however, was selectively augmented in the prefrontal cortex and attenuated in the nucleus accumbens at 14 days, but not one day, after amphetamine treatment. Changes in GABA(B) receptor coupling to G-proteins in rats sensitized to amphetamine, but not in similarly treated but unsensitized rats, lead us to suggest that altered GABA(B) receptor functioning may contribute to the expression of amphetamine-induced behavioral sensitization.

  19. High-resolution metabolic profiling towards G protein-coupled receptors: rapid and comprehensive screening of histamine H₄ receptor ligands.

    PubMed

    Kool, J; Rudebeck, A F; Fleurbaaij, F; Nijmeijer, S; Falck, D; Smits, R A; Vischer, H F; Leurs, R; Niessen, W M A

    2012-10-12

    In the past years we developed high-resolution screening platforms involving separation of bioactive mixtures and on-line or at-line bioassays for a wide variety of biological targets with parallel mass spectrometric detection and identification. In the current research, we make a major step forward in the development of at-line bioassays by implementation of radioligand receptor binding and functional cellular assays to evaluate bioactvity and selectivity. We demonstrate a new platform for high-resolution metabolic profiling of lead compounds for functional activity and selectivity toward the human histamine H(4) receptor (hH(4)R), a member of the large family of membrane-bound G protein-coupled receptors. In this platform analytical chemistry, cell biology and pharmacology are merged. The methodology is based on chromatographic separation of metabolic mixtures by HPLC coupled to high-resolution fractionation onto (multiple) microtiter well plates for complementary assaying. The methodology was used for efficient and rapid metabolic profiling of the drug clozapine and three selective hH(4)R lead compounds. With this new platform metabolites with undesired alterations in target selectivity profiles can be readily identified. Moreover, the parallel identification of metabolite structures, with accurate-mass measurements and MS/MS, allowed identification of liable metabolic 'hotspots' for further lead optimization and plays a central role in the workflow and in this study. The methodology can be easily adapted for use with other receptor screening formats. The efficient combination of pharmacological assays with analytical techniques by leveraging high-resolution at-line fractionation as a linking technology will allow implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.

  20. Identification of the Orphan G Protein-coupled Receptor GPR31 as a Receptor for 12-(S)-Hydroxyeicosatetraenoic Acid*

    PubMed Central

    Guo, Yande; Zhang, Wenliang; Giroux, Craig; Cai, Yinlong; Ekambaram, Prasanna; Dilly, Ashok-kumar; Hsu, Andrew; Zhou, Senlin; Maddipati, Krishna Rao; Liu, Jingjing; Joshi, Sangeeta; Tucker, Stephanie C.; Lee, Menq-Jer; Honn, Kenneth V.

    2011-01-01

    Hydroxy fatty acids are critical lipid mediators involved in various pathophysiologic functions. We cloned and identified GPR31, a plasma membrane orphan G protein-coupled receptor that displays high affinity for the human 12-lipoxygenase-derived product 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE). Thus, GPR31 is named 12-(S)-HETE receptor (12-HETER) in this study. The cloned 12-HETER demonstrated high affinity binding for 12-(S)-[3H]HETE (Kd = 4.8 ± 0.12 nm). Also, 12-(S)-HETE efficiently and selectively stimulated GTPγS coupling in the membranes of 12-HETER-transfected cells (EC50 = 0.28 ± 1.26 nm). Activating GTPγS coupling with 12-(S)-HETE proved to be both regio- and stereospecific. Also, 12-(S)-HETE/12-HETER interactions lead to activation of ERK1/2, MEK, and NFκB. Moreover, knocking down 12-HRTER specifically inhibited 12-(S)-HETE-stimulated cell invasion. Thus, 12-HETER represents the first identified high affinity receptor for the 12-(S)-HETE hydroxyl fatty acids. PMID:21712392

  1. Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, beta 2-adrenergic, and m2 muscarinic cholinergic receptors.

    PubMed

    Gurevich, V V; Dion, S B; Onorato, J J; Ptasienski, J; Kim, C M; Sterne-Marr, R; Hosey, M M; Benovic, J L

    1995-01-13

    Arrestins play an important role in quenching signal transduction initiated by G protein-coupled receptors. To explore the specificity of arrestin-receptor interaction, we have characterized the ability of various wild-type arrestins to bind to rhodopsin, the beta 2-adrenergic receptor (beta 2AR), and the m2 muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found to be the most selective arrestin since it discriminated best between the three different receptors tested (highest binding to rhodopsin) as well as between the phosphorylation and activation state of the receptor (> 10-fold higher binding to the phosphorylated light-activated form of rhodopsin compared to any other form of rhodopsin). While beta-arrestin and arrestin 3 were also found to preferentially bind to the phosphorylated activated form of a given receptor, they only modestly discriminated among the three receptors tested. To explore the structural characteristics important in arrestin function, we constructed a series of truncated and chimeric arrestins. Analysis of the binding characteristics of the various mutant arrestins suggests a common molecular mechanism involved in determining receptor binding selectivity. Structural elements that contribute to arrestin binding include: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of a receptor, without directly participating in receptor interaction; 2) a basic N-terminal domain that directly participates in receptor interaction and appears to serve a regulatory role via intramolecular interaction with the C-terminal acidic region; and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity. A comparative structure-function model of all arrestins and a kinetic model of beta-arrestin and arrestin 3 interaction with receptors are proposed.

  2. Structural organization of G-protein-coupled receptors

    NASA Astrophysics Data System (ADS)

    Lomize, Andrei L.; Pogozheva, Irina D.; Mosberg, Henry I.

    1999-07-01

    Atomic-resolution structures of the transmembrane 7-α-helical domains of 26 G-protein-coupled receptors (GPCRs) (including opsins, cationic amine, melatonin, purine, chemokine, opioid, and glycoprotein hormone receptors and two related proteins, retinochrome and Duffy erythrocyte antigen) were calculated by distance geometry using interhelical hydrogen bonds formed by various proteins from the family and collectively applied as distance constraints, as described previously [Pogozheva et al., Biophys. J., 70 (1997) 1963]. The main structural features of the calculated GPCR models are described and illustrated by examples. Some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane α-bundle: the formation of extensive networks of interhelical H-bonds and sulfur-aromatic clusters that are spatially organized as 'polarity gradients' the close packing of side-chains throughout the transmembrane domain; and the formation of interhelical disulfide bonds in some receptors and a plausible Zn2+ binding center in retinochrome. Other features of the models are related to biological function and evolution of GPCRs: the formation of a common 'minicore' of 43 evolutionarily conserved residues; a multitude of correlated replacements throughout the transmembrane domain; an Na+-binding site in some receptors, and excellent complementarity of receptor binding pockets to many structurally dissimilar, conformationally constrained ligands, such as retinal, cyclic opioid peptides, and cationic amine ligands. The calculated models are in good agreement with numerous experimental data.

  3. Peptide ligand recognition by G protein-coupled receptors

    PubMed Central

    Krumm, Brian E.

    2015-01-01

    The past few years have seen spectacular progress in the structure determination of G protein-coupled receptors (GPCRs). We now have structural representatives from classes A, B, C, and F. Within the rhodopsin-like class A, most structures belong to the α group, whereas fewer GPCR structures are available from the β, γ, and δ groups, which include peptide GPCRs such as the receptors for neurotensin (β group), opioids, chemokines (γ group), and protease-activated receptors (δ group). Structural information on peptide GPCRs is restricted to complexes with non-peptidic drug-like antagonists with the exception of the chemokine receptor CXCR4 that has been crystallized in the presence of a cyclic peptide antagonist. Notably, the neurotensin receptor 1 is to date the only peptide GPCR whose structure has been solved in the presence of a peptide agonist. Although limited in number, the current peptide GPCR structures reveal great diversity in shape and electrostatic properties of the ligand binding pockets, features that play key roles in the discrimination of ligands. Here, we review these aspects of peptide GPCRs in view of possible models for peptide agonist binding. PMID:25852552

  4. Selectivity of Odorant Receptors in Insects

    DTIC Science & Technology

    2012-07-13

    characterization of the Aedes aegypti odorant receptor gene family. Insect Mol. Biol. 16, 525–537. Bohbot, J. D., and Dickens, J. C. (2009). Characterization of an...enantioselective odorant receptor in the yellow fever mosquito Aedes aegypti . PLoS ONE 4, e7032. doi: 10.1371/journal.pone.0007032 Bohbot, J. D., and...Dickens, J. C. (2011). Functional characteri- zation of the octenol receptor neuron on the maxillary palps of the yellow fever mosquito, Aedes aegypti

  5. On the g-protein-coupled receptor heteromers and their allosteric receptor-receptor interactions in the central nervous system: focus on their role in pain modulation.

    PubMed

    Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Rivera, Alicia; Van Craenenbroeck, Kathleen; Tarakanov, Alexander O; Agnati, Luigi F; Fuxe, Kjell

    2013-01-01

    The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via β -arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia.

  6. On the G-Protein-Coupled Receptor Heteromers and Their Allosteric Receptor-Receptor Interactions in the Central Nervous System: Focus on Their Role in Pain Modulation

    PubMed Central

    Borroto-Escuela, Dasiel O.; Romero-Fernandez, Wilber; Rivera, Alicia; Van Craenenbroeck, Kathleen; Tarakanov, Alexander O.; Agnati, Luigi F.; Fuxe, Kjell

    2013-01-01

    The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via β-arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia. PMID:23956775

  7. Selectivity of oxomemazine for the M1 muscarinic receptors.

    PubMed

    Lee, S W; Woo, C W; Kim, J G

    1994-12-01

    The binding characteristics of pirenzepine and oxomemazine to muscarinic receptor were studied to evaluate the selectivity of oxomemazine for the muscarinic receptor subtypes in rat cerebral microsomes. Equilibrium dissociation constant (KD) of (-)-[3H]quinuclidinyl benzilate([3H]QNB) determined from saturation isotherms was 64 pM. Analysis of the pirenzepine inhibition curve of [3H]QNB binding to cerebral microsome indicated the presence of two receptor subtypes with high (Ki = 16 nM, M1 receptor) and low (Ki = 400 nM, M3 receptor) affinity for pirenzepine. Oxomemazine also identified two receptor subtypes with about 20-fold difference in the affinity for high (Ki = 84 nM, OH receptor) and low (Ki = 1.65 microM, OL receptor) affinity sites. The percentage populations of M1 and M3 receptors to the total receptors were 61:39, and those of OH and OL receptors 39:61, respectively. Both pirenzepine and oxomemazine increased the KD value for [3H]QNB without affecting the binding site concentrations and Hill coefficient for the [3H]QNB binding. Oxomemazine had a 10-fold higher affinity at M1 receptors than at M3 receptors, and pirenzepine a 8-fold higher affinity at OH receptors than at OL receptors. Analysis of the shallow competition binding curves of oxomemazine for M1 receptors and pirenzepine for OL receptors yielded that 69% of M1 receptors were of OH receptors and the remaining 31% of OL receptors, and that 29% of OL receptors were of M1 receptors and 71% of M3 receptors. However, M3 for oxomemazine and OH for pirenzepine were composed of a uniform population. These results suggest that oxomemazine could be classified as a selective drug for M1 receptors and also demonstrate that rat cerebral microsomes contain three different subtypes of M1, M3 and the other site which is different from M1, M2 and M3 receptors.

  8. G-protein coupled receptors of the renin-angiotensin system: new targets against breast cancer?

    PubMed Central

    Rodrigues-Ferreira, Sylvie; Nahmias, Clara

    2015-01-01

    G-protein coupled receptors (GPCRs) constitute the largest family of membrane receptors, with high potential for drug discovery. These receptors can be activated by a panel of different ligands including ions, hormones, small molecules, and vasoactive peptides. Among those, angiotensins [angiotensin II (AngII) and angiotensin 1–7] are the major biologically active products of the classical and alternative renin-angiotensin system (RAS). These peptides bind and activate three different subtypes of GPCRs, namely AT1, AT2, and Mas receptors, to regulate cardiovascular functions. Over the past decade, the contribution of several RAS components in tumorigenesis has emerged as a novel important concept, AngII being considered as harmful and Ang1–7 as protective against cancer. Development of selective ligands targeting each RAS receptor may provide novel and efficient targeted therapeutic strategies against cancer. In this review, we focus on breast cancer to summarize current knowledge on angiotensin receptors (AT1, AT2, and Mas), and discuss the potential use of angiotensin receptor agonists and antagonists in clinics. PMID:25741281

  9. G-protein coupled receptors of the renin-angiotensin system: new targets against breast cancer?

    PubMed

    Rodrigues-Ferreira, Sylvie; Nahmias, Clara

    2015-01-01

    G-protein coupled receptors (GPCRs) constitute the largest family of membrane receptors, with high potential for drug discovery. These receptors can be activated by a panel of different ligands including ions, hormones, small molecules, and vasoactive peptides. Among those, angiotensins [angiotensin II (AngII) and angiotensin 1-7] are the major biologically active products of the classical and alternative renin-angiotensin system (RAS). These peptides bind and activate three different subtypes of GPCRs, namely AT1, AT2, and Mas receptors, to regulate cardiovascular functions. Over the past decade, the contribution of several RAS components in tumorigenesis has emerged as a novel important concept, AngII being considered as harmful and Ang1-7 as protective against cancer. Development of selective ligands targeting each RAS receptor may provide novel and efficient targeted therapeutic strategies against cancer. In this review, we focus on breast cancer to summarize current knowledge on angiotensin receptors (AT1, AT2, and Mas), and discuss the potential use of angiotensin receptor agonists and antagonists in clinics.

  10. A highly selective receptor for zwitterionic proline.

    PubMed

    Temprano, Álvaro G; Monleón, Laura M; Rubio, Omayra H; Rubio, Luis Simón; Pérez, Asunción B; Sanz, Francisca; Morán, Joaquín R

    2016-01-28

    A chiral chromane receptor has been synthesized which mimics the oxyanion hole of the enzymes. In this receptor H-bonds and cation-π interactions team up to generate an apolar host-guest complex with zwitterionic proline. This complex allows the extraction of only proline to a chloroform phase, while no other natural amino acids are extracted. Due to the chiral nature of the receptor, enantioselective extraction from the aqueous proline solution to a chloroform phase takes place. L-Proline provided an easy method to resolve the receptor racemic mixture, while anisotropic effects, NOE and CD studies revealed the absolute configuration of the receptor. Modelling studies also support the proposed structures. The presence of an oxyanion-hole motif in this structure was corroborated by X-ray diffraction studies.

  11. G protein-coupled receptors function as logic gates for nanoparticle binding and cell uptake

    PubMed Central

    Hild, Wolfgang; Pollinger, Klaus; Caporale, Andrea; Cabrele, Chiara; Keller, Max; Pluym, Nicola; Buschauer, Armin; Rachel, Reinhard; Tessmar, Joerg; Breunig, Miriam; Goepferich, Achim

    2010-01-01

    More selective interactions of nanoparticles with cells would substantially increase their potential for diagnostic and therapeutic applications. Thus, it would not only be highly desirable that nanoparticles can be addressed to any cell with high target specificity and affinity, but that we could unequivocally define whether they rest immobilized on the cell surface as a diagnostic tag, or if they are internalized to serve as a delivery vehicle for drugs. To date no class of targets is known that would allow direction of nanoparticle interactions with cells alternatively into one of these mutually exclusive events. Using MCF-7 breast cancer cells expressing the human Y1-receptor, we demonstrate that G protein-coupled receptors provide us with this option. We show that quantum dots carrying a surface-immobilized antagonist remain with nanomolar affinity on the cell surface, and particles carrying an agonist are internalized upon receptor binding. The receptor functions like a logic “and-gate” that grants cell access only to those particles that carry a receptor ligand “and” where the ligand is an agonist. We found that agonist- and antagonist-modified nanoparticles bind to several receptor molecules at a time. This multiligand binding leads to five orders of magnitude increased-receptor affinities, compared with free ligand, in displacement studies. More than 800 G protein-coupled receptors in humans provide us with the paramount advantage that targeting of a plethora of cells is possible, and that switching from cell recognition to cell uptake is simply a matter of nanoparticle surface modification with the appropriate choice of ligand type. PMID:20498042

  12. G protein-coupled receptors function as logic gates for nanoparticle binding and cell uptake.

    PubMed

    Hild, Wolfgang; Pollinger, Klaus; Caporale, Andrea; Cabrele, Chiara; Keller, Max; Pluym, Nicola; Buschauer, Armin; Rachel, Reinhard; Tessmar, Joerg; Breunig, Miriam; Goepferich, Achim

    2010-06-08

    More selective interactions of nanoparticles with cells would substantially increase their potential for diagnostic and therapeutic applications. Thus, it would not only be highly desirable that nanoparticles can be addressed to any cell with high target specificity and affinity, but that we could unequivocally define whether they rest immobilized on the cell surface as a diagnostic tag, or if they are internalized to serve as a delivery vehicle for drugs. To date no class of targets is known that would allow direction of nanoparticle interactions with cells alternatively into one of these mutually exclusive events. Using MCF-7 breast cancer cells expressing the human Y(1)-receptor, we demonstrate that G protein-coupled receptors provide us with this option. We show that quantum dots carrying a surface-immobilized antagonist remain with nanomolar affinity on the cell surface, and particles carrying an agonist are internalized upon receptor binding. The receptor functions like a logic "and-gate" that grants cell access only to those particles that carry a receptor ligand "and" where the ligand is an agonist. We found that agonist- and antagonist-modified nanoparticles bind to several receptor molecules at a time. This multiligand binding leads to five orders of magnitude increased-receptor affinities, compared with free ligand, in displacement studies. More than 800 G protein-coupled receptors in humans provide us with the paramount advantage that targeting of a plethora of cells is possible, and that switching from cell recognition to cell uptake is simply a matter of nanoparticle surface modification with the appropriate choice of ligand type.

  13. Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs

    PubMed Central

    Hines, John; Gough, Jonathan D.; Corson, Timothy W.; Crews, Craig M.

    2013-01-01

    Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinase-signaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short- and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects. PMID:23674677

  14. Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs.

    PubMed

    Hines, John; Gough, Jonathan D; Corson, Timothy W; Crews, Craig M

    2013-05-28

    Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinase-signaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short- and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects.

  15. Characterization of signaling pathways coupled to melatonin receptors in gastrointestinal smooth muscle.

    PubMed

    Ahmed, Rashad; Mahavadi, Sunila; Al-Shboul, Othman; Bhattacharya, Sayak; Grider, John R; Murthy, Karnam S

    2013-06-10

    Melatonin, a close derivative of serotonin, is involved in physiological regulation of circadian rhythms. In the gastrointestinal (GI) system, melatonin exhibits endocrine, paracrine and autocrine actions and is implicated in the regulation of GI motility. However, it is not known whether melatonin can also act directly on GI smooth muscle cells. The aim of the present study was to determine the expression of melatonin receptors in smooth muscle and identify their signaling pathways. MT1, but not MT2 receptors are expressed in freshly dispersed and cultured gastric smooth muscle cells. Melatonin selectively activated Gq and stimulated phosphoinositide (PI) hydrolysis in freshly dispersed and cultured muscle cells. PI hydrolysis was blocked by the expression of Gq, but not Gi minigene in cultured muscle cells. Melatonin also caused rapid increase in cytosolic Ca(2+) as determined by epifluorescence microscopy in fura-2 loaded single smooth muscle cells, and induced rapid contraction. Melatonin-induced PI hydrolysis and contraction were blocked by a non-selective MT1/MT2 antagonist luzindole (1 μM), but not by a selective MT2 antagonist 4P-PDOT (100 nM), and by the PLC inhibitor U73122. MT2 selective agonist IIK7 (100 nM) had no effect on PI hydrolysis and contraction. We conclude that rabbit gastric smooth muscle cells express melatonin MT1 receptors coupled to Gq. Activation of these receptors causes stimulation of PI hydrolysis and increase in cytosolic Ca(2+), and elicits muscle contraction.

  16. Functional selectivity of dopamine D1 receptor agonists in regulating the fate of internalized receptors *

    PubMed Central

    Ryman-Rasmussen, Jessica P.; Griffith, Adam; Oloff, Scott; Vaidehi, Nagarajan; Brown, Justin T.; Goddard, William A.; Mailman, Richard B.

    2007-01-01

    Recently, we demonstrated that D1 agonists can cause functionally selective effects when the endpoints of receptor internalization and adenylate cyclase activation are compared. The present study was designed to probe the phenomenon of functional selectivity at the D1 receptor further by testing the hypothesis that structurally dissimilar agonists with efficacies at these endpoints that equal or exceed those of dopamine would differ in ability to influence receptor fate after internalization, a functional endpoint largely unexplored for the D1 receptor. We selected two novel agonists of therapeutic interest that meet these criteria (the isochroman A-77636, and the isoquinoline dinapsoline), and compared the fates of the D1 receptor after internalization in response to these two compounds with that of dopamine. We found that dopamine caused the receptor to be rapidly recycled to the cell surface within 1 h of removal. Conversely, A-77636 caused the receptor to be retained intracellularly up to 48 h after agonist removal. Most surprisingly, the D1 receptor recovered to the cell surface 48 h after removal of dinapsoline. Taken together, these data indicate that these agonists target the D1 receptor to different intracellular trafficking pathways, demonstrating that the phenomenon of functional selectivity at the D1 receptor is operative for cellular events that are temporally downstream of immediate receptor activation. We hypothesize that these differential effects result from interactions of the synthetic ligands with aspects of the D1 receptor that are distal from the ligand binding domain. PMID:17067639

  17. Rabies virus infection selectively impairs membrane receptor functions in neuronal model cells.

    PubMed

    Koschel, K; Halbach, M

    1979-03-01

    A persistent infection with rabies virus (HEP-Flury) was established in the CNS-derived hybrid cell line 108CC15 which possesses specific membrane receptors for prostaglandins, catecholamines and acetylcholine. We report a differential virus influence on the specific receptor response to PGE, isoproterenol and acetycholine as indicated by typical changes of the intracellular cyclic AMP levels. As the adenylate cyclase activity was unchanged in infected cells in vitro, a selective virus influence on specific receptors themselves or their coupling to the cAMP synthesizing system must be considered.

  18. G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes

    PubMed Central

    Marchese, Adriano; Paing, May M.; Temple, Brenda R.S.; Trejo, JoAnn

    2010-01-01

    The heptahelical G protein–coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine- and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs. PMID:17995450

  19. Minireview: Nutrient Sensing by G Protein-Coupled Receptors

    PubMed Central

    Wauson, Eric M.; Lorente-Rodríguez, Andrés

    2013-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that recognize molecules in the extracellular milieu and transmit signals inside cells to regulate their behaviors. Ligands for many GPCRs are hormones or neurotransmitters that direct coordinated, stereotyped adaptive responses. Ligands for other GPCRs provide information to cells about the extracellular environment. Such information facilitates context-specific decision making that may be cell autonomous. Among ligands that are important for cellular decisions are amino acids, required for continued protein synthesis, as metabolic starting materials and energy sources. Amino acids are detected by a number of class C GPCRs. One cluster of amino acid-sensing class C GPCRs includes umami and sweet taste receptors, GPRC6A, and the calcium-sensing receptor. We have recently found that the umami taste receptor heterodimer T1R1/T1R3 is a sensor of amino acid availability that regulates the activity of the mammalian target of rapamycin. This review focuses on an array of findings on sensing amino acids and sweet molecules outside of neurons by this cluster of class C GPCRs and some of the physiologic processes regulated by them. PMID:23820899

  20. Heterodimerization and Surface Localization of G Protein Coupled Receptors

    PubMed Central

    Minneman, Kenneth P.

    2007-01-01

    G protein coupled receptors (GPCRs) are one of the largest human gene families, and are targets for many important therapeutic drugs. Over the last few years, there has been a major paradigm shift in our understanding of how these receptors function. Formerly, GPCRs were thought to exist as monomers that, upon agonist occupation, activated a heterotrimeric G protein to alter the concentrations of specific second messengers. Until recently, this relatively linear cascade has been the standard paradigm for signaling by these molecules. However, it is now clear that this model is not adequate to explain many aspects of GPCR function. We now know that many, if not most, GPCRs form homo- and/or hetero-oligomeric complexes and interact directly with intracellular proteins in addition to G proteins. It now appears that many GPCRs may not function independently, but might more accurately be described as subunits of large multi-protein signaling complexes. These observations raise many important new questions; some of which include: 1) How many functionally and pharmacologically distinct receptor subtypes exist in vivo? 2) Which GPCRs physically associate, and in what stochiometries? 3) What are the roles of individual subunits in binding ligand and activating responses? 4) Are the pharmacological or signaling properties of GPCR heterodimers different from monomers? Since these receptors are the targets for a large number of clinically useful compounds, such information is likely to be of direct therapeutic importance, both in understanding how existing drugs work, but also in discovering novel compounds to treat disease. PMID:17011524

  1. Cross-Pharmacology Analysis of G Protein-Coupled Receptors

    PubMed Central

    Briansó, Ferran; Carrascosa, Maria C.; Oprea, Tudor I.; Mestres, Jordi

    2013-01-01

    The degree of applicability of chemogenomic approaches to protein families depends on the accuracy and completeness of pharmacological data and the corresponding level of pharmacological similarity observed among their protein members. The recent public domain availability of pharmacological data for thousands of small molecules on 204 G protein-coupled receptors (GPCRs) provides a firm basis for an in-depth cross-pharmacology analysis of this superfamily. The number of protein targets included in the cross-pharmacology profile of the different GPCRs changes significantly upon varying the ligand similarity and binding affinity criteria. However, with the exception of muscarinic receptors, aminergic GPCRs distinguish themselves from the rest of the members in the family by their remarkably high levels of pharmacological similarity among them. Clusters of non-GPCR targets related by cross-pharmacology with particular GPCRs are identified and the implications for unwanted side-effects, as well as for repurposing opportunities, discussed. PMID:21851335

  2. Therapeutic antibodies directed at G protein-coupled receptors.

    PubMed

    Hutchings, Catherine J; Koglin, Markus; Marshall, Fiona H

    2010-01-01

    G protein-coupled receptors (GPCRs) are one of the most important classes of targets for small molecule drug discovery, but many current GPCRs of interest are proving intractable to small molecule discovery and may be better approached with bio-therapeutics. GPCRs are implicated in a wide variety of diseases where antibody therapeutics are currently used. These include inflammatory diseases such as rheumatoid arthritis and Crohn disease, as well as metabolic disease and cancer. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigen because GPCRs are often expressed at low levels in cells and are very unstable when purified. A number of new developments in over-expressing receptors, as well as formulating stable pure protein, are contributing to the growing interest in targeting GPCRs with antibodies. This review discusses the opportunities for targeting GPCRs with antibodies using these approaches and describes the therapeutic antibodies that are currently in clinical development.

  3. Therapeutic antibodies directed at G protein-coupled receptors

    PubMed Central

    Hutchings, Catherine J; Koglin, Markus

    2010-01-01

    G protein-coupled receptors (GPCRs) are one of the most important classes of targets for small molecule drug discovery, but many current GPCRs of interest are proving intractable to small molecule discovery and may be better approached with bio-therapeutics. GPCRs are implicated in a wide variety of diseases where antibody therapeutics are currently used. These include inflammatory diseases such as rheumatoid arthritis and Crohn disease, as well as metabolic disease and cancer. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigen because GPCRs are often expressed at low levels in cells and are very unstable when purified. A number of new developments in overexpressing receptors, as well as formulating stable pure protein, are contributing to the growing interest in targeting GPCRs with antibodies. This review discusses the opportunities for targeting GPCRs with antibodies using these approaches and describes the therapeutic antibodies that are currently in clinical development. PMID:20864805

  4. Anatomical profiling of G protein-coupled receptor expression

    PubMed Central

    Regard, Jean B.; Sato, Isaac T.; Coughlin, Shaun R.

    2008-01-01

    Summary G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane signaling molecules and regulate a host of physiological and disease processes. To better understand the functions of GPCRs in vivo, we quantified transcript levels of 353 non-odorant GPCRs in 41 adult mouse tissues. Cluster analysis placed many GPCRs into anticipated anatomical and functional groups and predicted novel roles for less studied receptors. From one such prediction, we showed that the Gpr91 ligand succinate can regulate lipolysis in white adipose tissue suggesting that signaling by this citric acid cycle intermediate may regulate energy homeostasis. We also showed that pairwise analysis of GPCR expression across tissues may help predict drug side effects. This resource will aid studies to understand GPCR function in vivo and may assist in the identification of therapeutic targets. PMID:18984166

  5. Enhancement of G Protein-Coupled Receptor Surface Expression

    PubMed Central

    Dunham, Jill H.; Hall, Randy A.

    2009-01-01

    G protein-coupled receptors (GPCRs) mediate physiological responses to a diverse array of stimuli and are the molecular targets for numerous therapeutic drugs. GPCRs primarily signal from the plasma membrane, but when expressed in heterologous cells many GPCRs exhibit poor trafficking to the cell surface. Multiple approaches have been taken to enhance GPCR surface expression in heterologous cells, including addition/deletion of receptor sequences, co-expression with interacting proteins, and treatment with pharmacological chaperones. In addition to allowing for enhanced surface expression of certain GPCRs in heterologous cells, these approaches have also shed light on the control of GPCR trafficking in vivo and in some cases have led to new therapeutic approaches for treating human diseases that result from defects in GPCR trafficking. PMID:19679364

  6. Tyrosine kinase receptor transactivation associated to G protein-coupled receptors.

    PubMed

    Almendro, Vanessa; García-Recio, Susana; Gascón, Pedro

    2010-09-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, cell differentiation and oncogenesis among others. Some of the effects of GPCRs are known to be mediated by the activation of MAPK pathways. Several GPCRs are also able to transactivate receptors with tyrosine kinase activity (TKR) such as EGFR and HER2 and thus to control DNA synthesis and cell proliferation. The interaction between these receptors not only plays an important physiological role but its disregulation can induce pathological states such as cancer. For this reason, the crosstalk between these two types of receptors can be considered a possible mechanism for cell transformation, tumor progression, reactivation of the metastatic disease, and the acquisition of resistance to therapies targeting TKR receptors. The transactivation of some TKRs by GPCRs is related to the lost of response of TKRs to inhibitors of TK activity, mainly by the activation of the c-Src protein which can directly phosphorylate and activate the cytoplasmic domain of a TKR. For these reason, the dual inhibition of GPCRs and TKRs in some types of cancer has been proposed as a better strategy to kill tumor cells. Increased understanding of the mechanisms that interconnect the two pathways regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

  7. Selective Estrogen Receptor Modulators: Cannabinoid Receptor Inverse Agonists with Differential CB1 and CB2 Selectivity

    PubMed Central

    Franks, Lirit N.; Ford, Benjamin M.; Prather, Paul L.

    2016-01-01

    Selective estrogen receptor modulators (SERMs) are used to treat estrogen receptor (ER)-positive breast cancer and osteoporosis. Interestingly, tamoxifen and newer classes of SERMs also exhibit cytotoxic effects in cancers devoid of ERs, indicating a non-estrogenic mechanism of action. Indicative of a potential ER-independent target, reports demonstrate that tamoxifen binds to cannabinoid receptors (CBRs) with affinity in the low μM range and acts as an inverse agonist. To identify cannabinoids with improved pharmacological properties relative to tamoxifen, and further investigate the use of different SERM scaffolds for future cannabinoid drug development, this study characterized the affinity and activity of SERMs in newer structural classes at CBRs. Fourteen SERMs from five structurally distinct classes were screened for binding to human CBRs. Compounds from four of five SERM classes examined bound to CBRs. Subsequent studies fully characterized CBR affinity and activity of one compound from each class. Ospemifine (a triphenylethylene) selectively bound to CB1Rs, while bazedoxifine (an indole) bound to CB2Rs with highest affinity. Nafoxidine (a tetrahydronaphthalene) and raloxifene (RAL; a benzothiaphene) bound to CB1 and CB2Rs non-selectively. All four compounds acted as inverse agonists at CB1 and CB2Rs, reducing basal G-protein activity with IC50 values in the nM to low μM range. Ospemifine, bazedoxifene and RAL also acted as inverse agonists to elevate basal intracellular cAMP levels in intact CHO-hCB2 cells. The four SERMs examined also acted as CB1 and CB2R antagonists in the cAMP assay, producing rightward shifts in the concentration-effect curve of the CBR agonist CP-55,940. In conclusion, newer classes of SERMs exhibit improved pharmacological characteristics (e.g., in CBR affinity and selectivity) relative to initial studies with tamoxifen, and thus suggest that different SERM scaffolds may be useful for development of safe and selective drugs acting

  8. In situ functionalized fluorescent nanoparticles for efficient receptor coupling

    NASA Astrophysics Data System (ADS)

    Salman, Abbas Abdulameer; Heidelberg, Thorsten

    2014-05-01

    An oligoethylene glycol-based propargyl phosphonate was applied to functionalize the surface of LaPO4:Ce,Tb nanoparticles in situ during the particle synthesis. The application of the surface modification reagent did not alter either size (5-7 nm in diameter) or morphology of the nanocrystalline core, but provided efficient anchor groups for subsequent coupling of a carbohydrate model receptor under mild reaction conditions. The biofunctionalization efficiency was quantified by thermogravimetric analysis and confirmed by a photometric assay. A calculation-based estimation suggested an average number of about 20 biomarkers per nanoparticles and a surface density of about 1 marker per 6 nm-2.

  9. Selectivity of odorant receptors in insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect olfactory receptors (ORs) detect chemical signals, shape neuronal physiology and regulate behavior. Although ORs have been categorized as generalists and specialists based on their ligand spectrum, both electrophysiological studies and recent pharmacological investigations show that ORs spec...

  10. Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics*

    PubMed Central

    Zhang, Ping; Leger, Andrew J.; Baleja, James D.; Rana, Rajashree; Corlin, Tiffany; Nguyen, Nga; Koukos, Georgios; Bohm, Andrew; Covic, Lidija; Kuliopulos, Athan

    2015-01-01

    G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class. PMID:25934391

  11. Discovery AND Therapeutic Promise OF Selective Androgen Receptor Modulators

    PubMed Central

    Chen, Jiyun; Kim, Juhyun; Dalton, James T.

    2007-01-01

    Androgens are essential for male development and the maintenance of male secondary characteristics, such as bone mass, muscle mass, body composition, and spermatogenesis. The main disadvantages of steroidal androgens are their undesirable physicochemical and pharmacokinetic properties. The recent discovery of nonsteroidal selective androgen receptor modulators (SARMs) provides a promising alternative for testosterone replacement therapies with advantages including oral bioavailability, flexibility of structural modification, androgen receptor specificity, tissue selectivity, and the lack of steroid-related side effects. PMID:15994457

  12. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

    PubMed Central

    Cattaneo, Fabio; Guerra, Germano; Parisi, Melania; De Marinis, Marta; Tafuri, Domenico; Cinelli, Mariapia; Ammendola, Rosario

    2014-01-01

    G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we

  13. Angiotensin II receptor subtypes are coupled with distinct signal-transduction mechanisms in neurons and astrocytes from rat brain

    SciTech Connect

    Sumners, C.; Wei Tang; Zelezna, B.; Raizada, M.K. )

    1991-09-01

    Both neurons and astrocytes contain specific receptors for angiotensin II (AII). The authors used selective ligands for the AT{sub 1} and AT{sub 2} types of AII receptors to investigate the expression of functional receptor subtypes in astrocyte cultures and neuron cultures from 1-day-old (neonatal) rat brain. In astrocyte cultures, competition of {sup 125}I-labeled AII ({sup 125}I-AII) specific binding with AT{sub 1} (DuP753) or AT{sub 2} {l brace}PD123177, CGP42112A, (Phe(p-NH{sub 2}){sup 6})AII{r brace} selective receptor ligands revealed a potency series of AII > DuP753 > > > CGP42112A > (Phe(p-NH{sub 2}){sup 6})AII > PD123177. These results suggest a predominance of the AT{sub 1} receptor subtype in neonatal astrocytes. {sup 125}I-AII specific binding to neonate neuronal cultures was reduced 73-84% by 1 {mu} MPD123177, and the residual {sup 125}I-AII specific binding was eliminated by DuP753. The results suggest that astrocyte cultures from neonatal rat brains contain predominantly AT{sub 1} receptors that are coupled to a stimulation of inositophospholipid hydrolysis. In contrast, neuron cultures from neonatal rat brain contain mostly AT{sub 2} receptors that are coupled to a reduction in basal cGMP levels, but a smaller population of AT{sub 1} receptors is also present in these neurons.

  14. Chemical modification of Class II G-protein coupled receptor ligands

    PubMed Central

    Chapter, Megan C.; White, Caitlin M.; De Ridder, Angela; Chadwick, Wayne; Martin, Bronwen; Maudsley, Stuart

    2009-01-01

    Recent research and clinical data have begun to demonstrate the huge potential therapeutic importance of ligands that modulate the activity of the secretin-like, Class II, G-protein coupled receptors (GPCRs). Ligands that can modulate the activity of these Class II GPCRs may have important clinical roles in the treatment of a wide variety of conditions such as osteoporosis, diabetes, amyotrophic lateral sclerosis and autism spectrum disorders. While these receptors present important new therapeutic targets, the large glycoprotein nature of their cognate ligands poses many problems with respect to therapeutic peptidergic drug design. These native peptides often exhibit poor bioavailability, metabolic instability, poor receptor selectivity and resultant low potencies in vivo. Recently, increased attention has been paid to the structural modification of these peptides to enhance their therapeutic efficacy. Successful modification strategies have included D-amino acid substitutions, selective truncation, and fatty acid acylation of the peptide. Through these and other processes, these novel peptide ligand analogs can demonstrate enhanced receptor subtype selectivity, directed signal transduction pathway activation, resistance to proteolytic degradation, and improved systemic bioavailability. In the future, it is likely, through additional modification strategies such as addition of circulation-stabilizing transferrin moieties, that the therapeutic pharmacopeia of drugs targeted towards Class II secretin-like receptors may rival that of the Class I rhodopsin-like receptors that currently provide the majority of clinically used GPCR-based therapeutics. Currently, Class II-based drugs include synthesized analogues of vasoactive intestinal peptide for type 2 diabetes or parathyroid hormone for osteoporosis. PMID:19686775

  15. On the origin of ion selectivity in the Cys-loop receptor family.

    PubMed

    Sine, Steven M; Wang, Hai-Long; Hansen, Scott; Taylor, Palmer

    2010-01-01

    Agonist binding to Cys-loop receptors promotes a large transmembrane ion flux of several million cations or anions per second. To investigate structural bases for the dynamics (MD) simulations, X-ray crystallography, and single channel recording. MD simulations of the muscle nicotinic receptor, imbedded in a lipid bilayer with an applied transmembrane potential, reveal single cation translocation events during transient periods of channel hydration. During the simulation trajectory, cations paused for prolonged periods near several rings of anionic residues projecting from the lumen of the extracellular domain of the receptor, but subsequently the cation moved rapidly through the hydrophobic transmembrane region as the constituent alpha-helices exhibited back and forth rocking motions. Cocrystallization of acetylcholine binding protein with sulfate ions revealed coordination of five sulfates with residues from one of these charged rings; in cation-selective Cys-loop receptors this ring contains negatively charged residues, whereas in anion-selective receptors it contains positively charged residues. In the muscle nicotinic receptor, charge reversal of residues of this ring decreases unitary conductance by up to 80%. Thus in Cys-loop receptors, a series of charged rings along the ion translocation pathway concentrates hydrated ions relative to bulk solution, giving rise to charge selectivity, and then subtle motions of the hydrophobic transmembrane, coupled with transient periods of water filling, enable rapid ion flux.

  16. Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

    PubMed Central

    Cassill, J A; Whitney, M; Joazeiro, C A; Becker, A; Zuker, C S

    1991-01-01

    G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades. Images PMID:1662381

  17. Application of RGS box proteins to evaluate G-protein selectivity in receptor-promoted signaling.

    PubMed

    Hains, Melinda D; Siderovski, David P; Harden, T Kendall

    2004-01-01

    Regulator of G-protein signaling (RGS) domains bind directly to GTP-bound Galpha subunits and accelerate their intrinsic GTPase activity by up to several thousandfold. The selectivity of RGS proteins for individual Galpha subunits has been illustrated. Thus, the expression of RGS proteins can be used to inhibit signaling pathways activated by specific G protein-coupled receptors (GPCRs). This article describes the use of specific RGS domain constructs to discriminate among G(i/o), Gq-and G(12/13)-mediated activation of phospholipase C (PLC) isozymes in COS-7 cells. Overexpression of the N terminus of GRK2 (amino acids 45-178) or p115 RhoGEF (amino acids 1-240) elicited selective inhibition of Galphaq- or Galpha(12/13)-mediated signaling to PLC activation, respectively. In contrast, RGS2 overexpression was found to inhibit PLC activation by both G(i/o)- and Gq-coupled GPCRs. RGS4 exhibited dramatic receptor selectivity in its inhibitory actions; of the G(i/o)- and Gq-coupled GPCRs tested (LPA1, LPA2, P2Y1, S1P3), only the Gq-coupled lysophosphatidic acid-activated LPA2 receptor was found to be inhibited by RGS4 overexpression.

  18. Synthesis, biological and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase

    PubMed Central

    Wang, Lei; Desmoulin, Sita Kugel; Cherian, Christina; Polin, Lisa; White, Kathryn; Kushner, Juiwanna; Fulterer, Andreas; Chang, Min-Hwang; Mitchell, Shermaine; Stout, Mark; Romero, Michael F.; Hou, Zhanjun; Matherly, Larry H.; Gangjee, Aleem

    2011-01-01

    2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1–3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with L-glutamate diethyl ester, followed by saponification, afforded 1–3. Compound 3 selectively inhibited proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including human tumor cells KB and IGROV1 much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1, 2 and 4-atom bridge lengths for the activity of this series. PMID:21879757

  19. Chemical biology methods for investigating G protein-coupled receptor signaling.

    PubMed

    Huber, Thomas; Sakmar, Thomas P

    2014-09-18

    G protein-coupled receptors (GPCRs) are targets for a quarter of prescription drugs. Despite recent progress in structural biology of GPCRs, only few key conformational states in the signal transduction process have been elucidated. Agonist ligands frequently display functional selectivity where activated receptors are biased to either G protein- or arrestin-mediated downstream signaling pathways. Selective manipulation of individual steps in the GPCR activation scheme requires precise information about the kinetics of ligand binding and the dynamics of downstream signaling. One approach is to obtain time-resolved information using receptors tagged with fluorescent or structural probes. Recent advances allow for site-specific introduction of genetically encoded unnatural amino acids into expressed GPCRs. We describe how bioorthogonal functional groups on GPCRs enable the mapping of receptor-ligand interactions and how bioorthogonal chemical reactions can be used to introduce fluorescent labels for single-molecule fluorescence applications to study the kinetics and conformational dynamics of GPCR signaling complexes ("signalosomes").

  20. Novel antigen design for the generation of antibodies to G-protein-coupled receptors.

    PubMed

    Larsson, K; Hofström, C; Lindskog, C; Hansson, M; Angelidou, P; Hökfelt, T; Uhlén, M; Wernérus, H; Gräslund, T; Hober, S

    2011-07-29

    Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.

  1. Phosphorylation of G protein-coupled receptors: from the barcode hypothesis to the flute model.

    PubMed

    Yang, Zhao; Yang, Fan; Zhang, Daolai; Liu, Zhixin; Lin, Amy; Liu, Chuan; Xiao, Peng; Yu, Xiao; Sun, Jin-Peng

    2017-02-28

    Seven transmembrane G protein-coupled receptors (GPCRs) are often phosphorylated at the C-terminus and on intracellular loops in response to various extracellular stimuli. Phosphorylation of GPCRs by GPCR kinases (GRKs) and certain other kinases can promote the recruitment of arrestin molecules. The arrestins critically regulate GPCR functions, not only by mediating receptor desensitization and internalization, but also by redirecting signalling to G protein-independent pathways via interactions with numerous downstream effector molecules. Accumulating evidence over the past decade has given rise to the phospho-barcode hypothesis, which states that ligand-specific phosphorylation patterns of a receptor direct its distinct functional outcomes. Our recent work, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, led to the flute model, which provides preliminary insight into the receptor phospho-coding mechanism, by which receptor phosphorylation patterns are recognized by an array of phosphate-binding pockets on arrestin and are translated into distinct conformations. These selective conformations are recognized by various effector molecules downstream of arrestin. The phospho-barcoding mechanism enables arrestin to recognize a wide range of phosphorylation patterns of GPCRs, contributing to their diverse functions.

  2. G-protein Coupled Estrogen Receptor, Estrogen Receptor α, and Progesterone Receptor Immunohistochemistry in the Hypothalamus of Aging Female Rhesus Macaques Given Long-Term Estradiol Treatment

    PubMed Central

    NAUGLE, MICHELLE M.; NGUYEN, LONG T.; MERCERON, TYLER K.; FILARDO, EDWARD; JANSSEN, WILLIAM G.M.; MORRISON, JOHN H.; RAPP, PETER R.; GORE, ANDREA C.

    2014-01-01

    Steroid hormone receptors are widely and heterogeneously expressed in the brain, and are regulated by age and gonadal hormones. Our goal was to quantify effects of aging, long-term estradiol (E2) treatment, and their interactions, on expression of G protein-coupled estrogen receptor (GPER), estrogen receptor α (ERα) and progesterone receptor (PR) immunoreactivity in two hypothalamic regions, the arcuate (ARC) and the periventricular area (PERI) of rhesus monkeys as a model of menopause and hormone replacement. Ovariectomized (OVX) rhesus macaques were young (~11 years) or aged (~25 years), given oil (vehicle) or E2 every 3 weeks for 2 years. Immunohistochemistry and stereologic analysis of ERα, PR, and GPER was performed. More effects were detected for GPER than the other two receptors. Specifically, GPER cell density in the ARC and PERI, and the percent of GPER-immunoreactive cells in the PERI, were greater in aged than in young monkeys. In addition, we mapped the qualitative distribution of GPER in the monkey hypothalamus and nearby regions. For ERα, E2 treated monkeys tended to have higher cell density than vehicle monkeys in the ARC. The percent of PR density in the PERI tended to be higher in E2 than vehicle monkeys of both ages. This study shows that the aged hypothalamus maintains expression of hormone receptors with age, and that long-term cyclic E2 treatment has few effects on their expression, although GPER was affected more than ERα or PR. This result is surprising in light of evidence for E2 regulation of the receptors studied here, and differences may be due to the selected regions, long-term nature of E2 treatment, among other possibilities. PMID:24862737

  3. G protein-coupled receptors and the regulation of autophagy

    PubMed Central

    Wauson, Eric M.; Dbouk, Hashem A.; Ghosh, Anwesha B.; Cobb, Melanie H.

    2014-01-01

    Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. While autophagy is generally accepted to be regulated in a cell autonomous fashion, recent studies suggest nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets. PMID:24751357

  4. G-protein—coupled receptors, hedgehog signaling and primary cilia

    PubMed Central

    Mukhopadhyay, Saikat; Rohatgi, Rajat

    2014-01-01

    The Hedgehog (Hh) pathway has become an important model to study diverse aspects of cell biology of the primary cilium, and reciprocally, the study of ciliary processes provides an opportunity to solve longstanding mysteries in the mechanism of vertebrate Hh signal transduction. The cilium is emerging as an unique compartment for G-protein—coupled receptor (GPCR) signaling in many systems. Two members of the GPCR family, Smoothened and Gpr161, play important roles in the Hh pathway. We review the current understanding of how these proteins may function to regulate Hh signaling and also highlight some of the critical unanswered questions being tackled by the field. Uncovering GPCR-regulated mechanisms important in Hh signaling may provide therapeutic strategies against the Hh pathway that plays important roles in development, regeneration and cancer. PMID:24845016

  5. Cell-free expression of G-protein-coupled receptors.

    PubMed

    Orbán, Erika; Proverbio, Davide; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.

  6. Molecular dynamics techniques for modeling G protein-coupled receptors.

    PubMed

    McRobb, Fiona M; Negri, Ana; Beuming, Thijs; Sherman, Woody

    2016-10-01

    G protein-coupled receptors (GPCRs) constitute a major class of drug targets and modulating their signaling can produce a wide range of pharmacological outcomes. With the growing number of high-resolution GPCR crystal structures, we have the unprecedented opportunity to leverage structure-based drug design techniques. Here, we discuss a number of advanced molecular dynamics (MD) techniques that have been applied to GPCRs, including long time scale simulations, enhanced sampling techniques, water network analyses, and free energy approaches to determine relative binding free energies. On the basis of the many success stories, including those highlighted here, we expect that MD techniques will be increasingly applied to aid in structure-based drug design and lead optimization for GPCRs.

  7. GPCRDB: an information system for G protein-coupled receptors.

    PubMed Central

    Horn, F; Weare, J; Beukers, M W; Hörsch, S; Bairoch, A; Chen, W; Edvardsen, O; Campagne, F; Vriend, G

    1998-01-01

    The GPCRDB is a G protein-coupled receptor (GPCR) database system aimed at the collection and dissemination of GPCR related data. It holds sequences, mutant data and ligand binding constants as primary (experimental) data. Computationally derived data such as multiple sequence alignments, three dimensional models, phylogenetic trees and two dimensional visualization tools are added to enhance the database's usefulness. The GPCRDB is an EU sponsored project aimed at building a generic molecular class specific database capable of dealing with highly heterogeneous data. GPCRs were chosen as test molecules because of their enormous importance for medical sciences and due to the availability of so much highly heterogeneous data. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm PMID:9399852

  8. Engineering therapeutic antibodies targeting G-protein-coupled receptors.

    PubMed

    Jo, Migyeong; Jung, Sang Taek

    2016-02-05

    G-protein-coupled receptors (GPCRs) are one of the most attractive therapeutic target classes because of their critical roles in intracellular signaling and their clinical relevance to a variety of diseases, including cancer, infection and inflammation. However, high conformational variability, the small exposed area of extracellular epitopes and difficulty in the preparation of GPCR antigens have delayed both the isolation of therapeutic anti-GPCR antibodies as well as studies on the structure, function and biochemical mechanisms of GPCRs. To overcome the challenges in generating highly specific anti-GPCR antibodies with enhanced efficacy and safety, various forms of antigens have been successfully designed and employed for screening with newly emerged systems based on laboratory animal immunization and high-throughput-directed evolution.

  9. Engineering therapeutic antibodies targeting G-protein–coupled receptors

    PubMed Central

    Jo, Migyeong; Jung, Sang Taek

    2016-01-01

    G-protein–coupled receptors (GPCRs) are one of the most attractive therapeutic target classes because of their critical roles in intracellular signaling and their clinical relevance to a variety of diseases, including cancer, infection and inflammation. However, high conformational variability, the small exposed area of extracellular epitopes and difficulty in the preparation of GPCR antigens have delayed both the isolation of therapeutic anti-GPCR antibodies as well as studies on the structure, function and biochemical mechanisms of GPCRs. To overcome the challenges in generating highly specific anti-GPCR antibodies with enhanced efficacy and safety, various forms of antigens have been successfully designed and employed for screening with newly emerged systems based on laboratory animal immunization and high-throughput-directed evolution. PMID:26846450

  10. Peptide drugs to target G protein-coupled receptors.

    PubMed

    Bellmann-Sickert, Kathrin; Beck-Sickinger, Annette G

    2010-09-01

    Major indications for use of peptide-based therapeutics include endocrine functions (especially diabetes mellitus and obesity), infectious diseases, and cancer. Whereas some peptide pharmaceuticals are drugs, acting as agonists or antagonists to directly treat cancer, others (including peptide diagnostics and tumour-targeting pharmaceuticals) use peptides to 'shuttle' a chemotherapeutic agent or a tracer to the tumour and allow sensitive imaging or targeted therapy. Significant progress has been made in the last few years to overcome disadvantages in peptide design such as short half-life, fast proteolytic cleavage, and low oral bioavailability. These advances include peptide PEGylation, lipidisation or multimerisation; the introduction of peptidomimetic elements into the sequences; and innovative uptake strategies such as liposomal, capsule or subcutaneous formulations. This review focuses on peptides targeting G protein-coupled receptors that are promising drug candidates or that have recently entered the pharmaceutical market.

  11. G-protein-coupled receptors, Hedgehog signaling and primary cilia.

    PubMed

    Mukhopadhyay, Saikat; Rohatgi, Rajat

    2014-09-01

    The Hedgehog (Hh) pathway has become an important model to study the cell biology of primary cilia, and reciprocally, the study of ciliary processes provides an opportunity to solve longstanding mysteries in the mechanism of vertebrate Hh signal transduction. The cilium is emerging as an unique compartment for G-protein-coupled receptor (GPCR) signaling in many systems. Two members of the GPCR family, Smoothened and Gpr161, play important roles in the Hh pathway. We review the current understanding of how these proteins may function to regulate Hh signaling and also highlight some of the critical unanswered questions being tackled by the field. Uncovering GPCR-regulated mechanisms important in Hh signaling may provide therapeutic strategies against the Hh pathway that plays important roles in development, regeneration and cancer.

  12. Lysophospholipids and their G protein-coupled receptors in atherosclerosis.

    PubMed

    Li, Ya-Feng; Li, Rong-Shan; Samuel, Sonia B; Cueto, Ramon; Li, Xin-Yuan; Wang, Hong; Yang, Xiao-Feng

    2016-01-01

    Lysophospholipids (LPLs) are bioactive lipid-derived signaling molecules generated by the enzymatic and chemical processes of regiospecific phospholipases on substrates such as membrane phospholipids (PLs) and sphingolipids (SLs). They play a major role as extracellular mediators by activating G-protein coupled receptors (GPCRs) and stimulating diverse cellular responses from their signaling pathways. LPLs are involved in various pathologies of the vasculature system including coronary heart disease and hypertension. Many studies suggest the importance of LPLs in their association with the development of atherosclerosis, a chronic and severe vascular disease. This paper focuses on the pathophysiological effects of different lysophospholipids on atherosclerosis, which may promote the pathogenesis of myocardial infarction and strokes. Their atherogenic biological activities take place in vascular endothelial cells, vascular smooth muscle cells, fibroblasts, monocytes and macrophages, dendritic cells, T-lymphocytes, platelets, etc.

  13. Lysophospholipids and their G protein-coupled receptors in atherosclerosis

    PubMed Central

    Li, Ya-Feng; Li, Rong-Shan; Samuel, Sonia B.; Cueto, Ramon; Li, Xin-Yuan; Wang, Hong; Yang, Xiao-Feng

    2015-01-01

    Lysophospholipids (LPLs) are bioactive lipid-derived signaling molecules generated by the enzymatic and chemical processes of regiospecific phospholipases on substrates such as membrane phospholipids (PLs) and sphingolipids (SLs). They play a major role as extracellular mediators by activating G-protein coupled receptors (GPCRs) and stimulating diverse cellular responses from their signaling pathways. LPLs are involved in various pathologies of the vasculature system including coronary heart disease and hypertension. Many studies suggest the importance of LPLs in their association with the development of atherosclerosis, a chronic and severe vascular disease. This paper focuses on the pathophysiological effects of different lysophospholipids on atherosclerosis, which may promote the pathogenesis of myocardial infarction and strokes. Their atherogenic biological activities take place in vascular endothelial cells, vascular smooth muscle cells, fibroblasts, monocytes and macrophages, dendritic cells, T-lymphocytes, platelets, etc. PMID:26594106

  14. Pathway-selective antagonism of proteinase activated receptor 2

    PubMed Central

    Suen, J Y; Cotterell, A; Lohman, R J; Lim, J; Han, A; Yau, M K; Liu, L; Cooper, M A; Vesey, D A; Fairlie, D P

    2014-01-01

    Background and Purpose Proteinase activated receptor 2 (PAR2) is a GPCR associated with inflammation, metabolism and disease. Clues to understanding how to block PAR2 signalling associated with disease without inhibiting PAR2 activation in normal physiology could be provided by studies of biased signalling. Experimental Approach PAR2 ligand GB88 was profiled for PAR2 agonist and antagonist properties by several functional assays associated with intracellular G-protein-coupled signalling in vitro in three cell types and with PAR2-induced rat paw oedema in vivo. Key Results In HT29 cells, GB88 was a PAR2 antagonist in terms of Ca2+ mobilization and PKC phosphorylation, but a PAR2 agonist in attenuating forskolin-induced cAMP accumulation, increasing ERK1/2 phosphorylation, RhoA activation, myosin phosphatase phosphorylation and actin filament rearrangement. In CHO-hPAR2 cells, GB88 inhibited Ca2+ release, but activated Gi/o and increased ERK1/2 phosphorylation. In human kidney tubule cells, GB88 inhibited cytokine secretion (IL6, IL8, GM-CSF, TNF-α) mediated by PAR2. A rat paw oedema induced by PAR2 agonists was also inhibited by orally administered GB88 and compared with effects of locally administered inhibitors of G-protein coupled pathways. Conclusions and Implications GB88 is a biased antagonist of PAR2 that selectively inhibits PAR2/Gq/11/Ca2+/PKC signalling, leading to anti-inflammatory activity in vivo, while being an agonist in activating three other PAR2-activated pathways (cAMP, ERK, Rho) in human cells. These findings highlight opportunities to design drugs to block specific PAR2-linked signalling pathways in disease, without blocking beneficial PAR2 signalling in normal physiology, and to dissect PAR2-associated mechanisms of disease in vivo. PMID:24821440

  15. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    SciTech Connect

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  16. G-Protein-Coupled Receptors in Adult Neurogenesis

    PubMed Central

    Doze, Van A.

    2012-01-01

    The importance of adult neurogenesis has only recently been accepted, resulting in a completely new field of investigation within stem cell biology. The regulation and functional significance of adult neurogenesis is currently an area of highly active research. G-protein-coupled receptors (GPCRs) have emerged as potential modulators of adult neurogenesis. GPCRs represent a class of proteins with significant clinical importance, because approximately 30% of all modern therapeutic treatments target these receptors. GPCRs bind to a large class of neurotransmitters and neuromodulators such as norepinephrine, dopamine, and serotonin. Besides their typical role in cellular communication, GPCRs are expressed on adult neural stem cells and their progenitors that relay specific signals to regulate the neurogenic process. This review summarizes the field of adult neurogenesis and its methods and specifies the roles of various GPCRs and their signal transduction pathways that are involved in the regulation of adult neural stem cells and their progenitors. Current evidence supporting adult neurogenesis as a model for self-repair in neuropathologic conditions, adult neural stem cell therapeutic strategies, and potential avenues for GPCR-based therapeutics are also discussed. PMID:22611178

  17. Adhesion family of G protein-coupled receptors and cancer.

    PubMed

    Lin, Hsi-Hsien

    2012-01-01

    The adhesion-class G protein-coupled receptors (adhesion-GPCRs) constitute the second largest GPCR sub-family in humans. Adhesion-GPCRs are defined by the chimeric structure of an unusually large extracellular cell-adhesion domain and a GPCR-like seven-pass transmembrane domain. Adhesion-GPCRs are hence expected to display both cellular adhesion and signaling functions in many biological systems. Adhesion-GPCRs are normally expressed in the central nervous, immune, and reproductive systems in a cell type- or tissue-restricted fashion. However, aberrant expression of distinct adhesion-GPCR molecules has been identified in various human cancers with some of the receptors closely associated with cancer development. Tumor-associated adhesion-GPCRs are thought to involve in tumorigenesis by affecting the growth of tumor cells, angiogenesis, tumor cell migration, invasion and metastasis either positively or negatively. Furthermore, some adhesion-GPCRs are considered potential biomarkers for specific types of cancers. In this review article, the expressional characteristics and functional role of cancer-associated adhesion-GPCRs are discussed in depth.

  18. G protein-coupled receptors participate in cytokinesis.

    PubMed

    Zhang, Xin; Bedigian, Anne V; Wang, Wenchao; Eggert, Ulrike S

    2012-10-01

    Cytokinesis, the last step during cell division, is a highly coordinated process that involves the relay of signals from both the outside and inside of the cell. We have a basic understanding of how cells regulate internal events, but how cells respond to extracellular cues is less explored. In a systematic RNAi screen of G protein-coupled receptors (GPCRs) and their effectors, we found that some GPCRs are involved in cytokinesis. RNAi knockdown of these GPCRs caused increased binucleated cell formation, and live cell imaging showed that most formed midbodies but failed at the abscission stage. OR2A4 (olfactory receptor, family 2, subfamily A, member 4) localized to cytokinetic structures in cells and its knockdown caused cytokinesis failure at an earlier stage, likely due to effects on the actin cytoskeleton. Identifying the downstream components that transmit GPCR signals during cytokinesis will be the next step and we show that GIPC1 (GIPC PDZ domain containing family, member 1), an adaptor protein for GPCRs, may play a part. RNAi knockdown of GIPC1 significantly increased binucleated cell formation. Understanding the molecular details of GPCRs and their interaction proteins in cytokinesis regulation will give us important clues about GPCRs signaling as well as how cells communicate with their environment during division.

  19. Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs.

    PubMed

    Pediani, John D; Ward, Richard J; Godin, Antoine G; Marsango, Sara; Milligan, Graeme

    2016-06-17

    Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·μm(-2) human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior.

  20. Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs*

    PubMed Central

    Pediani, John D.; Ward, Richard J.; Godin, Antoine G.; Marsango, Sara

    2016-01-01

    Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·μm−2 human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior. PMID:27080256

  1. Solution structures and molecular interactions of selective melanocortin receptor antagonists.

    PubMed

    Lee, Chul-Jin; Yun, Ji-Hye; Lim, Sung-Kil; Lee, Weontae

    2010-12-01

    The solution structures and inter-molecular interaction of the cyclic melanocortin antagonists SHU9119, JKC363, HS014, and HS024 with receptor molecules have been determined by NMR spectroscopy and molecular modeling. While SHU9119 is known as a nonselective antagonist, JKC363, HS014, and HS024 are selective for the melanocortin subtype-4 receptor (MC4R) involved in modulation of food intake. Data from NMR and molecular dynamics suggest that the conformation of the Trp9 sidechain in the three MC4R-selective antagonists is quite different from that of SHU9119. This result strongly supports the concept that the spatial orientation of the hydrophobic aromatic residue is more important for determining selectivity than the presence of a basic, "arginine-like" moiety responsible for biological activity. We propose that the conformation of hydrophobic residues of MCR antagonists is critical for receptor-specific selectivity.

  2. Revisiting automated G-protein coupled receptor modeling: the benefit of additional template structures for a neurokinin-1 receptor model.

    PubMed

    Kneissl, Benny; Leonhardt, Bettina; Hildebrandt, Andreas; Tautermann, Christofer S

    2009-05-28

    The feasibility of automated procedures for the modeling of G-protein coupled receptors (GPCR) is investigated on the example of the human neurokinin-1 (NK1) receptor. We use a combined method of homology modeling and molecular docking and analyze the information content of the resulting docking complexes regarding the binding mode for further refinements. Moreover, we explore the impact of different template structures, the bovine rhodopsin structure, the human beta(2) adrenergic receptor, and in particular a combination of both templates to include backbone flexibility in the target conformational space. Our results for NK1 modeling demonstrate that model selection from a set of decoys can in general not solely rely on docking experiments but still requires additional mutagenesis data. However, an enrichment factor of 2.6 in a nearly fully automated approach indicates that reasonable models can be created automatically if both available templates are used for model construction. Thus, the recently resolved GPCR structures open new ways to improve the model building fundamentally.

  3. Hyodeoxycholic acid derivatives as liver X receptor α and G-protein-coupled bile acid receptor agonists

    PubMed Central

    De Marino, Simona; Carino, Adriana; Masullo, Dario; Finamore, Claudia; Marchianò, Silvia; Cipriani, Sabrina; Di Leva, Francesco Saverio; Catalanotti, Bruno; Novellino, Ettore; Limongelli, Vittorio; Fiorucci, Stefano; Zampella, Angela

    2017-01-01

    Bile acids are extensively investigated for their potential in the treatment of human disorders. The liver X receptors (LXRs), activated by oxysterols and by a secondary bile acid named hyodeoxycholic acid (HDCA), have been found essential in the regulation of lipid homeostasis in mammals. Unfortunately, LXRα activates lipogenic enzymes causing accumulation of lipid in the liver. In addition to LXRs, HDCA has been also shown to function as ligand for GPBAR1, a G protein coupled receptor for secondary bile acids whose activation represents a promising approach to liver steatosis. In the present study, we report a library of HDCA derivatives endowed with modulatory activity on the two receptors. The lead optimization of HDCA moiety was rationally driven by the structural information on the binding site of the two targets and results from pharmacological characterization allowed the identification of hyodeoxycholane derivatives with selective agonistic activity toward LXRα and GPBAR1 and notably to the identification of the first example of potent dual LXRα/GPBAR1 agonists. The new chemical entities might hold utility in the treatment of dyslipidemic disorders. PMID:28233865

  4. Hyodeoxycholic acid derivatives as liver X receptor α and G-protein-coupled bile acid receptor agonists

    NASA Astrophysics Data System (ADS)

    de Marino, Simona; Carino, Adriana; Masullo, Dario; Finamore, Claudia; Marchianò, Silvia; Cipriani, Sabrina; di Leva, Francesco Saverio; Catalanotti, Bruno; Novellino, Ettore; Limongelli, Vittorio; Fiorucci, Stefano; Zampella, Angela

    2017-02-01

    Bile acids are extensively investigated for their potential in the treatment of human disorders. The liver X receptors (LXRs), activated by oxysterols and by a secondary bile acid named hyodeoxycholic acid (HDCA), have been found essential in the regulation of lipid homeostasis in mammals. Unfortunately, LXRα activates lipogenic enzymes causing accumulation of lipid in the liver. In addition to LXRs, HDCA has been also shown to function as ligand for GPBAR1, a G protein coupled receptor for secondary bile acids whose activation represents a promising approach to liver steatosis. In the present study, we report a library of HDCA derivatives endowed with modulatory activity on the two receptors. The lead optimization of HDCA moiety was rationally driven by the structural information on the binding site of the two targets and results from pharmacological characterization allowed the identification of hyodeoxycholane derivatives with selective agonistic activity toward LXRα and GPBAR1 and notably to the identification of the first example of potent dual LXRα/GPBAR1 agonists. The new chemical entities might hold utility in the treatment of dyslipidemic disorders.

  5. Synthetic FXR agonist GW4064 is a modulator of multiple G protein-coupled receptors.

    PubMed

    Singh, Nidhi; Yadav, Manisha; Singh, Abhishek Kumar; Kumar, Harish; Dwivedi, Shailendra Kumar Dhar; Mishra, Jay Sharan; Gurjar, Anagha; Manhas, Amit; Chandra, Sharat; Yadav, Prem Narayan; Jagavelu, Kumaravelu; Siddiqi, Mohammad Imran; Trivedi, Arun Kumar; Chattopadhyay, Naibedya; Sanyal, Sabyasachi

    2014-05-01

    The synthetic nuclear bile acid receptor (farnesoid X receptor [FXR]) agonist GW4064 is extensively used as a specific pharmacological tool to illustrate FXR functions. We noticed that GW4064 activated empty luciferase reporters in FXR-deficient HEK-293T cells. We postulated that this activity of GW4064 might be routed through as yet unknown cellular targets and undertook an unbiased exploratory approach to identify these targets. Investigations revealed that GW4064 activated cAMP and nuclear factor for activated T-cell response elements (CRE and NFAT-RE, respectively) present on these empty reporters. Whereas GW4064-induced NFAT-RE activation involved rapid intracellular Ca(2+) accumulation and NFAT nuclear translocation, CRE activation involved soluble adenylyl cyclase-dependent cAMP accumulation and Ca(2+)-calcineurin-dependent nuclear translocation of transducers of regulated CRE-binding protein 2. Use of dominant negative heterotrimeric G-protein minigenes revealed that GW4064 caused activation of Gαi/o and Gq/11 G proteins. Sequential pharmacological inhibitor-based screening and radioligand-binding studies revealed that GW4064 interacted with multiple G protein-coupled receptors. Functional studies demonstrated that GW4064 robustly activated H1 and H4 and inhibited H2 histamine receptor signaling events. We also found that MCF-7 breast cancer cells, reported to undergo GW4064-induced apoptosis in an FXR-dependent manner, did not express FXR, and the GW4064-mediated apoptosis, also apparent in HEK-293T cells, could be blocked by selective histamine receptor regulators. Taken together, our results demonstrate identification of histamine receptors as alternate targets for GW4064, which not only necessitates cautious interpretation of the biological functions attributed to FXR using GW4064 as a pharmacological tool but also provides a basis for the rational designing of new pharmacophores for histamine receptor modulation.

  6. Denatured G-protein coupled receptors as immunogens to generate highly specific antibodies.

    PubMed

    Talmont, Franck; Moulédous, Lionel; Boué, Jérôme; Mollereau, Catherine; Dietrich, Gilles

    2012-01-01

    G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (μ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.

  7. γ-Aminobutyric Acid B Receptor Mediated Inhibition of Gonadotropin-Releasing Hormone Neurons Is Suppressed by Kisspeptin-G Protein-Coupled Receptor 54 Signaling

    PubMed Central

    Zhang, Chunguang; Bosch, Martha A.; Rønnekleiv, Oline K.; Kelly, Martin J.

    2009-01-01

    γ-Aminobutyric acid (GABA) is one of the most important neurotransmitters that regulate the excitability of GnRH neurons. Numerous studies have shown that GABA activates Cl− currents in GnRH neurons, and these effects are antagonized by GABAA receptor antagonists. The GABAB receptor is a heterodimer composed of GABAB R1 and R2, and although both subunits have been localized in GnRH neurons, nothing is known about the cellular signaling of this Gαi,o-coupled receptor in GnRH neurons. Using whole-cell recordings from mouse enhanced green fluorescent protein-GnRH neurons, we found that the GABAB receptor agonist baclofen hyperpolarized GnRH neurons through activation of an inwardly rectifying K+ current in a concentration-dependent manner. The effects of baclofen were antagonized by the selective GABAB receptor antagonist CGP 52432 with a Ki (inhibitory constant) of 85 nm. Furthermore, in the presence of the GABAA receptor antagonist picrotoxin, GABA hyperpolarized GnRH neurons in a similar manner. Treatment with 17β-estradiol as compared with oil vehicle did not significantly alter either the EC50 for the baclofen-induced response (0.8 ± 0.1 vs. 1.0 ± 0.1 μm, respectively) or the maximal outward current (10.8 ± 1.7 pA vs. 11.4 ± 0.6 pA, respectively) in GnRH neurons. However, the outward current (and membrane hyperpolarization) was abrogated by submaximal concentrations of the G protein-coupled receptor 54 (GPR54) agonist kisspeptin-10 in both groups, indicating that Gαq-coupled (GPR54) can desensitize the GABAB receptor-mediated response. Therefore, the activation of GABAB receptors in GnRH neurons may provide increased inhibitory tone during estrogen-negative feedback states that is attenuated by kisspeptin during positive feedback. PMID:19164470

  8. 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.

  9. Exploiting Receptor Competition to Enhance Nanoparticle Binding Selectivity

    NASA Astrophysics Data System (ADS)

    Angioletti-Uberti, Stefano

    2017-02-01

    Nanoparticles functionalized with multiple ligands can be programed to bind biological targets depending on the receptors they express, providing a general mechanism exploited in various technologies, from selective drug delivery to biosensing. For binding to be highly selective, ligands should exclusively interact with specific targeted receptors, because the formation of bonds with other, untargeted ones would lead to nonspecific binding and potentially harmful behavior. This poses a particular problem for multivalent nanoparticles, because even very weak bonds can collectively lead to strong binding. A statistical mechanical model is used here to describe how competition between different receptors together with multivalent effects can be harnessed to design ligand-functionalized nanoparticles insensitive to the presence of untargeted receptors, preventing nonspecific binding.

  10. Structural Basis for Hormone Recognition by the Human CRFR2[alpha] G Protein-coupled Receptor

    SciTech Connect

    Pal, Kuntal; Swaminathan, Kunchithapadam; Xu, H. Eric; Pioszak, Augen A.

    2012-05-09

    The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2{alpha} isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2{alpha} ECD bound to each of the Ucn peptides. The CRFR2{alpha} ECD forms the same fold observed for the CRFR1 and mouse CRFR2{beta} ECDs but contains a unique N-terminal {alpha}-helix formed by its pseudo signal peptide. The CRFR2{alpha} ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the {alpha}-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2{alpha} Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.

  11. Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors

    NASA Astrophysics Data System (ADS)

    Woods, Kristina N.; Pfeffer, Jürgen; Dutta, Arpana; Klein-Seetharaman, Judith

    2016-11-01

    G protein-coupled receptors are a large family of membrane proteins activated by a variety of structurally diverse ligands making them highly adaptable signaling molecules. Despite recent advances in the structural biology of this protein family, the mechanism by which ligands induce allosteric changes in protein structure and dynamics for its signaling function remains a mystery. Here, we propose the use of terahertz spectroscopy combined with molecular dynamics simulation and protein evolutionary network modeling to address the mechanism of activation by directly probing the concerted fluctuations of retinal ligand and transmembrane helices in rhodopsin. This approach allows us to examine the role of conformational heterogeneity in the selection and stabilization of specific signaling pathways in the photo-activation of the receptor. We demonstrate that ligand-induced shifts in the conformational equilibrium prompt vibrational resonances in the protein structure that link the dynamics of conserved interactions with fluctuations of the active-state ligand. The connection of vibrational modes creates an allosteric association of coupled fluctuations that forms a coherent signaling pathway from the receptor ligand-binding pocket to the G-protein activation region. Our evolutionary analysis of rhodopsin-like GPCRs suggest that specific allosteric sites play a pivotal role in activating structural fluctuations that allosterically modulate functional signals.

  12. Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors.

    PubMed

    Woods, Kristina N; Pfeffer, Jürgen; Dutta, Arpana; Klein-Seetharaman, Judith

    2016-11-16

    G protein-coupled receptors are a large family of membrane proteins activated by a variety of structurally diverse ligands making them highly adaptable signaling molecules. Despite recent advances in the structural biology of this protein family, the mechanism by which ligands induce allosteric changes in protein structure and dynamics for its signaling function remains a mystery. Here, we propose the use of terahertz spectroscopy combined with molecular dynamics simulation and protein evolutionary network modeling to address the mechanism of activation by directly probing the concerted fluctuations of retinal ligand and transmembrane helices in rhodopsin. This approach allows us to examine the role of conformational heterogeneity in the selection and stabilization of specific signaling pathways in the photo-activation of the receptor. We demonstrate that ligand-induced shifts in the conformational equilibrium prompt vibrational resonances in the protein structure that link the dynamics of conserved interactions with fluctuations of the active-state ligand. The connection of vibrational modes creates an allosteric association of coupled fluctuations that forms a coherent signaling pathway from the receptor ligand-binding pocket to the G-protein activation region. Our evolutionary analysis of rhodopsin-like GPCRs suggest that specific allosteric sites play a pivotal role in activating structural fluctuations that allosterically modulate functional signals.

  13. Vibrational resonance, allostery, and activation in rhodopsin-like G protein-coupled receptors

    PubMed Central

    Woods, Kristina N.; Pfeffer, Jürgen; Dutta, Arpana; Klein-Seetharaman, Judith

    2016-01-01

    G protein-coupled receptors are a large family of membrane proteins activated by a variety of structurally diverse ligands making them highly adaptable signaling molecules. Despite recent advances in the structural biology of this protein family, the mechanism by which ligands induce allosteric changes in protein structure and dynamics for its signaling function remains a mystery. Here, we propose the use of terahertz spectroscopy combined with molecular dynamics simulation and protein evolutionary network modeling to address the mechanism of activation by directly probing the concerted fluctuations of retinal ligand and transmembrane helices in rhodopsin. This approach allows us to examine the role of conformational heterogeneity in the selection and stabilization of specific signaling pathways in the photo-activation of the receptor. We demonstrate that ligand-induced shifts in the conformational equilibrium prompt vibrational resonances in the protein structure that link the dynamics of conserved interactions with fluctuations of the active-state ligand. The connection of vibrational modes creates an allosteric association of coupled fluctuations that forms a coherent signaling pathway from the receptor ligand-binding pocket to the G-protein activation region. Our evolutionary analysis of rhodopsin-like GPCRs suggest that specific allosteric sites play a pivotal role in activating structural fluctuations that allosterically modulate functional signals. PMID:27849063

  14. Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway.

    PubMed Central

    Price, L A; Kajkowski, E M; Hadcock, J R; Ozenberger, B A; Pausch, M H

    1995-01-01

    A detailed analysis of structural and functional aspects of G-protein-coupled receptors, as well as discovery of novel pharmacophores that exert their effects on members of this class of receptors, will be facilitated by development of a yeast-based bioassay. To that end, yeast strains that functionally express the rat somatostatin receptor subtype 2 (SSTR2) were constructed. High-affinity binding sites for somatostatin ([125I-Tyr-11]S-14) comparable to those in native tissues were detected in yeast membrane extracts at levels equivalent to the alpha-mating pheromone receptor (Ste2p). Somatostatin-dependent growth of strains modified by deletion of genes encoding components of the pheromone response pathway was detected through induction of a pheromone-responsive HIS3 reporter gene, enabling cells to grow on medium lacking histidine. Dose-dependent growth responses to S-14 and related SSTR2 subtype-selective agonists that were proportional to the affinity of the ligands for SSTR2 were observed. The growth response required SSTR2, G alpha proteins, and an intact signal transduction pathway. The sensitivity of the bioassay was affected by intracellular levels of the G alpha protein. A mutation in the SST2 gene, which confers supersensitivity to pheromone, was found to significantly enhance the growth response to S-14. In sst2 delta cells, SSTR2 functionally interacted with both a chimeric yeast/mammalian G alpha protein and the yeast G alpha protein, Gpa1p; to promote growth. These yeast strains should serve as a useful in vivo reconstitution system for examination of molecular interactions of the G-protein-coupled receptors and G proteins. PMID:7565771

  15. [Interest of selective progesterone receptor modulators in endometriosis].

    PubMed

    Merviel, P; Lourdel, E; Sanguin, S; Gagneur, O; Cabry, R; Nasreddine, A

    2013-09-01

    The SPRM (selective progesterone receptor modulators) are agonists and/or antagonists of progesterone receptor. They are responsible for anovulation, amenorrhea and a lower prostaglandin levels, which leads to an improvement in pain and regression of lesions in endometriosis. On the endometrium, a particular aspect, the progesterone receptor modulator-associated endometrial changes (PAEC), raises additional studies to verify its harmlessness. However, due to the lack of hypoestrogenism and metabolic effects with these drugs, it is very likely that the SPRM will in the near future an important place in the treatment of endometriosis.

  16. Designing of a fluoride selective receptor through molecular orbital engineering

    NASA Astrophysics Data System (ADS)

    Mishra, Rakesh K.; Kumar, Virendra; Diwan, Uzra; Upadhyay, K. K.; Roy Chowdhury, P. K.

    2012-11-01

    The stepwise substitution of appropriate groups over the 3-[(2,4-dinitro-phenyl)-hydrazono]-butyric acid ethyl ester (R3) lead receptor R1 which showed selectivity towards fluoride in DMSO. The UV-vis and 1H NMR titration studies revealed the details of the binding between receptor R1 and fluoride. The receptor R1 also recognized fluoride in a toothpaste solution to as low as 50 ppm. The theoretical simulations of recognition event at Density Functional Theory (DFT) level using B3LYP/6-31G** basis set and polarizable continuum model (PCM) approach lead a semi-quantitative match with the experimental results.

  17. Structural basis for selective activation of ABA receptors

    SciTech Connect

    Peterson, Francis C.; Burgie, E. Sethe; Park, Sang-Youl; Jensen, Davin R.; Weiner, Joshua J.; Bingman, Craig A.; Chang, Chia-En A.; Cutler, Sean R.; Phillips, Jr., George N.; Volkman, Brian F.

    2010-11-01

    Changing environmental conditions and lessening fresh water supplies have sparked intense interest in understanding and manipulating abscisic acid (ABA) signaling, which controls adaptive responses to drought and other abiotic stressors. We recently discovered a selective ABA agonist, pyrabactin, and used it to discover its primary target PYR1, the founding member of the PYR/PYL family of soluble ABA receptors. To understand pyrabactin's selectivity, we have taken a combined structural, chemical and genetic approach. We show that subtle differences between receptor binding pockets control ligand orientation between productive and nonproductive modes. Nonproductive binding occurs without gate closure and prevents receptor activation. Observations in solution show that these orientations are in rapid equilibrium that can be shifted by mutations to control maximal agonist activity. Our results provide a robust framework for the design of new agonists and reveal a new mechanism for agonist selectivity.

  18. Deletion of G-protein-coupled receptor 55 promotes obesity by reducing physical activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cannabinoid receptor 1 (CB1) is the best-characterized cannabinoid receptor, and CB1 antagonists are used in clinical trials to treat obesity. Because of the wide range of CB1 functions, the side effects of CB1 antagonists pose serious concerns. G-protein-coupled receptor 55 (GPR55) is an atypical c...

  19. Molecular Recognition of Corticotropin releasing Factor by Its G protein-coupled Receptor CRFR1

    SciTech Connect

    Pioszak, Augen A.; Parker, Naomi R.; Suino-Powell, Kelly; Xu, H. Eric

    2009-01-15

    The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observed for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.

  20. Coupling of human delta-opioid receptor to retinal rod transducin in Chinese hamster ovary cells.

    PubMed

    Varga, E V; Stropova, D; Kim, T; Wang, M; Roeske, W R; Yamamura, H I

    2000-01-01

    Reverse transcription-polymerase chain reaction was used to identify the pertussis toxin (Ptx)-sensitive G protein alpha-subunit pool in Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. We detected the presence of mRNA for G(ialpha2), G(ialpha3), and G(oalpha) in both cell lines. G(ialpha1) and G(alphaz) mRNAs were not detected. We also found a homolog of the retinal rod transducin (G(talpha1)) in CHO, and the mouse cone transducin (G(talpha2)) in B82 cells. The presence of the transducin alpha-subunit proteins in CHO and B82 cells was confirmed by immunoprecipitation with specific antibodies. To test the interaction of heterologously expressed receptors with transducin in CHO cells, a Ptx-insensitive (C347S) rod transducin mutant was transfected into a CHO cell line stably expressing the human delta-opioid receptor (hDOR/CHO). (+)-4-[(alphaR)-alpha-((2S,2R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, a selective delta-opioid receptor agonist, stimulated guanosine-5'-O-(3-[(35)S]thio)triphosphate binding by 293 +/- 36% after Ptx pretreatment in the mutant cell line with an EC(50) value of 54 +/- 32 nM, showing that transducin can functionally couple to the human delta-opioid receptors in these cells.

  1. [Roles of G protein-coupled estrogen receptor in the male reproductive system].

    PubMed

    Chen, Kai-hong; Zhang, Xian; Jiang, Xue-wu

    2016-02-01

    The G protein-coupled estrogen receptor (GPER), also known as G protein-coupled receptor 30 (GPR30), was identified in the recent years as a functional membrane receptor different from the classical nuclear estrogen receptors. This receptor is widely expressed in the cortex, cerebellum, hippocampus, heart, lung, liver, skeletal muscle, and the urogenital system. It is responsible for the mediation of nongenomic effects associated with estrogen and its derivatives, participating in the physiological activities of the body. The present study reviews the molecular structure, subcellular localization, signaling pathways, distribution, and function of GPER in the male reproductive system.

  2. G Protein–Coupled Receptor Oligomerization Revisited: Functional and Pharmacological Perspectives

    PubMed Central

    Casadó, Vicent; Devi, Lakshmi A.; Filizola, Marta; Jockers, Ralf; Lohse, Martin J.; Milligan, Graeme; Pin, Jean-Philippe; Guitart, Xavier

    2014-01-01

    Most evidence indicates that, as for family C G protein–coupled receptors (GPCRs), family A GPCRs form homo- and heteromers. Homodimers seem to be a predominant species, with potential dynamic formation of higher-order oligomers, particularly tetramers. Although monomeric GPCRs can activate G proteins, the pentameric structure constituted by one GPCR homodimer and one heterotrimeric G protein may provide a main functional unit, and oligomeric entities can be viewed as multiples of dimers. It still needs to be resolved if GPCR heteromers are preferentially heterodimers or if they are mostly constituted by heteromers of homodimers. Allosteric mechanisms determine a multiplicity of possible unique pharmacological properties of GPCR homomers and heteromers. Some general mechanisms seem to apply, particularly at the level of ligand-binding properties. In the frame of the dimer-cooperativity model, the two-state dimer model provides the most practical method to analyze ligand–GPCR interactions when considering receptor homomers. In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to different intrinsic efficacy of ligands for different signaling pathways (functional selectivity). This gives a rationale for the use of GPCR oligomers, and particularly heteromers, as novel targets for drug development. Herein, we review the functional and pharmacological properties of GPCR oligomers and provide some guidelines for the application of discrete direct screening and high-throughput screening approaches to the discovery of receptor-heteromer selective compounds. PMID:24515647

  3. G protein-coupled receptor 35: an emerging target in inflammatory and cardiovascular disease

    PubMed Central

    Divorty, Nina; Mackenzie, Amanda E.; Nicklin, Stuart A.; Milligan, Graeme

    2015-01-01

    G protein-coupled receptor 35 (GPR35) is an orphan receptor, discovered in 1998, that has garnered interest as a potential therapeutic target through its association with a range of diseases. However, a lack of pharmacological tools and the absence of convincingly defined endogenous ligands have hampered the understanding of function necessary to exploit it therapeutically. Although several endogenous molecules can activate GPR35 none has yet been confirmed as the key endogenous ligand due to reasons that include lack of biological specificity, non-physiologically relevant potency and species ortholog selectivity. Recent advances have identified several highly potent synthetic agonists and antagonists, as well as agonists with equivalent potency at rodent and human orthologs, which will be useful as tool compounds. Homology modeling and mutagenesis studies have provided insight into the mode of ligand binding and possible reasons for the species selectivity of some ligands. Advances have also been made in determining the role of the receptor in disease. In the past, genome-wide association studies have associated GPR35 with diseases such as inflammatory bowel disease, type 2 diabetes, and coronary artery disease. More recent functional studies have implicated it in processes as diverse as heart failure and hypoxia, inflammation, pain transduction and synaptic transmission. In this review, we summarize the progress made in understanding the molecular pharmacology, downstream signaling and physiological function of GPR35, and discuss its emerging potential applications as a therapeutic target. PMID:25805994

  4. Cross-Electrophile Coupling: Principles of Reactivity and Selectivity

    PubMed Central

    2015-01-01

    A critical overview of the catalytic joining of two different electrophiles, cross-electrophile coupling (XEC), is presented with an emphasis on the central challenge of cross-selectivity. Recent synthetic advances and mechanistic studies have shed light on four possible methods for overcoming this challenge: (1) employing an excess of one reagent; (2) electronic differentiation of starting materials; (3) catalyst–substrate steric matching; and (4) radical chain processes. Each method is described using examples from the recent literature. PMID:24820397

  5. International Union of Basic and Clinical Pharmacology. XCVII. G Protein–Coupled Estrogen Receptor and Its Pharmacologic Modulators

    PubMed Central

    2015-01-01

    Estrogens are critical mediators of multiple and diverse physiologic effects throughout the body in both sexes, including the reproductive, cardiovascular, endocrine, nervous, and immune systems. As such, alterations in estrogen function play important roles in many diseases and pathophysiological conditions (including cancer), exemplified by the lower prevalence of many diseases in premenopausal women. Estrogens mediate their effects through multiple cellular receptors, including the nuclear receptor family (ERα and ERβ) and the G protein–coupled receptor (GPCR) family (GPR30/G protein–coupled estrogen receptor [GPER]). Although both receptor families can initiate rapid cell signaling and transcriptional regulation, the nuclear receptors are traditionally associated with regulating gene expression, whereas GPCRs are recognized as mediating rapid cellular signaling. Estrogen-activated pathways are not only the target of multiple therapeutic agents (e.g., tamoxifen, fulvestrant, raloxifene, and aromatase inhibitors) but are also affected by a plethora of phyto- and xeno-estrogens (e.g., genistein, coumestrol, bisphenol A, dichlorodiphenyltrichloroethane). Because of the existence of multiple estrogen receptors with overlapping ligand specificities, expression patterns, and signaling pathways, the roles of the individual receptors with respect to the diverse array of endogenous and exogenous ligands have been challenging to ascertain. The identification of GPER-selective ligands however has led to a much greater understanding of the roles of this receptor in normal physiology and disease as well as its interactions with the classic estrogen receptors ERα and ERβ and their signaling pathways. In this review, we describe the history and characterization of GPER over the past 15 years focusing on the pharmacology of steroidal and nonsteroidal compounds that have been employed to unravel the biology of this most recently recognized estrogen receptor. PMID

  6. Type-1, but Not Type-5, Metabotropic Glutamate Receptors are Coupled to Polyphosphoinositide Hydrolysis in the Retina.

    PubMed

    Romano, Maria Rosaria; Di Menna, Luisa; Scarselli, Pamela; Mascio, Giada; Madonna, Michele; Notartomaso, Serena; Puliti, Aldamaria; Bruno, Valeria; Battaglia, Giuseppe; Nicoletti, Ferdinando

    2016-04-01

    mGlu1 and mGlu5 metabotropic glutamate receptors are expressed in the vertebrate retina, and are co-localized in some retinal neurons. It is believed that both receptors are coupled to polyphosphoinositide (PI) hydrolysis in the retina and their function may diverge in some cells because of a differential engagement of downstream signaling molecules. Here, we show that it is only the mGlu1 receptor that is coupled to PI hydrolysis in the retina. We used either bovine retinal slices or intact mouse retinas challenged with the mixed mGlu1/5 receptor agonist, DHPG. In both models, DHPG-stimulated PI hydrolysis was abrogated by the selective mGlu1 receptor antagonist, JNJ16259685, but was insensitive to the mGlu5 receptor antagonist, MPEP. In addition, the PI response to DHPG was unchanged in the retina of mGlu5(-/-) mice but was abolished in the retina of crv4 mice lacking mGlu1 receptors. Stimulation of the mitogen-activated protein kinase pathway by DHPG in intact mouse retinas were also entirely mediated by mGlu1 receptors. Our data provide the first example of a tissue in which a biochemically detectable PI response is mediated by mGlu1, but not mGlu5, receptors. Hence, bovine retinal slices might be used as a model for the functional screening of mGlu1 receptor ligands. In addition, the mGlu1 receptor caters the potential as a drug target in the experimental treatment of degenerative disorders of the retina.

  7. Discovery, synthesis, and molecular pharmacology of selective positive allosteric modulators of the δ-opioid receptor.

    PubMed

    Burford, Neil T; Livingston, Kathryn E; Canals, Meritxell; Ryan, Molly R; Budenholzer, Lauren M L; Han, Ying; Shang, Yi; Herbst, John J; O'Connell, Jonathan; Banks, Martyn; Zhang, Litao; Filizola, Marta; Bassoni, Daniel L; Wehrman, Tom S; Christopoulos, Arthur; Traynor, John R; Gerritz, Samuel W; Alt, Andrew

    2015-05-28

    Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, β-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.

  8. Tyrosine sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3.

    PubMed

    Zhu, John Z; Millard, Christopher J; Ludeman, Justin P; Simpson, Levi S; Clayton, Daniel J; Payne, Richard J; Widlanski, Theodore S; Stone, Martin J

    2011-03-08

    The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.

  9. Induction of RAGE Shedding by Activation of G Protein-Coupled Receptors

    PubMed Central

    Metz, Verena V.; Kojro, Elzbieta; Rat, Dorothea; Postina, Rolf

    2012-01-01

    The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimes disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cβ, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cβ, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca2+ signaling, PKCα/PKCβI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNA-mediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount of

  10. Induction of RAGE shedding by activation of G protein-coupled receptors.

    PubMed

    Metz, Verena V; Kojro, Elzbieta; Rat, Dorothea; Postina, Rolf

    2012-01-01

    The multiligand Receptor for Advanced Glycation End products (RAGE) is involved in various pathophysiological processes, including diabetic inflammatory conditions and Alzheimers disease. Full-length RAGE, a cell surface-located type I membrane protein, can proteolytically be converted by metalloproteinases ADAM10 and MMP9 into a soluble RAGE form. Moreover, administration of recombinant soluble RAGE suppresses activation of cell surface-located RAGE by trapping RAGE ligands. Therefore stimulation of RAGE shedding might have a therapeutic value regarding inflammatory diseases. We aimed to investigate whether RAGE shedding is inducible via ligand-induced activation of G protein-coupled receptors (GPCRs). We chose three different GPCRs coupled to distinct signaling cascades: the V2 vasopressin receptor (V2R) activating adenylyl cyclase, the oxytocin receptor (OTR) linked to phospholipase Cβ, and the PACAP receptor (subtype PAC1) coupled to adenylyl cyclase, phospholipase Cβ, calcium signaling and MAP kinases. We generated HEK cell lines stably coexpressing an individual GPCR and full-length RAGE and then investigated GPCR ligand-induced activation of RAGE shedding. We found metalloproteinase-mediated RAGE shedding on the cell surface to be inducible via ligand-specific activation of all analyzed GPCRs. By using specific inhibitors we have identified Ca(2+) signaling, PKCα/PKCβI, CaMKII, PI3 kinases and MAP kinases to be involved in PAC1 receptor-induced RAGE shedding. We detected an induction of calcium signaling in all our cell lines coexpressing RAGE and different GPCRs after agonist treatment. However, we did not disclose a contribution of adenylyl cyclase in RAGE shedding induction. Furthermore, by using a selective metalloproteinase inhibitor and siRNA-mediated knock-down approaches, we show that ADAM10 and/or MMP9 are playing important roles in constitutive and PACAP-induced RAGE shedding. We also found that treatment of mice with PACAP increases the amount

  11. Muscarinic receptor subtype selectivity of novel heterocyclic QNB analogues

    SciTech Connect

    Baumgold, J.; Cohen, V.I.; Paek, R.; Reba, R.C. )

    1991-01-01

    In an effort at synthesizing centrally-active subtype-selective antimuscarinic agents, the authors derivatized QNB (quinuclidinyl benzilate), a potent muscarinic antagonist, by replacing one of the phenyl groups with less lipophilic heterocyclic moieties. The displacement of ({sup 3}H)-N-methyl scopolamine binding by these novel compounds to membranes from cells expressing ml - m4 receptor subtypes was determined. Most of the novel 4-bromo-QNB analogues were potent and slightly selective for ml receptors. The 2-thienyl derivative was the most potent, exhibiting a 2-fold greater potency than BrQNB at ml receptors, and a 4-fold greater potency than BrQNB at ml receptors, and a 4-fold greater potency at m2 receptors. This compound was also considerably less lipophilic than BrQNB as determined from its retention time on C18 reverse phase HPLC. This compound may therefore be useful both for pharmacological studies and as a candidate for a radioiodinated SPECT imaging agent for ml muscarinic receptors in human brain.

  12. Investigation of orexin-2 selective receptor antagonists: Structural modifications resulting in dual orexin receptor antagonists.

    PubMed

    Skudlarek, Jason W; DiMarco, Christina N; Babaoglu, Kerim; Roecker, Anthony J; Bruno, Joseph G; Pausch, Mark A; O'Brien, Julie A; Cabalu, Tamara D; Stevens, Joanne; Brunner, Joseph; Tannenbaum, Pamela L; Wuelfing, W Peter; Garson, Susan L; Fox, Steven V; Savitz, Alan T; Harrell, Charles M; Gotter, Anthony L; Winrow, Christopher J; Renger, John J; Kuduk, Scott D; Coleman, Paul J

    2017-03-15

    In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX2R subtype and culminating in the discovery of 23, a highly potent, OX2R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX1R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.

  13. Alpha-Bulges in G Protein-Coupled Receptors

    PubMed Central

    van der Kant, Rob; Vriend, Gert

    2014-01-01

    Agonist binding is related to a series of motions in G protein-coupled receptors (GPCRs) that result in the separation of transmembrane helices III and VI at their cytosolic ends and subsequent G protein binding. A large number of smaller motions also seem to be associated with activation. Most helices in GPCRs are highly irregular and often contain kinks, with extensive literature already available about the role of prolines in kink formation and the precise function of these kinks. GPCR transmembrane helices also contain many α-bulges. In this article we aim to draw attention to the role of these α-bulges in ligand and G-protein binding, as well as their role in several aspects of the mobility associated with GPCR activation. This mobility includes regularization and translation of helix III in the extracellular direction, a rotation of the entire helix VI, an inward movement of the helices near the extracellular side, and a concerted motion of the cytosolic ends of the helices that makes their orientation appear more circular and that opens up space for the G protein to bind. In several cases, α-bulges either appear or disappear as part of the activation process. PMID:24806342

  14. Receptor-coupled effector systems and their interactions

    SciTech Connect

    Wiener, E.C.

    1988-01-01

    We investigated the modulation of intracellular signal generation by receptor-coupled effector systems in B lymphocytes, and whether these alterations are consistent with the effects of prostaglandins. TPA (12-O-tetradecanoyl phorbol-13-acetate) and sn-1,2,-dioctanoylglycerol (diC{sub 8}) substitute for lipid derived signals which activate protein kinase C. Pretreating splenocytes from athymic nude mice with 100nM TPA or 5 {mu}M diC{sub 8} potentiated the forskolin-induced increased in cAMP (measured by radioimmunoassay) 2.5 and 3.0 times (respectively), but they decreased the PGE{sub 1}-induced cAMP rise 48% and 35% (respectively). Goat anti-mouse IgM, which activates diacylglycerol production, potentiated the forskolin-induced cAMP increase by 76%, but reduced that of PGE{sub 1} by 30%. Rabbit anti-mouse IgG, its F(ab{prime}){sub 2} fragment, or goat anti-mouse IGM induced increases in the cytosolic free (Ca{sup 2+}), (Ca{sup 2+}){sub i}, which TPA inhibited. In contrast, TPA potential antibody-induced {sup 3}H-thymidine (85x) and {sup 3}H-uridine (30x) uptake in B lymphocytes.

  15. GRK2: multiple roles beyond G protein-coupled receptor desensitization

    PubMed Central

    Evron, Tama; Daigle, Tanya L.; Caron, Marc G.

    2012-01-01

    G protein-coupled receptor kinases (GRKs) regulate numerous G protein-coupled receptors (GPCRs) by phosphorylating the intracellular domain of the active receptor, resulting in receptor desensitization and internalization. GRKs also regulate GPCR trafficking in a phosphorylation-independent manner via direct protein-protein interactions. Emerging evidence suggests that GRK2, the most widely studied member of this family of kinases, modulates multiple cellular responses in various physiological contexts by either phosphorylating non-receptor substrates or by directly interacting with signaling molecules. In this review, we discuss traditional and newly discovered roles of GRK2 in receptor internalization and signaling as well as its impact on non-receptor substrates. We also discuss novel exciting roles of GRK2 in the regulation of dopamine receptor signaling and in the activation and trafficking of the atypical GPCR, Smoothened (Smo). PMID:22277298

  16. Selection for Genes Encoding Secreted Proteins and Receptors

    NASA Astrophysics Data System (ADS)

    Klein, Robert D.; Gu, Qimin; Goddard, Audrey; Rosenthal, Arnon

    1996-07-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

  17. Ligand-Driven T Cell Receptor Selection in Celiac Disease.

    PubMed

    Singh, Nishant K; Baker, Brian M

    2016-10-04

    Recognition of antigens by T cell receptors (TCRs) underlies cellular immunity. By comparing how different TCRs recognize the key antigens associated with celiac disease, Petersen et al. (2016), in this issue of Structure, show how celiac antigen properties select immunologically distinct yet structurally and physically compatible TCRs, ultimately driving autoimmunity.

  18. Selective nonpeptidic fluorescent ligands for oxytocin receptor: design, synthesis, and application to time-resolved FRET binding assay.

    PubMed

    Karpenko, Iuliia A; Margathe, Jean-François; Rodriguez, Thiéric; Pflimlin, Elsa; Dupuis, Elodie; Hibert, Marcel; Durroux, Thierry; Bonnet, Dominique

    2015-03-12

    The design and the synthesis of the first high-affinity fluorescent ligands for oxytocin receptor (OTR) are described. These compounds enabled the development of a TR-FRET based assay for OTR, readily amenable to high throughput screening. The validation of the assay was achieved by competition experiments with both peptide and nonpeptide OTR ligands as competitors. These probes represent the first selective fluorescent ligands for the oxytocin G protein-coupled receptor.

  19. Novel RNAi-mediated approach to G protein-coupled receptor deorphanization: proof of principle and characterization of a planarian 5-HT receptor.

    PubMed

    Zamanian, Mostafa; Agbedanu, Prince N; Wheeler, Nicolas J; McVeigh, Paul; Kimber, Michael J; Day, Tim A

    2012-01-01

    G protein-coupled receptors (GPCRs) represent the largest known superfamily of membrane proteins extending throughout the Metazoa. There exists ample motivation to elucidate the functional properties of GPCRs given their role in signal transduction and their prominence as drug targets. In many target organisms, these efforts are hampered by the unreliable nature of heterologous receptor expression platforms. We validate and describe an alternative loss-of-function approach for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. Our efforts are focused on the phylum Platyhelminthes, given the heavy health burden exacted by pathogenic flatworms, as well as the role of free-living flatworms as model organisms for the study of developmental biology. RNA interference (RNAi) was used in conjunction with a biochemical endpoint assay to monitor cAMP modulation in response to the translational suppression of individual receptors. As proof of principle, this approach was used to confirm the neuropeptide GYIRFamide as the cognate ligand for the planarian neuropeptide receptor GtNPR-1, while revealing its endogenous coupling to Gα(i/o). The method was then extended to deorphanize a novel Gα(s)-coupled planarian serotonin receptor, DtSER-1. A bioinformatics protocol guided the selection of receptor candidates mediating 5-HT-evoked responses. These results provide functional data on a neurotransmitter central to flatworm biology, while establishing the great potential of an RNAi-based deorphanization protocol. Future work can help optimize and adapt this protocol for higher-throughput platforms as well as other phyla.

  20. Ligands Raise the Constraint That Limits Constitutive Activation in G Protein-coupled Opioid Receptors*

    PubMed Central

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

    2013-01-01

    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. PMID:23836900

  1. 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.

  2. [Prospects for use of peptides and their derivatives, structurally corresponding to the G protein-coupled receptors, in medicine].

    PubMed

    Shpakov, A O; Shpakova, E A

    2015-01-01

    The regulation of signaling pathways involved in the control of many physiological functions is carried out via the heterotrimeric G protein-coupled receptors (GPCR). The search of effective and selective regulators of GPCR and intracellular signaling cascades coupled with them is one of the important problems of modern fundamental and clinical medicine. Recently data suggest that synthetic peptides and their derivatives, structurally corresponding to the intracellular and transmembrane regions of GPCR, can interact with high efficiency and selectivity with homologous receptors and influence, thus, the functional activity of intracellular signaling cascades and fundamental cellular processes controlled by them. GPCR-peptides are active in both in vitro and in vivo. They regulate hematopoiesis, angiogenesis and cell proliferation, inhibit tumor growth and metastasis, and prevent the inflammatory diseases and septic shock. These data show greatest prospects in the development of the new generations of drugs based on GPCR-derived peptides, capable of regulating the important functions of the organism.

  3. 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

  4. G-protein-coupled receptor kinase 2 terminates G-protein-coupled receptor function in steroid hormone 20-hydroxyecdysone signaling

    PubMed Central

    Zhao, Wen-Li; Wang, Di; Liu, Chun-Yan; Zhao, Xiao-Fan

    2016-01-01

    G-protein-coupled receptors (GPCRs) transmit extracellular signals across the cell membrane. GPCR kinases (GRKs) desensitize GPCR signals in the cell membrane. However, the role and mechanism of GRKs in the desensitization of steroid hormone signaling are unclear. In this study, we propose that GRK2 is phosphorylated by protein kinase C (PKC) in response to induction by the steroid hormone 20-hydroxyecdysone (20E), which determines its translocation to the cell membrane of the lepidopteran Helicoverpa armigera. GRK2 protein expression is increased during the metamorphic stage because of induction by 20E. Knockdown of GRK2 in larvae causes accelerated pupation, an increase in 20E-response gene expression, and advanced apoptosis and metamorphosis. 20E induces translocation of GRK2 from the cytoplasm to the cell membrane via steroid hormone ecdysone-responsive GPCR (ErGPCR-2). GRK2 is phosphorylated by PKC on serine 680 after induction by 20E, which leads to the translocation of GRK2 to the cell membrane. GRK2 interacts with ErGPCR-2. These data indicate that GRK2 terminates the ErGPCR-2 function in 20E signaling in the cell membrane by a negative feedback mechanism. PMID:27412951

  5. CNTF variants with increased biological potency and receptor selectivity define a functional site of receptor interaction.

    PubMed Central

    Saggio, I; Gloaguen, I; Poiana, G; Laufer, R

    1995-01-01

    Human CNTF is a neurocytokine that elicits potent neurotrophic effects by activating a receptor complex composed of the ligand-specific alpha-receptor subunit (CNTFR alpha) and two signal transducing proteins, which together constitute a receptor for leukemia inhibitory factor (LIFR). At high concentrations, CNTF can also activate the LIFR and possibly other cross-reactive cytokine receptors in the absence of CNTFR alpha. To gain a better understanding of its structure-function relationships and to develop analogs with increased receptor specificity, the cytokine was submitted to affinity maturation using phage display technology. Variants with greatly increased CNTFR alpha affinity were selected from a phage-displayed library of CNTF variants carrying random amino acid substitutions in the putative D helix. Selected variants contained substitutions of the wild-type Gln167 residue, either alone or in combination with neighboring mutations. These results provide evidence for an important functional role of the mutagenized region in CNTFR alpha binding. Affinity enhancing mutations conferred to CNTF increased potency to trigger biological effects mediated by CNTFR alpha and enhanced neurotrophic activity on chicken ciliary neurons. In contrast, the same mutations did not potentiate the CNTFR alpha-independent receptor actions of CNTF. These CNTF analogs thus represent receptor-specific superagonists, which should help to elucidate the mechanisms underlying the pleiotropic actions of the neurocytokine. PMID:7621819

  6. A Molecular and Chemical Perspective in Defining Melatonin Receptor Subtype Selectivity

    PubMed Central

    Chan, King Hang; Wong, Yung Hou

    2013-01-01

    Melatonin is primarily synthesized and secreted by the pineal gland during darkness in a normal diurnal cycle. In addition to its intrinsic antioxidant property, the neurohormone has renowned regulatory roles in the control of circadian rhythm and exerts its physiological actions primarily by interacting with the G protein-coupled MT1 and MT2 transmembrane receptors. The two melatonin receptor subtypes display identical ligand binding characteristics and mediate a myriad of signaling pathways, including adenylyl cyclase inhibition, phospholipase C stimulation and the regulation of other effector molecules. Both MT1 and MT2 receptors are widely expressed in the central nervous system as well as many peripheral tissues, but each receptor subtype can be linked to specific functional responses at the target tissue. Given the broad therapeutic implications of melatonin receptors in chronobiology, immunomodulation, endocrine regulation, reproductive functions and cancer development, drug discovery and development programs have been directed at identifying chemical molecules that bind to the two melatonin receptor subtypes. However, all of the melatoninergics in the market act on both subtypes of melatonin receptors without significant selectivity. To facilitate the design and development of novel therapeutic agents, it is necessary to understand the intrinsic differences between MT1 and MT2 that determine ligand binding, functional efficacy, and signaling specificity. This review summarizes our current knowledge in differentiating MT1 and MT2 receptors and their signaling capacities. The use of homology modeling in the mapping of the ligand-binding pocket will be described. Identification of conserved and distinct residues will be tremendously useful in the design of highly selective ligands. PMID:24018885

  7. Homology modelling of CB1 receptor and selection of potential inhibitor against Obesity

    PubMed Central

    Shrinivasan, Mahesh; Skariyachan, Sinosh; Aparna, Vaka; Kolte, Vinod Rama

    2012-01-01

    Obesity and patient morbidity has become a health concern worldwide. Obesity is associated with over activity of the endocannabinoid system, which is involved in the regulation of appetite, lipogenesis and insulin resistance. Hypothalamic cannabinoid-1 receptor (CB1R) inverse agonists reduce body weight and improve cardiometabolic abnormalities in experimental and human obesity but displayed neuropsychiatric side effects. Hence, there is a need to develop therapeutics which employs blocking peripheral CB1 receptors and still achieve substantial weight loss. In view of the same, adipose tissue CB1 receptors are employed for this study since it is more specific in reducing visceral fat. Computer aided structure based virtual screening finds application to screen novel inhibitors and develop highly selective and potential drug. The rational drug design requires crystal structure for the CB1 receptor. However, the structure for the CB1 receptor is not available in its native form. Thus, we modelled the crystal structure using a lipid G-Protein coupled receptor (PDB: 3V2W, chain A) as template. Furthermore, we have screened a herbal ligand Quercetin [- 2- (3, 4-dihydroxyphenyl) - 3, 5, 7-trihydroxychromen-4-one] a flavonol present in Mimosa pudica based on its better pharmacokinetics and bioavailability profile. This ligand was selected as an ideal lead molecule. The docking of quercetin with CB1 receptor showed a binding energy of -6.56 Kcal/mol with 4 hydrogen bonds, in comparison to the known drug Rimonabant. This data finds application in proposing antagonism of CB1 receptor with Quercetin, for controlling obesity. PMID:22829723

  8. Lighting up G protein-coupled purinergic receptors with engineered fluorescent ligands

    PubMed Central

    Ciruela, Francisco; Fernández-Dueñas, Víctor; Jacobson, Kenneth A.

    2015-01-01

    The use of G protein-coupled receptors fluorescent ligands is undergoing continuous expansion. In line with this, fluorescent agonists and antagonists of high affinity for G protein-coupled adenosine and P2Y receptors have been shown to be useful pharmacological probe compounds. Fluorescent ligands for A1R, A2AR, and A3R (adenosine receptors) and P2Y2R, P2Y4R, P2Y6R, and P2Y14R (nucleotide receptors) have been reported. Such ligands have been successfully applied to drug discovery and to GPCR characterization by flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer and scanning confocal microscopy. Here we summarize recently reported and readily available representative fluorescent ligands of purinergic receptors. In addition, we pay special attention on the use of this family of fluorescent ligands revealing two main aspects of purinergic receptor biology, namely ligand binding and receptor oligomerization. PMID:25890205

  9. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    PubMed Central

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

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

    PubMed

    Handlon, Anthony L; Schaller, Lee T; Leesnitzer, Lisa M; Merrihew, Raymond V; Poole, Chuck; Ulrich, John C; Wilson, Joseph W; Cadilla, Rodolfo; Turnbull, Philip

    2016-01-14

    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%).

  11. 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

  12. Biased G Protein-Coupled Receptor Signaling: New Player in Modulating Physiology and Pathology

    PubMed Central

    Bologna, Zuzana; Teoh, Jian-peng; Bayoumi, Ahmed S.; Tang, Yaoliang; Kim, Il-man

    2017-01-01

    G protein-coupled receptors (GPCRs) are a family of cell-surface proteins that play critical roles in regulating a variety of pathophysiological processes and thus are targeted by almost a third of currently available therapeutics. It was originally thought that GPCRs convert extracellular stimuli into intracellular signals through activating G proteins, whereas β-arrestins have important roles in internalization and desensitization of the receptor. Over the past decade, several novel functional aspects of β-arrestins in regulating GPCR signaling have been discovered. These previously unanticipated roles of β-arrestins to act as signal transducers and mediators of G protein-independent signaling have led to the concept of biased agonism. Biased GPCR ligands are able to engage with their target receptors in a manner that preferentially activates only G protein- or β-arrestin-mediated downstream signaling. This offers the potential for next generation drugs with high selectivity to therapeutically relevant GPCR signaling pathways. In this review, we provide a summary of the recent studies highlighting G protein- or β-arrestin-biased GPCR signaling and the effects of biased ligands on disease pathogenesis and regulation. PMID:28035079

  13. Molecular manipulation of G-protein-coupled receptors: a new avenue into drug discovery.

    PubMed

    Sautel, M; Milligan, G

    2000-09-01

    During the past 10 years or so, associated with the introduction of molecular biology techniques to G protein-coupled receptor (GPCR) research, outstanding progress has been made in understanding the mechanisms of action of these key proteins and their physiological functions. in-vivo manipulation of levels of GPCRs using transgenic and gene knock-out approaches have been particularly successful in assessing the roles of specific GPCRs in animal physiology. Drug discovery is aiming to produce highly specific compounds based on subtle definition of receptor subtypes which can best be studied using heterologous expression of wild type or mutated forms of cDNA or genes encoding these proteins. Furthermore, new therapeutic opportunities may be provided by investigation of orphan receptors, the natural ligands for which remain unidentified. Some human diseases have been shown to be associated with rare mutations of GPCRs and the possibility that widely distributed polymorphisms in GPCR genes may allow selective therapeutic strategies for population subgroups is driving the development of the science of pharmacogenetics.

  14. Alpha/sub 1/ receptor coupling events initiated by methoxy-substituted tolazoline partial agonists

    SciTech Connect

    Wick, P.; Keung, A.; Deth, R.

    1986-03-01

    A series of mono- and dimethyoxy substituted tolazoline derivatives, known to be partial agonists at the alpha/sub 1/ receptor, were compared with the ..cap alpha../sub 1/ selective full agonist phenylephrine (PE) on isolated strips of rabbit aorta Agonist activity was evaluated in contraction, /sup 45/Ca influx, /sup 45/Ca efflux, and /sup 32/P-Phospholipid labelling studies. Maximum contractile responses for the 2-, 3-, and 3, 5- methoxy substituted tolazoline derivatives (10/sup -5/M) were 53.8, 67.6 and 99.7% of the PE (10/sup -5/M) response respectively. These same partial agonists caused a stimulation of /sup 45/Ca influx to the extent of 64, 86, and 95% of the PE response respectively. In /sup 45/Ca efflux studies, (a measure of the intracellular Ca/sup +2/ release) the tolazolines caused: 30%, 63%, and 78% of the PE stimulated level. /sup 32/P-Phosphatidic acid (PA) labelling was measured as an index of PI turnover after ..cap alpha../sub 1/ receptor stimulation. Compared to PE, the 2-, 3-, and 3,5- methoxy substituted tolazoline derivatives caused 22, 46, and 72% PA labelling. The above values are all in reasonable accord with the rank order or agonist activity shown in maximum contractile responses. The results of this investigation suggest that partial agonists stimulate ..cap alpha.. receptor coupling events at a level which is quantitatively comparable to their potencies in causing contraction of arterial smooth muscle.

  15. 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.

  16. 2-Dialkynyl derivatives of (N)-methanocarba nucleosides: 'Clickable' A(3) adenosine receptor-selective agonists.

    PubMed

    Tosh, Dilip K; Chinn, Moshe; Yoo, Lena S; Kang, Dong Wook; Luecke, Hans; Gao, Zhan-Guo; Jacobson, Kenneth A

    2010-01-15

    We modified a series of (N)-methanocarba nucleoside 5'-uronamides to contain dialkyne groups on an extended adenine C2 substituent, as synthetic intermediates leading to potent and selective A(3) adenosine receptor (AR) agonists. The proximal alkyne was intended to promote receptor recognition, and the distal alkyne reacted with azides to form triazole derivatives (click cycloaddition). Click chemistry was utilized to couple an octadiynyl A(3)AR agonist to azido-containing fluorescent, chemically reactive, biotinylated, and other moieties with retention of selective binding to the A(3)AR. A bifunctional thiol-reactive crosslinking reagent was introduced. The most potent and selective novel compound was a 1-adamantyl derivative (K(i) 6.5nM), although some of the click products had K(i) values in the range of 200-400nM. Other potent, selective derivatives (K(i) at A(3)AR innM) were intended as possible receptor affinity labels: 3-nitro-4-fluorophenyl (10.6), alpha-bromophenacyl (9.6), thiol-reactive isothiazolone (102), and arylisothiocyanate (37.5) derivatives. The maximal functional effects in inhibition of forskolin-stimulated cAMP were measured, indicating that this class of click adducts varied from partial to full A(3)AR agonist compared to other widely used agonists. Thus, this strategy provides a general chemical approach to linking potent and selective A(3)AR agonists to reporter groups of diverse structure and to carrier moieties.

  17. Selective Pinacol-Coupling Reaction using a Continuous Flow System.

    PubMed

    Sotto, Nicolas; Cazorla, Clément; Villette, Carole; Billamboz, Muriel; Len, Christophe

    2016-11-18

    The first continuous flow pinacol coupling reaction of carbonyl compounds was successfully achieved within only 2 min during a single pass through a cartridge filled with zinc(0). The optimized method allowed the efficient production of gram-scale value-added compounds with high productivity. The developed methodology is efficient for aromatic or α,β-unsaturated aldehydes but gives moderate results for more stable acetophenone derivatives. Moreover, the flow method displayed better results in terms of yield and selectivity in comparison to the corresponding batch methodology.

  18. Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

    PubMed Central

    Orr, Anna G.; Hsiao, Edward C.; Wang, Max M.; Ho, Kaitlyn; Kim, Daniel H.; Wang, Xin; Guo, Weikun; Kang, Jing; Yu, Gui-Qiu; Adame, Anthony; Devidze, Nino; Dubal, Dena B.; Masliah, Eliezer; Conklin, Bruce R.; Mucke, Lennart

    2014-01-01

    Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer’s disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice, and increased the levels of Arc/Arg3.1, an immediate-early gene required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Similar to humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss. PMID:25622143

  19. Accelerated structure-based design of chemically diverse allosteric modulators of a muscarinic G protein-coupled receptor

    PubMed Central

    Miao, Yinglong; Goldfeld, Dahlia Anne; Moo, Ee Von; Sexton, Patrick M.; Christopoulos, Arthur; McCammon, J. Andrew; Valant, Celine

    2016-01-01

    Design of ligands that provide receptor selectivity has emerged as a new paradigm for drug discovery of G protein-coupled receptors, and may, for certain families of receptors, only be achieved via identification of chemically diverse allosteric modulators. Here, the extracellular vestibule of the M2 muscarinic acetylcholine receptor (mAChR) is targeted for structure-based design of allosteric modulators. Accelerated molecular dynamics (aMD) simulations were performed to construct structural ensembles that account for the receptor flexibility. Compounds obtained from the National Cancer Institute (NCI) were docked to the receptor ensembles. Retrospective docking of known ligands showed that combining aMD simulations with Glide induced fit docking (IFD) provided much-improved enrichment factors, compared with the Glide virtual screening workflow. Glide IFD was thus applied in receptor ensemble docking, and 38 top-ranked NCI compounds were selected for experimental testing. In [3H]N-methylscopolamine radioligand dissociation assays, approximately half of the 38 lead compounds altered the radioligand dissociation rate, a hallmark of allosteric behavior. In further competition binding experiments, we identified 12 compounds with affinity of ≤30 μM. With final functional experiments on six selected compounds, we confirmed four of them as new negative allosteric modulators (NAMs) and one as positive allosteric modulator of agonist-mediated response at the M2 mAChR. Two of the NAMs showed subtype selectivity without significant effect at the M1 and M3 mAChRs. This study demonstrates an unprecedented successful structure-based approach to identify chemically diverse and selective GPCR allosteric modulators with outstanding potential for further structure-activity relationship studies. PMID:27601651

  20. Synthetic anabolic agents: steroids and nonsteroidal selective androgen receptor modulators.

    PubMed

    Thevis, Mario; Schänzer, Wilhelm

    2010-01-01

    The central role of testosterone in the development of male characteristics, as well as its beneficial effects on physical performance and muscle growth, has led to the search for synthetic alternatives with improved pharmacological profiles. Hundreds of steroidal analogs have been prepared with a superior oral bioavailability, which should also possess reduced undesirable effects. However, only a few entered the pharmaceutical market due to severe toxicological incidences that were mainly attributed to the lack of tissue selectivity. Prominent representatives of anabolic-androgenic steroids (AAS) are for instance methyltestosterone, metandienone and stanozolol, which are discussed as model compounds with regard to general pharmacological aspects of synthetic AAS. Recently, nonsteroidal alternatives to AAS have been developed that selectively activate the androgen receptor in either muscle tissue or bones. These so-called selective androgen receptor modulators (SARMs) are currently undergoing late clinical trials (IIb) and will be prohibited by the World Anti-Doping Agency from January 2008. Their entirely synthetic structures are barely related to steroids, but particular functional groups allow for the tissue-selective activation or inhibition of androgen receptors and, thus, the stimulation of muscle growth without the risk of severe undesirable effects commonly observed in steroid replacement therapies. Hence, these compounds possess a high potential for misuse in sports and will be the subject of future doping control assays.

  1. Highly selective and sensitive detection of neurotransmitters using receptor-modified single-walled carbon nanotube sensors

    NASA Astrophysics Data System (ADS)

    Kim, Byeongju; Song, Hyun Seok; Jin, Hye Jun; Park, Eun Jin; Lee, Sang Hun; Lee, Byung Yang; Park, Tai Hyun; Hong, Seunghun

    2013-07-01

    We present receptor-modified carbon nanotube sensors for the highly selective and sensitive detection of acetylcholine (ACh), one kind of neurotransmitter. Here, we successfully expressed the M1 muscarinic acetylcholine receptor (M1 mAChR), a family of G protein-coupled receptors (GPCRs), in E. coli and coated single-walled carbon nanotube (swCNT)-field effect transistors (FETs) with lipid membrane including the receptor, enabling highly selective and sensitive ACh detection. Using this sensor, we could detect ACh at 100 pM concentration. Moreover, we showed that this sensor could selectively detect ACh among other neurotransmitters. This is the first demonstration of the real-time detection of ACh using specific binding between ACh and M1 mAChR, and it may lead to breakthroughs for various applications such as disease diagnosis and drug screening.

  2. Systematic generation of in vivo G protein-coupled receptor mutants in the rat

    PubMed Central

    van Boxtel, R; Vroling, B; Toonen, P; Nijman, I J; van Roekel, H; Verheul, M; Baakman, C; Guryev, V; Vriend, G; Cuppen, E

    2011-01-01

    G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies. PMID:20531371

  3. Systematic generation of in vivo G protein-coupled receptor mutants in the rat.

    PubMed

    van Boxtel, R; Vroling, B; Toonen, P; Nijman, I J; van Roekel, H; Verheul, M; Baakman, C; Guryev, V; Vriend, G; Cuppen, E

    2011-10-01

    G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies.

  4. Mining flexible-receptor docking experiments to select promising protein receptor snapshots

    PubMed Central

    2010-01-01

    Background Molecular docking simulation is the Rational Drug Design (RDD) step that investigates the affinity between protein receptors and ligands. Typically, molecular docking algorithms consider receptors as rigid bodies. Receptors are, however, intrinsically flexible in the cellular environment. The use of a time series of receptor conformations is an approach to explore its flexibility in molecular docking computer simulations, but it is extensively time-consuming. Hence, selection of the most promising conformations can accelerate docking experiments and, consequently, the RDD efforts. Results We previously docked four ligands (NADH, TCL, PIF and ETH) to 3,100 conformations of the InhA receptor from M. tuberculosis. Based on the receptor residues-ligand distances we preprocessed all docking results to generate appropriate input to mine data. Data preprocessing was done by calculating the shortest interatomic distances between the ligand and the receptor’s residues for each docking result. They were the predictive attributes. The target attribute was the estimated free-energy of binding (FEB) value calculated by the AutodDock3.0.5 software. The mining inputs were submitted to the M5P model tree algorithm. It resulted in short and understandable trees. On the basis of the correlation values, for NADH, TCL and PIF we obtained more than 95% correlation while for ETH, only about 60%. Post processing the generated model trees for each of its linear models (LMs), we calculated the average FEB for their associated instances. From these values we considered a LM as representative if its average FEB was smaller than or equal the average FEB of the test set. The instances in the selected LMs were considered the most promising snapshots. It totalized 1,521, 1,780, 2,085 and 902 snapshots, for NADH, TCL, PIF and ETH respectively. Conclusions By post processing the generated model trees we were able to propose a criterion of selection of linear models which, in turn, is

  5. The effects of (-)-epicatechin on endothelial cells involve the G protein-coupled estrogen receptor (GPER).

    PubMed

    Moreno-Ulloa, Aldo; Mendez-Luna, David; Beltran-Partida, Ernesto; Castillo, Carmen; Guevara, Gustavo; Ramirez-Sanchez, Israel; Correa-Basurto, José; Ceballos, Guillermo; Villarreal, Francisco

    2015-10-01

    We have provided evidence that the stimulatory effects of (-)-epicatechin ((-)-EPI) on endothelial cell nitric oxide (NO) production may involve the participation of a cell-surface receptor. Thus far, such entity(ies) has not been fully elucidated. The G protein-coupled estrogen receptor (GPER) is a cell-surface receptor that has been linked to protective effects on the cardiovascular system and activation of intracellular signaling pathways (including NO production) similar to those reported with (-)-EPI. In bovine coronary artery endothelial cells (BCAEC) by the use of confocal imaging, we evidence the presence of GPER at the cell-surface and on F-actin filaments. Using in silico studies we document the favorable binding mode between (-)-EPI and GPER. Such binding is comparable to that of the GPER agonist, G1. By the use of selective blockers, we demonstrate that the activation of ERK 1/2 and CaMKII by (-)-EPI is dependent on the GPER/c-SRC/EGFR axis mimicking those effects noted with G1. We also evidence by the use of siRNA the role that GPER has on mediating ERK1/2 activation by (-)-EPI. GPER appears to be coupled to a non Gαi/o or Gαs, protein subtype. To extrapolate our findings to an ex vivo model, we employed phenylephrine pre-contracted aortic rings evidencing that (-)-EPI can mediate vasodilation through GPER activation. In conclusion, we provide evidence that suggests the GPER as a potential mediator of (-)-EPI effects and highlights the important role that GPER may have on cardiovascular system protection.

  6. Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors.

    PubMed Central

    Hermans, E; Challiss, R A

    2001-01-01

    In 1991 a new type of G-protein-coupled receptor (GPCR) was cloned, the type 1a metabotropic glutamate (mGlu) receptor, which, despite possessing the defining seven-transmembrane topology of the GPCR superfamily, bore little resemblance to the growing number of other cloned GPCRs. Subsequent studies have shown that there are eight mammalian mGlu receptors that, together with the calcium-sensing receptor, the GABA(B) receptor (where GABA is gamma-aminobutyric acid) and a subset of pheromone, olfactory and taste receptors, make up GPCR family C. Currently available data suggest that family C GPCRs share a number of structural, biochemical and regulatory characteristics, which differ markedly from those of the other GPCR families, most notably the rhodopsin/family A GPCRs that have been most widely studied to date. This review will focus on the group I mGlu receptors (mGlu1 and mGlu5). This subgroup of receptors is widely and differentially expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in the control of an array of key signalling events, including roles in the adaptative changes needed for long-term depression or potentiation of neuronal synaptic connectivity. In addition to playing critical physiological roles within the brain, the mGlu receptors are also currently the focus of considerable attention because of their potential as drug targets for the treatment of a variety of neurological and psychiatric disorders. PMID:11672421

  7. Nonsteroidal selective androgen receptor modulator Ostarine in cancer cachexia.

    PubMed

    Zilbermint, Mihail F; Dobs, Adrian S

    2009-10-01

    Cancer cachexia is a complex syndrome, affecting up to 60% of the approximately 1.4 million patients diagnosed with cancer each year in the USA. This condition is characterized by progressive deterioration of a patient's nutritional status, weight loss, anorexia, diminished quality of life and increased mortality and morbidity. Current therapy with progestational, anti-inflammatory and anabolic agents is often ineffective and has a large number of undesirable effects. The newly developed nonsteroidal selective androgen receptor modulator Ostarine has demonstrated promising results in Phase I and II clinical trials, increasing total lean body mass, enhancing functional performance and decreasing total tissue percent fat. This selective androgen receptor modulator may have the ability to perform as a potent anabolic agent with minimal side effects on other organs (prostate and hair follicles), thus presenting a new strategy in managing cancer cachexia. However, more extensive data is required before its efficacy is confirmed.

  8. Comparison of the Binding and Functional Properties of Two Structurally Different D2 Dopamine Receptor Subtype Selective Compounds

    PubMed Central

    2012-01-01

    We previously reported on the synthesis of substituted phenyl-4-hydroxy-1-piperidyl indole analogues with nanomolar affinity at D2 dopamine receptors, ranging from 10- to 100-fold selective for D2 compared to the D3 dopamine receptor subtype. More recently, we evaluated a panel of aripiprazole analogues, identifying several analogues that also exhibit D2 vs D3 dopamine receptor binding selectivity. These studies further characterize the intrinsic efficacy of the compound with the greatest binding selectivity from each chemical class, 1-((5-methoxy-1H-indol-3-yl)methyl)-4-(4-(methylthio)phenyl)piperidin-4-ol (SV 293) and 7-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (SV-III-130s), using an adenylyl cyclase inhibition assay, a G-protein-coupled inward-rectifying potassium (GIRK) channel activation assay, and a cell based phospho-MAPK (pERK1/2) assay. SV 293 was found to be a neutral antagonist at D2 dopamine receptors using all three assays. SV-III-130s is a partial agonist using an adenylyl cyclase inhibition assay but an antagonist in the GIRK and phospho ERK1/2 assays. To define the molecular basis for the binding selectivity, the affinity of these two compounds was evaluated using (a) wild type human D2 and D3 receptors and (b) a panel of chimeric D2/D3 dopamine receptors. Computer-assisted modeling techniques were used to dock these compounds to the human D2 and D3 dopamine receptor subtypes. It is hoped that these studies on D2 receptor selective ligands will be useful in the future design of (a) receptor selective ligands used to define the function of D2-like receptor subtypes, (b) novel pharmacotherapeutic agents, and/or (c) in vitro and in vivo imaging agents. PMID:23259040

  9. Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

    PubMed

    Wilkinson, Trevor C I

    2016-06-15

    The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed.

  10. Role of antibodies in developing drugs that target G-protein-coupled receptor dimers.

    PubMed

    Hipser, Chris; Bushlin, Ittai; Gupta, Achla; Gomes, Ivone; Devi, Lakshmi A

    2010-01-01

    G-protein-coupled receptors are important molecular targets in drug discovery. These receptors play a pivotal role in physiological signaling pathways and are targeted by nearly 50% of currently available drugs. Mounting evidence suggests that G-protein-coupled receptors form dimers, and various studies have shown that dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological implications of dimerization in vivo have not been well explored because detection of GPCR dimers in endogenous systems has been a challenging task. One exciting new approach to this challenge is the generation of antibodies against specific G-protein-coupled receptor dimers. Such antibodies could be used as tools for characterization of heteromer-specific function; as reagents for their purification, tissue localization, and regulation in vivo; and as probes for mapping their functional domains. In addition, such antibodies could serve as alternative ligands for G-protein-coupled receptor heteromers. Thus, heteromer-specific antibodies represent novel tools for the exploration and manipulation of G-protein-coupled receptor-dimer pharmacology.

  11. [Bone and Men's Health. Bone selective androgen receptor modulators].

    PubMed

    Furuya, Kazuyuki

    2010-02-01

    Androgen, one of the sex steroid hormones shows various biological activities on the corresponding various tissues. Many efforts to produce novel drug materials maintaining a desired biological activity with an adequate tissue selectivity, which is so-called selective androgen receptor modulators (SARMs) , are being performed. As one of such efforts, studies on SARMs against bone tissues which possess a significant potential to stimulate a bone formation with reducing undesirable androgenic virilizing activities are in progress all over the world. This review focuses on the research and development activities of such SARMs and discuses their usefulness for the treatment of osteoporosis.

  12. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1: Expression and action in brain.

    PubMed

    Morland, Cecilie; Lauritzen, Knut Husø; Puchades, Maja; Holm-Hansen, Signe; Andersson, Krister; Gjedde, Albert; Attramadal, Håvard; Storm-Mathisen, Jon; Bergersen, Linda Hildegard

    2015-07-01

    We have proposed that lactate is a "volume transmitter" in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes the cerebral neocortex and the hippocampus, where it can be stimulated by physiological concentrations of lactate and by the HCAR1 agonist 3,5-dihydroxybenzoate to reduce cAMP levels. Cerebral HCAR1 is concentrated on the postsynaptic membranes of excitatory synapses and also is enriched at the blood-brain barrier. In synaptic spines and in adipocytes, HCAR1 immunoreactivity is also located on subplasmalemmal vesicular organelles, suggesting trafficking to and from the plasma membrane. Through activation of HCAR1, lactate can act as a volume transmitter that links neuronal activity, cerebral blood flow, energy metabolism, and energy substrate availability, including a glucose- and glycogen-saving response. HCAR1 may contribute to optimizing the cAMP concentration. For instance, in the prefrontal cortex, excessively high cAMP levels are implicated in impaired cognition in old age, fatigue, stress, and schizophrenia and in the deposition of phosphorylated tau protein in Alzheimer's disease. HCAR1 could serve to ameliorate these conditions and might also act through downstream mechanisms other than cAMP. Lactate exits cells through monocarboxylate transporters in an equilibrating manner and through astrocyte anion channels activated by depolarization. In addition to locally produced lactate, lactate produced by exercising muscle as well as exogenous HCAR1 agonists, e.g., from fruits and berries, might activate the receptor on cerebral blood vessels and brain cells.

  13. Identification of Anabolic Selective Androgen Receptor Modulators with Reduced Activities in Reproductive Tissues and Sebaceous Glands

    PubMed Central

    Schmidt, Azriel; Harada, Shun-Ichi; Kimmel, Donald B.; Bai, Chang; Chen, Fang; Rutledge, Su Jane; Vogel, Robert L.; Scafonas, Angela; Gentile, Michael A.; Nantermet, Pascale V.; McElwee-Witmer, Sheila; Pennypacker, Brenda; Masarachia, Patricia; Sahoo, Soumya P.; Kim, Yuntae; Meissner, Robert S.; Hartman, George D.; Duggan, Mark E.; Rodan, Gideon A.; Towler, Dwight A.; Ray, William J.

    2009-01-01

    Androgen replacement therapy is a promising strategy for the treatment of frailty; however, androgens pose risks for unwanted effects including virilization and hypertrophy of reproductive organs. Selective Androgen Receptor Modulators (SARMs) retain the anabolic properties of androgens in bone and muscle while having reduced effects in other tissues. We describe two structurally similar 4-aza-steroidal androgen receptor (AR) ligands, Cl-4AS-1, a full agonist, and TFM-4AS-1, which is a SARM. TFM-4AS-1 is a potent AR ligand (IC50, 38 nm) that partially activates an AR-dependent MMTV promoter (55% of maximal response) while antagonizing the N-terminal/C-terminal interaction within AR that is required for full receptor activation. Microarray analyses of MDA-MB-453 cells show that whereas Cl-4AS-1 behaves like 5α-dihydrotestosterone (DHT), TFM-4AS-1 acts as a gene-selective agonist, inducing some genes as effectively as DHT and others to a lesser extent or not at all. This gene-selective agonism manifests as tissue-selectivity: in ovariectomized rats, Cl-4AS-1 mimics DHT while TFM-4AS-1 promotes the accrual of bone and muscle mass while having reduced effects on reproductive organs and sebaceous glands. Moreover, TFM-4AS-1 does not promote prostate growth and antagonizes DHT in seminal vesicles. To confirm that the biochemical properties of TFM-4AS-1 confer tissue selectivity, we identified a structurally unrelated compound, FTBU-1, with partial agonist activity coupled with antagonism of the N-terminal/C-terminal interaction and found that it also behaves as a SARM. TFM-4AS-1 and FTBU-1 represent two new classes of SARMs and will allow for comparative studies aimed at understanding the biophysical and physiological basis of tissue-selective effects of nuclear receptor ligands. PMID:19846549

  14. G-Protein–Coupled Receptors Signaling Pathways in New Antiplatelet Drug Development

    PubMed Central

    Gurbel, Paul A.; Kuliopulos, Athan; Tantry, Udaya S.

    2016-01-01

    Platelet G-protein–coupled receptors influence platelet function by mediating the response to various agonists, including ADP, thromboxane A2, and thrombin. Blockade of the ADP receptor, P2Y12, in combination with cyclooxygenase-1 inhibition by aspirin has been among the most widely used pharmacological strategies to reduce cardiovascular event occurrence in high-risk patients. The latter dual pathway blockade strategy is one of the greatest advances in the field of cardiovascular medicine. In addition to P2Y12, the platelet thrombin receptor, protease activated receptor-1, has also been recently targeted for inhibition. Blockade of protease activated receptor-1 has been associated with reduced thrombotic event occurrence when added to a strategy using P2Y12 and cyclooxygenase-1 inhibition. At this time, the relative contributions of these G-protein–coupled receptor signaling pathways to in vivo thrombosis remain incompletely defined. The observation of treatment failure in ≈10% of high-risk patients treated with aspirin and potent P2Y12 inhibitors provides the rationale for targeting novel pathways mediating platelet function. Targeting intracellular signaling downstream from G-protein–coupled receptor receptors with phosphotidylionisitol 3-kinase and Gq inhibitors are among the novel strategies under investigation to prevent arterial ischemic event occurrence. Greater understanding of the mechanisms of G-protein–coupled receptor–mediated signaling may allow the tailoring of antiplatelet therapy. PMID:25633316

  15. Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response.

    PubMed

    Alvaro, Christopher G; Thorner, Jeremy

    2016-04-08

    The DNAs encoding the receptors that respond to the peptide mating pheromones of the budding yeastSaccharomyces cerevisiaewere isolated in 1985, and were the very first genes for agonist-binding heterotrimeric G protein-coupled receptors (GPCRs) to be cloned in any organism. Now, over 30 years later, this yeast and its receptors continue to provide a pathfinding experimental paradigm for investigating GPCR-initiated signaling and its regulation, as described in this retrospective overview.

  16. High content screening for G protein-coupled receptors using cell-based protein translocation assays.

    PubMed

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne; Linde, Viggo; Pedersen, Hans-Christian; Krog-Jensen, Christian; Rosenkilde, Mette M; Pagliaro, Len

    2005-06-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.

  17. RGS2 modulates coupling between GABAB receptors and GIRK channels in dopamine neurons of the ventral tegmental area.

    PubMed

    Labouèbe, Gwenaël; Lomazzi, Marta; Cruz, Hans G; Creton, Cyril; Luján, Rafael; Li, Meng; Yanagawa, Yuchio; Obata, Kunihiko; Watanabe, Masahiko; Wickman, Kevin; Boyer, Stephanie B; Slesinger, Paul A; Lüscher, Christian

    2007-12-01

    Agonists of GABA(B) receptors exert a bi-directional effect on the activity of dopamine (DA) neurons of the ventral tegmental area, which can be explained by the fact that coupling between GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK) channels is significantly weaker in DA neurons than in GABA neurons. Thus, low concentrations of agonists preferentially inhibit GABA neurons and thereby disinhibit DA neurons. This disinhibition might confer reinforcing properties on addictive GABA(B) receptor agonists such as gamma-hydroxybutyrate (GHB) and its derivatives. Here we show that, in DA neurons of mice, the low coupling efficiency reflects the selective expression of heteromeric GIRK2/3 channels and is dynamically modulated by a member of the regulator of G protein signaling (RGS) protein family. Moreover, repetitive exposure to GHB increases the GABA(B) receptor-GIRK channel coupling efficiency through downregulation of RGS2. Finally, oral self-administration of GHB at a concentration that is normally rewarding becomes aversive after chronic exposure. On the basis of these results, we propose a mechanism that might underlie tolerance to GHB.

  18. PEGylated Dendritic Unimolecular Micelles as Versatile Carriers for Ligands of G Protein-Coupled Receptors

    PubMed Central

    Kim, Yoonkyung; Hechler, Béatrice; Gao, Zhan-Guo; Gachet, Christian; Jacobson, Kenneth A.

    2009-01-01

    Despite its widespread application in nanomedicine, poly(ethylene glycol) (PEG) is seldom used for covalent modification of ligands for G protein-coupled receptors (GPCRs) due to potential steric complications. In order to study the influence of PEG chains on the biological activity of GPCR ligands bound to a common macromolecular carrier, we prepared a series of G3 polyamidoamine (PAMAM) dendrimers derivatized with Alexa Fluor 488, varying numbers of PEG550/PEG750/PEG2000, and nucleoside moieties derived from the A2A adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxylethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine). These dendrimer conjugates were purified by size exclusion chromatography and characterized by 1H NMR and MALDI MS. In radioligand binding assays, some PAMAM-PEG conjugates showed enhanced subtype-selectivity at the human A2A AR compared to monomeric ligands of comparable affinity. The functional potency was measured in the A2A AR-mediated activation of adenylate cyclase and inhibition of ADP-induced platelet aggregation. Interestingly, the dendrimer conjugate 10c bearing 11 PEG750 chains (out of theo. 32 amino end groups) and 14 nucleoside moieties was 5-fold more potent in A2A AR–mediated stimulation of cyclic AMP formation than 10d with four PEG2000 chains and 21 nucleosides, although the binding affinities of these two compounds were similar. Thus, a relatively small (≤10 nm) multivalent ligand 10c modified for water solubility maintained high potency and displayed increased A2A AR binding selectivity over the monomeric nucleosides. Longer PEG chains reduced affinity at the A2A AR. The current study demonstrates the feasiblity of using short PEG chains in the design of carriers that target ligand-receptor interactions. PMID:19785401

  19. G protein-coupled receptors: extranuclear mediators for the non-genomic actions of steroids.

    PubMed

    Wang, Chen; Liu, Yi; Cao, Ji-Min

    2014-09-01

    Steroids hormones possess two distinct actions, a delayed genomic effect and a rapid non-genomic effect. Rapid steroid-triggered signaling is mediated by specific receptors localized most often to the plasma membrane. The nature of these receptors is of great interest and accumulated data suggest that G protein-coupled receptors (GPCRs) are appealing candidates. Increasing evidence regarding the interaction between steroids and specific membrane proteins, as well as the involvement of G protein and corresponding downstream signaling, have led to identification of physiologically relevant GPCRs as steroid extranuclear receptors. Examples include G protein-coupled receptor 30 (GPR30) for estrogen, membrane progestin receptor for progesterone, G protein-coupled receptor family C group 6 member A (GPRC6A) and zinc transporter member 9 (ZIP9) for androgen, and trace amine associated receptor 1 (TAAR1) for thyroid hormone. These receptor-mediated biological effects have been extended to reproductive development, cardiovascular function, neuroendocrinology and cancer pathophysiology. However, although great progress have been achieved, there are still important questions that need to be answered, including the identities of GPCRs responsible for the remaining steroids (e.g., glucocorticoid), the structural basis of steroids and GPCRs' interaction and the integration of extranuclear and nuclear signaling to the final physiological function. Here, we reviewed the several significant developments in this field and highlighted a hypothesis that attempts to explain the general interaction between steroids and GPCRs.

  20. Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.

    PubMed

    Couve, A; Thomas, P; Calver, A R; Hirst, W D; Pangalos, M N; Walsh, F S; Smart, T G; Moss, S J

    2002-05-01

    GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.

  1. Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2

    PubMed Central

    Aragay, A. M.; Mellado, M.; Frade, J. M. R.; Martin, A. M.; Jimenez-Sainz, M. C.; Martinez-A, C.; Mayor, F.

    1998-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called β-adrenergic kinase 1 or βARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein β-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant βARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines. PMID:9501202

  2. The chemokine receptors CXCR1 and CXCR2 couple to distinct G protein-coupled receptor kinases to mediate and regulate leukocyte functions

    PubMed Central

    Raghuwanshi, Sandeep K.; Su, Yingjun; Singh, Vandana; Hayes, Katherine; Richmond, Ann; Richardson, Ricardo M.

    2012-01-01

    The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized and internalized. In this study we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, shRNA was used to inhibit GRK 2, 3, 5 and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6, respectively, increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization and internalization, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect in CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6−/−) displayed an increase in CXCR2-mediated G-protein activation, but in vitro exhibited a decrease in chemotaxis, receptor desensitization and internalization relative to wild type (GRK6+/+) cells. In contrast, neutrophil recruitment in vivo in GRK6−/− mice was increased in response to delivery of CXCL1 through the air-pouch model. In a wound closure assay, GRK6−/− mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure as compared to GRK6+/+ animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis. PMID:22869904

  3. Model-Based Discovery of Synthetic Agonists for the Zn(2+)-Sensing G-Protein-Coupled Receptor 39 (GPR39) Reveals Novel Biological Functions.

    PubMed

    Frimurer, Thomas M; Mende, Franziska; Graae, Anne-Sofie; Engelstoft, Maja S; Egerod, Kristoffer L; Nygaard, Rie; Gerlach, Lars-Ole; Hansen, Jakob Bondo; Schwartz, Thue W; Holst, Birgitte

    2017-02-09

    The G-protein-coupled receptor 39 (GPR39) is a G-protein-coupled receptor activated by Zn(2+). We used a homology model-based approach to identify small-molecule pharmacological tool compounds for the receptor. The method focused on a putative binding site in GPR39 for synthetic ligands and knowledge of ligand binding to other receptors with similar binding pockets to select iterative series of minilibraries. These libraries were cherry-picked from all commercially available synthetic compounds. A total of only 520 compounds were tested in vitro, making this method broadly applicable for tool compound development. The compounds of the initial library were inactive when tested alone, but lead compounds were identified using Zn(2+) as an allosteric enhancer. Highly selective, highly potent Zn(2+)-independent GPR39 agonists were found in subsequent minilibraries. These agonists identified GPR39 as a novel regulator of gastric somatostatin secretion.

  4. Phytoestrogens from Psoralea corylifolia reveal estrogen receptor-subtype selectivity.

    PubMed

    Xin, D; Wang, H; Yang, J; Su, Y-F; Fan, G-W; Wang, Y-F; Zhu, Y; Gao, X-M

    2010-02-01

    The seed of Psoralea corylifolia L. (PCL), a well-known traditional Chinese medicine, has been applied as a tonic or an aphrodisiac agent and commonly used as a remedy for bone fracture, osteomalacia and osteoporosis in China. In our study, the estrogen receptor subtype-selective activities of the extracts and compounds derived from PCL were analyzed using the HeLa cell assay. The different fractions including petroleum ether, CH(2)Cl(2) and EtOAc fractions of the EtOH extract of PCL showed significant activity in activating either ERalpha or ERbeta whereas the n-BuOH fraction showed no estrogenic activity. Further chromatographic purification of the active fractions yielded seven compounds including the two coumarins isopsoralen and psoralen, the four flavonoids isobavachalcone, bavachin, corylifol A and neobavaisoflavone, and the meroterpene phenol, bakuchiol. In reporter gene assay, the two coumarins (10(-8)-10(-5)M) acted as ERalpha-selective agonists while the other compounds (10(-9)-10(-6)M) activated both ERalpha and ERbeta. The estrogenic activities of all compounds could be completely suppressed by the pure estrogen antagonist, ICI 182,780, suggesting that the compounds exert their activities through ER. Only psoralen and isopsoralen as ERalpha agonists promoted MCF-7 cell proliferation significantly. Although all the compounds have estrogenic activity, they may exert different biological effects. In conclusion, both ER subtype-selective and nonselective activities in compounds derived from PCL suggested that PCL could be a new source for selective estrogen-receptor modulators.

  5. Selective inhibition of beta(2)-adrenergic receptor-mediated cAMP generation by activation of the P2Y(2) receptor in mouse pineal gland tumor cells.

    PubMed

    Suh, B C; Kim, J S; Namgung, U; Han, S; Kim, K T

    2001-06-01

    Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)-coupled P2Y(2) receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol-stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC-coupled receptors including P2Y(1) and bombesin receptors had little or no effect on the isoproterenol-stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E(2), or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y(2) and beta(2)-adrenergic receptors. The binding of [(3)H]CGP12177 to beta(2)-adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol-stimulated cAMP production, whereas phorbol 12-myristate 13-acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca(2+), however, had little effect on the ATP-induced inhibition of cAMP production, while it completely reversed the ionomycin-induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide-induced increase in intracellular Ca(2+) and inositol 1,4,5-trisphosphate production by 30-40%, suggesting that the ATP-mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin-sensitive G(i)-protein. We conclude that one of the functions of P2Y(2) receptors on the pineal gland is the selective inhibition of beta-adrenergic receptor-mediated signaling pathways via the inhibitory G-proteins.

  6. Relaxant Effects of the Selective Estrogen Receptor Modulator, Bazedoxifene, and Estrogen Receptor Agonists in Isolated Rabbit Basilar Artery.

    PubMed

    Castelló-Ruiz, María; Salom, Juan B; Fernández-Musoles, Ricardo; Burguete, María C; López-Morales, Mikahela A; Arduini, Alessandro; Jover-Mengual, Teresa; Hervás, David; Torregrosa, Germán; Alborch, Enrique

    2016-10-01

    We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves the consequences of ischemic stroke. Now we aimed to characterize the effects and mechanisms of action of bazedoxifene in cerebral arteries. Male rabbit isolated basilar arteries were used for isometric tension recording and quantitative polymerase chain reaction. Bazedoxifene relaxed cerebral arteries, as 17-β-estradiol, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [estrogen receptor (ER) α agonist], and G1 [G protein-coupled ER (GPER) agonist] did it (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol > bazedoxifene = G1 > 17-β-estradiol). 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERβ agonist) had no effect. Expression profile of genes encoding for ERα (ESR1), ERβ (ESR2), and GPER was GPER > ESR1 > ESR2. As to the endothelial mechanisms, endothelium removal, N-nitro-L-arginine methyl ester, and indomethacin, did not modify the relaxant responses to bazedoxifene. As to the K channels, both a high-K medium and the Kv blocker, 4-aminopyridine, inhibited the bazedoxifene-induced relaxations, whereas tetraethylammonium (nonselective K channel blocker), glibenclamide (selective KATP blocker) or iberiotoxin (selective KCa blocker) were without effect. Bazedoxifene also inhibited both Ca- and Bay K8644-elicited contractions. Therefore, bazedoxifene induces endothelium-independent relaxations of cerebral arteries through (1) activation of GPER and ERα receptors; (2) increase of K conductance through Kv channels; and (3) inhibition of Ca entry through L-type Ca channels. Such a profile is compatible with the beneficial effects of estrogenic compounds (eg, SERMs) on vascular function and, specifically, that concerning the brain. Therefore, bazedoxifene could be useful in the treatment of cerebral disorders in which the cerebrovascular function is compromised (eg, stroke).

  7. Persistence of evolutionary memory: primordial six-transmembrane helical domain mu opiate receptors selectively linked to endogenous morphine signaling.

    PubMed

    Kream, Richard M; Sheehan, Melinda; Cadet, Patrick; Mantione, Kirk J; Zhu, Wei; Casares, Federico; Stefano, George B

    2007-12-01

    Biochemical, molecular and pharmacological evidence for two unique six-transmembrane helical (TMH) domain opiate receptors expressed from the micro opioid receptor (MOR) gene have been shown. Designated micro3 and micro4 receptors, both protein species are Class A rhodopsin-like members of the superfamily of G-protein coupled receptors but are selectively tailored to mediate the cellular regulatory effects of endogenous morphine and related morphinan alkaloids via stimulation of nitric oxide (NO) production and release. Both micro3 and micro4 receptors lack an amino acid sequence of approximately 90 amino acids that constitute the extracellular N-terminal and TMH1 domains and part of the first intracellular loop of the micro1 receptor, but retain the empirically defined ligand binding pocket distributed across conserved TMH2, TMH3, and TMH7 domains of the micro1 sequence. Additionally, the receptor proteins are terminated by unique intracellular C-terminal amino acid sequences that serve as putative coupling or docking domains required for constitutive NO synthase activation. Because the recognition profile of micro3 and micro4 receptors is restricted to rigid benzylisoquinoline alkaloids typified by morphine and its extended family of chemical congeners, it is hypothesized that conformational stabilization provided by interaction of extended extracellular N-terminal protein domains and the extracellular loops is required for binding of endogenous opioid peptides as well as synthetic flexible opiate alkaloids.

  8. Quinoline derivatives: candidate drugs for a Class B G-protein coupled receptor, the Calcitonin gene-related peptide receptor, a cause of migraines

    PubMed Central

    Iftikhar, Hira; Ahmad, Iqra; Gan, Siew Hua; Shaik, Munvar Miya; Iftikhar, Naveed; Nawaz, Muhammad Sulaman; Greig, Nigel H.; Kamal, Mohammad A

    2016-01-01

    Class B G-protein coupled receptors are involved in a wide variety of diseases and are a major focus in drug design. Migraines are a common problem, and one of their major causative agents is class B G-protein coupled receptor, Calcitonin gene-related peptide (CGRP) receptor, a target for competitive drug discovery. The calcitonin receptor-like receptor generates complexes with a receptor activity-modifying protein, which determines the type of receptor protein formed. The CGRP receptor comprises a complex formed from the calcitonin receptor-like receptor and receptor activity-modifying protein 1. In this study, an in silico docking approach was used to target calcitonin receptor-like receptor in the bound form with receptor activity-modifying protein 1 (CGRP receptor), as well as in the unbound form. In both cases, the resulting inhibitors bound to the same cavity of the calcitonin receptor-like receptor. The twelve evaluated compounds were competitive inhibitors and showed efficient inhibitory activity against the CGRP receptor and Calcitonin receptor-like receptor. The two studied quinoline derivatives demonstrated potentially ideal inhibitory activity in terms of binding interactions and low range nano-molar inhibition constants. These compounds could prove helpful in designing drugs for the effective treatment of migraines. We propose that quinoline derivatives possess inhibitory activity by disturbing CGRP binding in the trigeminovascular system and may be considered for further preclinical appraisal for the treatment of migraines. PMID:25230231

  9. Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle.

    PubMed

    Willets, Jonathon M; Brighton, Paul J; Mistry, Rajendra; Morris, Gavin E; Konje, Justin C; Challiss, R A John

    2009-08-01

    Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration ([Ca(2+)](i)); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific > or =75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.

  10. Cyclic cholecystokinin analogues with high selectivity for central receptors.

    PubMed Central

    Charpentier, B; Pelaprat, D; Durieux, C; Dor, A; Reibaud, M; Blanchard, J C; Roques, B P

    1988-01-01

    Taking as a model the N-terminal folding of the cholecystokinin tyrosine-sulfated octapeptide [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] deduced from conformational studies, two cyclic cholecystokinin (CCK) analogues were synthesized by conventional peptide synthesis: Boc-D-Asp-Tyr(SO3H)-Ahx-D-Lys-Trp-Ahx-Asp-Phe-NH2 [compound I (Ahx, 2-aminohexanoic acid)] and Boc-gamma-D-Glu-Tyr(SO3H)-Ahx-D-Lys-Trp-Ahx-Asp-Phe-NH2 (compound II). The binding characteristics of these peptides were investigated on brain cortex membranes and pancreatic acini of guinea pig. Compounds I and II were competitive inhibitors of [3H]Boc[Ahx28,31]CCK-(27-33) binding to central CCK receptors and showed a high degree of selectivity for these binding sites (compound I: Ki for pancreas/Ki for brain, 179; compound II: Ki for pancreas/Ki for brain, 1979). This high selectivity was associated with a high affinity for central CCK receptors (compound I: Ki, 5.1 nM; compound II: Ki, 0.49 nM). Similar affinities and selectivities were found when 125I Bolton-Hunter-labeled CCK-8 was used as a ligand. Moreover, these compounds were only weakly active in the stimulation of amylase release from guinea pig pancreatic acini (EC50 greater than 10,000 nM) and were unable to induce contractions in the guinea pig ileum (to 10(-6) M). The two cyclic CCK analogues, therefore, appear to be synthetic ligands exhibiting both high affinity and high selectivity for central CCK binding sites. These compounds could help clarify the respective role of central and peripheral receptors for various CCK-8-induced pharmacological effects. PMID:3162318

  11. G protein-coupled receptors provide survival signals in prostate cancer.

    PubMed

    Yowell, Charles W; Daaka, Yehia

    2002-12-01

    Prostate cancer is the leading cause for noncutaneous cancer-related deaths among men in the United States. The disease is biologically characterized as being either androgen dependent or androgen independent. Whereas androgen-dependent prostate cancer can be successfully treated with androgen ablative therapy, to date no cure exists for androgen-independent disease. Mechanisms involved in the progression of prostate cancer to androgen independence are not known. Here we present evidence that in addition to growth factor receptor tyrosine kinases, G protein- coupled receptors can mediate survival signals in prostate cancer cells. The G protein- coupled receptors exert their effects by activating multiple intracellular signal transduction networks that promote prostate cancer cell survival, including the activation of c-Jun N-terminal kinase, protein kinase B (Akt) and nuclear factor-kB. Prostate-expressed G protein- coupled receptors and their downstream effectors may prove to be effective targets in the treatment of advanced prostate cancer.

  12. Functional characterisation of the Anopheles leucokinins and their cognate G-protein coupled receptor.

    PubMed

    Radford, Jonathan C; Terhzaz, Selim; Cabrero, Pablo; Davies, Shireen-A; Dow, Julian A T

    2004-12-01

    Identification of the Anopheles gambiae leucokinin gene from the completed A. gambiae genome revealed that this insect species contains three leucokinin peptides, named Anopheles leucokinin I-III. These peptides are similar to those identified in two other mosquito species, Aedes aegypti and Culex salinarius. Additionally, Anopheles leucokinin I displays sequence similarity to Drosophila melanogaster leucokinin. Using a combination of computational and molecular approaches, a full-length cDNA for a candidate leucokinin-like receptor was isolated from A. stephensi, a close relative of A. gambiae. Alignment of the known leucokinin receptors--all G protein-coupled receptors (GPCRs)--with this receptor, identified some key conserved regions within the receptors, notably transmembrane (TM) domains I, II, III, VI and VII. The Anopheles leucokinins and receptor were shown to be a functional receptor-ligand pair. All three Anopheles leucokinins caused a dose-dependent rise in intracellular calcium ([Ca2+]i) when applied to S2 cells co-expressing the receptor and an aequorin transgene, with a potency order of I>II>III. Drosophila leucokinin was also found to activate the Anopheles receptor with a similar EC50 value to Anopheles leucokinin I. However, when the Anopheles peptides were applied to the Drosophila receptor, only Anopheles leucokinin I and II elicited a rise in [Ca2+]i. This suggests that the Anopheles receptor has a broader specificity for leucokinin ligands than the Drosophila receptor. Antisera raised against the Anopheles receptor identified a doublet of approx. 65 and 72 kDa on western blots, consistent with the presence of four N-glycosylation sites within the receptor sequence, and the known glycosylation of the receptor in Drosophila. In Anopheles tubules, as in Drosophila, the receptor was localised to the stellate cells. Thus we provide the first identification of Anopheles mosquito leucokinins (Anopheles leucokinins) and a cognate leucokinin receptor

  13. Selection of the lamprey VLRC antigen receptor repertoire.

    PubMed

    Holland, Stephen J; Gao, Mingming; Hirano, Masayuki; Iyer, Lakshminarayan M; Luo, Ming; Schorpp, Michael; Cooper, Max D; Aravind, L; Mariuzza, Roy A; Boehm, Thomas

    2014-10-14

    The alternative adaptive immune system of jawless vertebrates is based on different isotypes of variable lymphocyte receptors (VLRs) that are composed of leucine-rich repeats (LRRs) and expressed by distinct B- and T-like lymphocyte lineages. VLRB is expressed by B-like cells, whereas VLRA and VLRC are expressed by two T-like lineages that develop in the thymoid, a thymus-like structure in lamprey larvae. In each case, stepwise combinatorial insertions of different types of short donor LRR cassettes into incomplete germ-line genes are required to generate functional VLR gene assemblies. It is unknown, however, whether the diverse repertoires of VLRs that are expressed by peripheral blood lymphocytes are shaped by selection after their assembly. Here, we identify signatures of selection in the peripheral repertoire of VLRC antigen receptors that are clonally expressed by one of the T-like cell types in lampreys. Selection strongly favors VLRC molecules containing four internal variable leucine-rich repeat (LRRV) modules, although VLRC assemblies encoding five internal modules are initially equally frequent. In addition to the length selection, VLRC molecules in VLRC(+) peripheral lymphocytes exhibit a distinct pattern of high entropy sites in the N-terminal LRR1 module, which is inserted next to the germ-line-encoded LRRNT module. This is evident in comparisons to VLRC gene assemblies found in the thymoid and to VLRC gene assemblies found in some VLRA(+) cells. Our findings are the first indication to our knowledge that selection operates on a VLR repertoire and provide a framework to establish the mechanism by which this selection occurs during development of the VLRC(+) lymphocyte lineage.

  14. Selective androgen receptor modulators in preclinical and clinical development

    PubMed Central

    Narayanan, Ramesh; Mohler, Michael L.; Bohl, Casey E.; Miller, Duane D.; Dalton, James T.

    2008-01-01

    Androgen receptor (AR) plays a critical role in the function of several organs including primary and accessory sexual organs, skeletal muscle, and bone, making it a desirable therapeutic target. Selective androgen receptor modulators (SARMs) bind to the AR and demonstrate osteo- and myo-anabolic activity; however, unlike testosterone and other anabolic steroids, these nonsteroidal agents produce less of a growth effect on prostate and other secondary sexual organs. SARMs provide therapeutic opportunities in a variety of diseases, including muscle wasting associated with burns, cancer, or end-stage renal disease, osteoporosis, frailty, and hypogonadism. This review summarizes the current standing of research and development of SARMs, crystallography of AR with SARMs, plausible mechanisms for their action and the potential therapeutic indications for this emerging class of drugs. PMID:19079612

  15. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors

    PubMed Central

    Culhane, Kelly J.; Liu, Yuting; Cai, Yingying; Yan, Elsa C. Y.

    2015-01-01

    Although family B G protein-coupled receptors (GPCRs) contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs. PMID:26594176

  16. Receptor component protein (RCP): a member of a multi-protein complex required for G-protein-coupled signal transduction.

    PubMed

    Prado, M A; Evans-Bain, B; Dickerson, I M

    2002-08-01

    The calcitonin-gene-related peptide (CGRP) receptor component protein (RCP) is a 148-amino-acid intracellular protein that is required for G-protein-coupled signal transduction at receptors for the neuropeptide CGRP. RCP works in conjunction with two other proteins to constitute a functional CGRP receptor: calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein 1 (RAMP1). CRLR has the stereotypical seven-transmembrane topology of a G-protein-coupled receptor; it requires RAMP1 for trafficking to the cell surface and for ligand specificity, and requires RCP for coupling to the cellular signal transduction pathway. We have made cell lines that expressed an antisense construct of RCP and determined that CGRP-mediated signal transduction was reduced, while CGRP binding was unaffected. Furthermore, signalling at two other endogenous G-protein-coupled receptors was unaffected, suggesting that RCP was specific for a limited subset of receptors.

  17. G protein-coupled receptors in child development, growth, and maturation.

    PubMed

    Latronico, Ana Claudia; Hochberg, Ze'ev

    2010-10-12

    G protein-coupled receptors (GPCRs) constitute a large family of cell membrane receptors that affect embryogenesis, development, and child physiology, and they are targets for approved drugs and those still in development. The sensitivity of GPCRs to their respective extracellular hormones, neurotransmitters, and environmental stimulants, as well as their interaction with other receptors and intracellular signaling proteins (such as receptor activity-modifying proteins), contribute to variations in child development, growth, and maturation. Here, we summarize current knowledge about the mechanisms of activation (in either the presence or absence of ligands) that lead to the sensitivities of GPCRs and their respective effects as seen throughout human developmental and maturational phases.

  18. Ultraviolet irradiation selectively disrupts the gamma-aminobutyric acid/benzodiazepine receptor-linked chloride ionophore

    SciTech Connect

    Evoniuk, G.; Moody, E.J.; Skolnick, P. )

    1989-05-01

    The ability of UV light to affect radioligand binding and 36Cl-uptake at the gamma-aminobutyric acidA (GABAA) receptor-chloride channel complex was examined. Exposure to 302 nm UV light produced a rapid (t1/2 = 4 min) reduction in (35S)t-butylbicyclo-phosphorothionate binding (assayed in the presence of 200 mM chloride) to sites associated with the GABAA receptor-coupled chloride ionophore. Saturation analysis revealed that this effect could be attributed entirely to a decrease in the maximum number of binding sites. Exposure to UV irradiation at lower (254 nm) and higher (366 nm) wavelengths also inhibited (35S)t-butylbicy-clophosphorothionate binding, but the respective rates of inactivation were 8- and 27-fold slower, compared with 302 nm. Other anion-dependent interactions at the GABAA receptor complex were disrupted in a similar manner. In the absence of permeant anion, (3H)flunitrazepam binding to benzodiazepine receptors was unaffected by 302 nm UV irradiation, whereas chloride-enhanced (3H)flunitrazepam binding was inhibited markedly. In the presence of 250-500 mM chloride, (3H)methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate binding to benzodiazepine receptors was also inhibited after UV exposure. Basal 36Cl- uptake into synaptoneurosomes was nearly doubled after 15 min of exposure to 302 nm light, whereas pentobarbital- and muscimol-stimulated 36Cl- uptake were reduced significantly. UV irradiation at 302 nm appears to disrupt selectively the anion-dependent functional interactions at the GABAA receptor complex. The apparent wavelength specificity suggests that the gating structure (channel) may contain tryptophan and/or tyrosine residues vital to the regulation of anion movement through the ionophore portion of this supramolecular receptor-ion channel complex.

  19. Functional receptor coupling to Gi is a mechanism of agonist-promoted desensitization of the beta2-adrenergic receptor.

    PubMed

    Tepe, N M; Liggett, S B

    2000-01-01

    The beta2-adrenergic receptor (beta2AR) couples to Gs activating adenylyl cyclase (AC) and increasing cAMP. Such signaling undergoes desensitization with continued agonist exposure. Beta2AR also couple to Gi after receptor phosphorylation by the cAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of beta2AR-Gi coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express beta2AR or beta2AR and Gialpha2, and then treated with vehicle or the agonist isoproterenol to evoke agonist-promoted beta2AR desensitization. Membrane AC activities showed that Gialpha2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist-stimulated AC was observed with the cells overexpressing Gialpha2. In the absence of such overexpression, beta2AR desensitization was 23+/-7%, while with 5-fold Gialpha2 overexpression desensitization was 58+/-5% (p<0.01, n=4). The effect of Gi on desensitization was receptor-specific, in that forskolin responses were not altered by G(i)alpha2 overexpression. Thus, acquired beta2AR coupling to Gi is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase Gi levels contribute to beta2AR dysfunction.

  20. Selection of DNA aptamers against rat liver X receptors

    SciTech Connect

    Surugiu-Waernmark, Ioana . E-mail: Ioana.Warnmark@tbiokem.lth.se; Waernmark, Anette; Toresson, Gudrun; Gustafsson, Jan-Ake; Buelow, Leif

    2005-07-01

    Liver X receptors alpha and beta (LXR{alpha}; LXR{beta}) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors. LXRs play an important role in the reverse cholesterol transport and govern the expression of many of the proteins that are indispensable for the regulation of normal cholesterol levels in the body. SELEX, an in vitro selection technology, was used on a single stranded DNA library harboring a 12 randomized nucleotide sequence in order to isolate aptamers showing affinity for LXR{alpha}. Enzyme-linked assays and surface plasmon resonance measurements showed that the selected aptamers had strong affinities for LXR{alpha} with apparent dissociation constants, K {sub d}s, in nanomolar range. All clones carried CG-repeats, indicating a probability for a similar manner of binding to LXR{alpha}. Very high cross-reactivities were observed when testing the aptamers with LXR{beta} (up to 700%) and RXR{alpha} (up to 50%). If instead we regard the aptamer sequences as selected against LXR{beta}, the cross-reactivities decrease considerably, to 17% for LXR{alpha} and 7% for RXR{alpha}. Therefore, in the future we are planning to use the obtained aptamers as binders for LXR{beta}.

  1. Supraspinal Gβγ-dependent stimulation of PLCβ3 originating from G inhibitory protein-μ opioid receptor-coupling is necessary for morphine induced acute hyperalgesia

    PubMed Central

    Bianchi, Enrica; Norcini, Monica; Smrcka, Alan; Ghelardini, Carla

    2009-01-01

    Although alterations in μ-opioid receptor signaling mediate excitatory effects of opiates in opioid tolerance, the molecular mechanism for the excitatory effect of acute low dose morphine, as it relates to μ-opioid receptor coupling, is presently unknown. A pronounced coupling of μ-opioid receptor to the α subunit of G inhibitory protein emerged in periaqueductal gray from mice systemically administered with morphine at a dose producing acute thermal hyperalgesia. This coupling was abolished in presence of the selective μ-opioid receptor receptor antagonist CTOP administered at the periaqueductal gray site, showing that the low dose morphine effect is triggered by μ-opioid receptor activated G inhibitory protein at supraspinal level. When Gβγ downstream signalling was blocked by intra-periaqueductal gray co-administration of M119, a compound that inhibits Gβγ dimer-dependent signaling, a complete prevention of low dose morphine induced acute thermal hyperalgesia was obtained. Phospholipase C β3, an enzyme necessary to morphine hyperalgesia, was revealed to be associated with Gβγ in periaqueductal gray. Although opioid administration induces a shift in μ-opioid receptor-G protein coupling from Gi to Gs after chronic administration, our data support that this condition is not realized in acute treatment providing evidence that a separate molecular mechanism underlies morphine induced acute excitatory effect. PMID:19656263

  2. Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes

    PubMed Central

    Cerrato, F; Fernández-Suárez, M E; Alonso, R; Alonso, M; Vázquez, C; Pastor, O; Mata, P; Lasunción, M A; Gómez-Coronado, D

    2015-01-01

    Background and Purpose Treatment with selective oestrogen receptor modulators (SERMs) reduces low-density lipoprotein (LDL) cholesterol levels. We assessed the effect of tamoxifen, raloxifene and toremifene and their combinations with lovastatin on LDL receptor activity in lymphocytes from normolipidaemic and familial hypercholesterolaemic (FH) subjects, and human HepG2 hepatocytes and MOLT-4 lymphoblasts. Experimental Approach Lymphocytes were isolated from peripheral blood, treated with different compounds, and 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanine perchlorate (DiI)-labelled LDL uptake was analysed by flow cytometry. Key Results Tamoxifen, toremifene and raloxifene, in this order, stimulated DiI-LDL uptake by lymphocytes by inhibiting LDL-derived cholesterol trafficking and subsequent down-regulation of LDL receptor expression. Differently to what occurred in HepG2 and MOLT-4 cells, only tamoxifen consistently displayed a potentiating effect with lovastatin in primary lymphocytes. The SERM-mediated increase in LDL receptor activity was not altered by the anti-oestrogen ICI 182 780 nor was it reproduced by 17β-oestradiol. However, the tamoxifen-active metabolite endoxifen was equally effective as tamoxifen. The SERMs produced similar effects on LDL receptor activity in heterozygous FH lymphocytes as in normal lymphocytes, although none of them had a potentiating effect with lovastatin in heterozygous FH lymphocytes. The SERMs had no effect in homozygous FH lymphocytes. Conclusions and Implications Clinically used SERMs up-regulate LDL receptors in primary human lymphocytes. There is a mild enhancement between SERMs and lovastatin of lymphocyte LDLR activity, the potentiation being greater in HepG2 and MOLT-4 cells. The effect of SERMs is independent of oestrogen receptors but is preserved in the tamoxifen-active metabolite endoxifen. This mechanism may contribute to the cholesterol-lowering action of SERMs. PMID:25395200

  3. Loss of Gi G-Protein-Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing.

    PubMed

    Wang, Liping; Hsiao, Edward C; Lieu, Shirley; Scott, Mark; O'Carroll, Dylan; Urrutia, Ashley; Conklin, Bruce R; Colnot, Celine; Nissenson, Robert A

    2015-10-01

    G-protein-coupled receptors (GPCRs) are key regulators of skeletal homeostasis and are likely important in fracture healing. Because GPCRs can activate multiple signaling pathways simultaneously, we used targeted disruption of G(i) -GPCR or activation of G(s) -GPCR pathways to test how each pathway functions in the skeleton. We previously demonstrated that blockade of G(i) signaling by pertussis toxin (PTX) transgene expression in maturing osteoblastic cells enhanced cortical and trabecular bone formation and prevented age-related bone loss in female mice. In addition, activation of G(s) signaling by expressing the G(s) -coupled engineered receptor Rs1 in maturing osteoblastic cells induced massive trabecular bone formation but cortical bone loss. Here, we test our hypothesis that the G(i) and G(s) pathways also have distinct functions in fracture repair. We applied closed, nonstabilized tibial fractures to mice in which endogenous G(i) signaling was inhibited by PTX, or to mice with activated G(s) signaling mediated by Rs1. Blockade of endogenous G(i) resulted in a smaller callus but increased bone formation in both young and old mice. PTX treatment decreased expression of Dkk1 and increased Lef1 mRNAs during fracture healing, suggesting a role for endogenous G(i) signaling in maintaining Dkk1 expression and suppressing Wnt signaling. In contrast, adult mice with activated Gs signaling showed a slight increase in the initial callus size with increased callus bone formation. These results show that G(i) blockade and G(s) activation of the same osteoblastic lineage cell can induce different biological responses during fracture healing. Our findings also show that manipulating the GPCR/cAMP signaling pathway by selective timing of G(s) and G(i) -GPCR activation may be important for optimizing fracture repair.

  4. Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling.

    PubMed

    Alvarez-Curto, Elisa; Inoue, Asuka; Jenkins, Laura; Raihan, Sheikh Zahir; Prihandoko, Rudi; Tobin, Andrew B; Milligan, Graeme

    2016-12-30

    G protein-coupled receptors (GPCRs) can initiate intracellular signaling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signaling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells together with the elimination of receptor phosphorylation sites to define the relative contribution of G proteins, arrestins, and receptor phosphorylation to the signaling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular Ca(2+) in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signaling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signaling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms, whereas a substantially enhanced ERK1/2 response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signaling research and employ these cells to reveal a previously unappreciated interplay of signaling pathways where receptor phosphorylation can impact on ERK1/2 signaling through a mechanism that is likely independent of arrestins.

  5. Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling*

    PubMed Central

    Alvarez-Curto, Elisa; Inoue, Asuka; Jenkins, Laura; Raihan, Sheikh Zahir; Prihandoko, Rudi; Tobin, Andrew B.

    2016-01-01

    G protein-coupled receptors (GPCRs) can initiate intracellular signaling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signaling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells together with the elimination of receptor phosphorylation sites to define the relative contribution of G proteins, arrestins, and receptor phosphorylation to the signaling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular Ca2+ in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signaling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signaling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms, whereas a substantially enhanced ERK1/2 response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signaling research and employ these cells to reveal a previously unappreciated interplay of signaling pathways where receptor phosphorylation can impact on ERK1/2 signaling through a mechanism that is likely independent of arrestins. PMID:27852822

  6. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors.

    PubMed

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-09-15

    G protein-coupled receptors are divided into three classes (A, B and C) based on homology of their seven transmembrane domains. Class C is the smallest class with 22 human receptor subtypes including eight metabotropic glutamate (mGlu1-8) receptors, two GABAB receptors (GABAB1 and GABAB2), three taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we review the existence of post-translational modifications in class C G protein-coupled receptors and their regulatory roles, with particular focus on glycosylation, phosphorylation, ubiquitination, SUMOylation, disulphide bonding and lipidation.

  7. Substituted Tetrahydroisoquinolines as Selective Antagonists for the Orexin 1 Receptor

    PubMed Central

    Perrey, David A.; German, Nadezhda A.; Gilmour, Brian P.; Li, Jun-Xu; Harris, Danni L.; Thomas, Brian F.; Zhang, Yanan

    2013-01-01

    Increasing evidence implicates the orexin 1 (OX1) receptor in reward processes, suggesting OX1 antagonism could be therapeutic in drug addiction. In a program to develop an OX1 selective antagonist, we designed and synthesized a series of substituted tetrahydroisoquinolines and determined their potency in OX1 and OX2 calcium mobilization assays. Structure-activity relationship (SAR) studies revealed limited steric tolerance and preference for electron deficiency at the 7-position. Pyridylmethyl groups were shown to be optimal for activity at the acetamide position. Computational studies resulted in a pharmacophore model and confirmed the SAR results. Compound 72 significantly attenuated the development of place preference for cocaine in rats. PMID:23941044

  8. Computational methods for studying G protein-coupled receptors (GPCRs).

    PubMed

    Kaczor, Agnieszka A; Rutkowska, Ewelina; Bartuzi, Damian; Targowska-Duda, Katarzyna M; Matosiuk, Dariusz; Selent, Jana

    2016-01-01

    The functioning of GPCRs is classically described by the ternary complex model as the interplay of three basic components: a receptor, an agonist, and a G protein. According to this model, receptor activation results from an interaction with an agonist, which translates into the activation of a particular G protein in the intracellular compartment that, in turn, is able to initiate particular signaling cascades. Extensive studies on GPCRs have led to new findings which open unexplored and exciting possibilities for drug design and safer and more effective treatments with GPCR targeting drugs. These include discovery of novel signaling mechanisms such as ligand promiscuity resulting in multitarget ligands and signaling cross-talks, allosteric modulation, biased agonism, and formation of receptor homo- and heterodimers and oligomers which can be efficiently studied with computational methods. Computer-aided drug design techniques can reduce the cost of drug development by up to 50%. In particular structure- and ligand-based virtual screening techniques are a valuable tool for identifying new leads and have been shown to be especially efficient for GPCRs in comparison to water-soluble proteins. Modern computer-aided approaches can be helpful for the discovery of compounds with designed affinity profiles. Furthermore, homology modeling facilitated by a growing number of available templates as well as molecular docking supported by sophisticated techniques of molecular dynamics and quantitative structure-activity relationship models are an excellent source of information about drug-receptor interactions at the molecular level.

  9. Effect of niflumic acid on electromechanical coupling by tachykinin NK1 receptor activation in rabbit colon.

    PubMed

    Patacchini, R; Santicioli, P; Maggi, C A

    1996-05-15

    We have investigated the effect of the Cl- channel blocker, niflumic acid, on the contractile response and electromechanical coupling activated by stimulation of the tachykinin NK1 receptor in the longitudinal muscle of rabbit proximal colon, in the presence of indomethacin (5 microM). The application of submaximal equieffective concentrations of the tachykinin NK1 receptor-selective agonist [Sar9]substance P sulfone (30 nM), of carbachol (300 nM) and KCl (40 mM), produced distinct phasic and tonic components of contraction. Niflumic acid (10-100 microM) preferentially and markedly inhibited the tonic component of the response to [Sar9]substance P sulfone and to carbachol, without affecting the response to KCl. Nifedipine (1 microM) abolished the response to KCl and greatly reduced the response to [Sar9]substance P sulfone and carbachol. The nifedipine-resistant response to [Sar9]substance P sulfone was attenuated by niflumic acid (100 microM), while that to carbachol was unaffected. In sucrose gap experiments, superfusion with niflumic acid (100 microM), in the presence of nifedipine (3 microM), produced membrane hyperpolarization, which was totally blocked by tetraethylammonium (10 mM). Niflumic acid inhibited both depolarization and contraction induced by [Sar9]substance P sulfone, both in the absence or in the presence of tetraethylammonium. The present findings support the idea that a niflumic acid-sensitive mechanism, probably an effect on Cl- channels, takes part in the post-receptorial events activated by tachykinin NK1 receptor stimulation in the longitudinal muscle of rabbit colon, and suggest that this mechanism would be more important for generating the sustained tonic than the phasic component of contraction.

  10. Direct molecular evolution of detergent-stable G protein-coupled receptors using polymer encapsulated cells.

    PubMed

    Scott, Daniel J; Plückthun, Andreas

    2013-02-08

    G protein-coupled receptors (GPCRs) are the largest class of pharmaceutical protein targets, yet drug development is encumbered by a lack of information about their molecular structure and conformational dynamics. Most mechanistic and structural studies as well as in vitro drug screening with purified receptors require detergent solubilization of the GPCR, but typically, these proteins exhibit only low stability in detergent micelles. We have developed the first directed evolution method that allows the direct selection of GPCRs stable in a chosen detergent from libraries containing over 100 million individual variants. The crucial concept was to encapsulate single Escherichia coli cells of a library, each expressing a different GPCR variant, to form detergent-resistant, semipermeable nano-containers. Unlike naked cells, these containers are not dissolved by detergents, allowing us to solubilize the GPCR proteins in situ while maintaining an association with the protein's genetic information, a prerequisite for directed evolution. The pore size was controlled to permit GPCR ligands to permeate but the solubilized receptor to remain within the nanocapsules. Fluorescently labeled ligands were used to bind to those GPCR variants inside the nano-containers that remained active in the detergent tested. With the use of fluorescence-activated cell sorting, detergent-stable mutants derived from two different family A GPCRs could be identified, some with the highest stability reported in short-chain detergents. In principle, this method (named cellular high-throughput encapsulation, solubilization and screening) is not limited to engineering stabilized GPCRs but could be used to stabilize other proteins for biochemical and structural studies.

  11. Optimization of 2-phenylcyclopropylmethylamines as selective serotonin 2C receptor agonists and their evaluation as potential antipsychotic agents.

    PubMed

    Cheng, Jianjun; Giguère, Patrick M; Onajole, Oluseye K; Lv, Wei; Gaisin, Arsen; Gunosewoyo, Hendra; Schmerberg, Claire M; Pogorelov, Vladimir M; Rodriguiz, Ramona M; Vistoli, Giulio; Wetsel, William C; Roth, Bryan L; Kozikowski, Alan P

    2015-02-26

    The discovery of a new series of compounds that are potent, selective 5-HT2C receptor agonists is described herein as we continue our efforts to optimize the 2-phenylcyclopropylmethylamine scaffold. Modifications focused on the alkoxyl substituent present on the aromatic ring led to the identification of improved ligands with better potency at the 5-HT2C receptor and excellent selectivity against the 5-HT2A and 5-HT2B receptors. ADMET studies coupled with a behavioral test using the amphetamine-induced hyperactivity model identified four compounds possessing drug-like profiles and having antipsychotic properties. Compound (+)-16b, which displayed an EC50 of 4.2 nM at 5-HT2C, no activity at 5-HT2B, and an 89-fold selectivity against 5-HT2A, is one of the most potent and selective 5-HT2C agonists reported to date. The likely binding mode of this series of compounds to the 5-HT2C receptor was also investigated in a modeling study, using optimized models incorporating the structures of β2-adrenergic receptor and 5-HT2B receptor.

  12. Complexing receptor pharmacology: modulation of family B G protein-coupled receptor function by RAMPs.

    PubMed

    Sexton, Patrick M; Morfis, Maria; Tilakaratne, Nanda; Hay, Debbie L; Udawela, Madhara; Christopoulos, George; Christopoulos, Arthur

    2006-07-01

    The most well-characterized subgroup of family B G protein-coupledreceptors (GPCRs) comprises receptors for peptide hormones, such as secretin, calcitonin (CT), glucagon, and vasoactive intestinal peptide (VIP). Recent data suggest that many of these receptors can interact with a novel family of GPCR accessory proteins termed receptor activity modifying proteins (RAMPs). RAMP interaction with receptors can lead to a variety of actions that include chaperoning of the receptor protein to the cell surface as is the case for the calcitonin receptor-like receptor (CLR) and the generation of novel receptor phenotypes. RAMP heterodimerization with the CLR and related CT receptor is required for the formation of specific CT gene-related peptide, adrenomedullin (AM) or amylin receptors. More recent work has revealed that the specific RAMP present in a heterodimer may modulate other functions such as receptor internalization and recycling and also the strength of activation of downstream signaling pathways. In this article we review our current state of knowledge of the consequence of RAMP interaction with family B GPCRs.

  13. Stimulation of α₁-adrenoceptor or angiotensin type 1 receptor enhances DNA synthesis in human-induced pluripotent stem cells via Gq-coupled receptor-dependent signaling pathways.

    PubMed

    Ishizuka, Toshiaki; Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro

    2013-08-15

    Stimulation of either α₁-adrenoceptor or angiotensin type 1 receptor (AT₁ receptor) induces proliferation of mouse induced pluripotent stem (iPS) cells. Both α₁-adrenoceptor and AT₁ receptor are guanine nucleotide-binding protein q polypeptide (Gq)-coupled receptors. However, it is not fully understood whether stimulation of these Gq-coupled receptors exert a similar effect in human iPS cells, i.e. proliferation of human iPS cells. In this study, we evaluated the involvement of α₁-adrenoceptor and AT₁ receptor in the DNA synthesis of human iPS cells. Treatment with either l-phenylephrine (a selective α₁-adrenoceptor agonist) or angiotensin II (Ang II) significantly increased DNA synthesis in human iPS cells. Enhanced DNA synthesis was significantly inhibited by pretreatment with protein kinase C (PKC) inhibitors, mitogen-activated protein kinase kinase (MEK) inhibitor, or phosphatidylinositol-3 phosphate kinase (PI3K) inhibitor. Treatment with either l-phenylephrine or Ang II significantly increased Akt and p44/42 MAPK phosphorylation. Short interfering RNA (siRNA) directed against Gq significantly inhibited DNA synthesis and phosphorylation of Akt and p44/42 MAPK enhanced by l-phenylephrine or Ang II. These results suggest that stimulation of α₁-adrenoceptor or AT₁ receptor may enhance DNA synthesis in human iPS cells via Gq-coupled receptor-dependent signaling pathways.

  14. Ligand-guided pathway selection in nickel-catalyzed couplings of enals and alkynes.

    PubMed

    Li, Wei; Montgomery, John

    2012-01-28

    Nickel-catalyzed couplings of enals and alkynes utilizing triethylborane as the reducing agent illustrate a significant dependence on ligand structure. Simple variation of monodentate phosphines allows selective access to alkylative couplings or reductive cycloadditions, while further variation of reaction conditions provides clean access to reductive couplings and redox-neutral couplings.

  15. Cloning of a novel G protein-coupled receptor, SLT, a subtype of the melanin-concentrating hormone receptor.

    PubMed

    Mori, M; Harada, M; Terao, Y; Sugo, T; Watanabe, T; Shimomura, Y; Abe, M; Shintani, Y; Onda, H; Nishimura, O; Fujino, M

    2001-05-25

    A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.

  16. Ligand-induced IFN gamma receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91).

    PubMed Central

    Greenlund, A C; Farrar, M A; Viviano, B L; Schreiber, R D

    1994-01-01

    Herein we report that interferon-gamma (IFN gamma) induces the rapid and reversible tyrosine phosphorylation of the IFN gamma receptor. Using a panel of receptor intracellular domain mutants, we show that a membrane-proximal LPKS sequence (residues 266-269) is required for ligand-induced tyrosine kinase activation and/or kinase-receptor association and biological responsiveness, and a functionally critical membrane-distal tyrosine residue (Y440) is a target of the activated enzyme. The biological significance of Y440 phosphorylation was demonstrated by showing that a receptor-derived nonapeptide corresponding to receptor residues 436-444 and containing phosphorylated Y440 bound specifically to p91, blocked p91 phosphorylation and inhibited the generation of an active p91-containing transcription factor complex. In contrast, nonphosphorylated wild-type, phosphorylated mutant, or phosphorylated irrelevant peptides did not. Moreover, the phosphorylated Y440-containing peptide did not interact with a related but distinct latent transcription factor (p113) which is activatible by IFN alpha but not IFN gamma. These results thus document the specific and inducible association of p91 with the phosphorylated IFN gamma receptor and thereby elucidate the mechanism by which ligand couples the IFN gamma receptor to its signal transduction system. Images PMID:8156998

  17. Molecular basis for agonist selectivity and activation of the orphan BRS-3-receptor

    PubMed Central

    Gonzalez, Nieves; Hocart, Simon J.; Portal-Nuñez, Sergio; Mantey, Samuel A.; Nakagawa, Tomoo; Zudaire, Enrique; Coy, David H.; Jensen, Robert T.

    2008-01-01

    Bombesin receptor subtype-3(BRS-3), a G protein-coupled orphan receptor, shares 51% identity with the mammalian bombesin(Bn) receptor for gastrin-releasing peptide(GRPR). There is increasing interest in BRS-3 because it is important in energy metabolism, glucose control,motility and tumor-growth. BRS-3 has low affinity for all Bn-related peptides, however, recently synthetic high-affinity agonists[D-Tyr6/D-Phe6,βAla11,Phe13,Nle14]Bn-(6–14) were described, but they are nonselective for BRS-3 over other Bn-receptors. Based on these peptides, three BRS-3 selective-ligands were developed: peptide#2,[D-Tyr6(R)-Apa11,Phe13,Nle14]Bn(6–14); peptide#3,[D-Tyr6,(R)-Apa11,4Cl-Phe13,Nle14]Bn(6–14); peptide #4,Ac-Phe-Trp-Ala-His(tBzl)-Nip-Gly-Arg-NH2. Their molecular determinants of selectivity/high affinity for BRS-3 are unknown. To address this we used a chimeric/site-mutagenesis approach. Substitution of extracellular domain2(EC2) of BRS-3 by the comparable GRPR domain decreased 26-,4,0-fold affinity for peptides#4,3,2. Substitution of EC3 decreased affinity 4-,11-,0-fold affinity for peptides#2,3,4. Ten point mutations in the EC2 and adjacent transmembrane regions (TM2) 2 and 3 of BRS-3 were made. His107(EC2-BRS-3) for lysine(H107K)(EC2-GRPR), decreased affinity(25-,0-fold) for peptide#4,1; however it could not be activated by either peptide. Its combination with Val101(TM2),Gly112(EC2),Arg127(TM3) resulted in complete loss-of-affinity of peptide#4. Receptor-modeling showed that each of these residues face inward and are within 4Å of the binding-pocket. These results demonstrate [Val101,His107,Gly112,Arg127] in the EC2/adjacent upper TMs of BRS-3 are critical for the high BRS3-selectivity of peptide#4. His107 in EC2 is essential for BRS-3 activation, suggesting amino-aromatic ligand/receptor interactions with peptide#4 are critical for both binding/ activation. Furthermore, these result demonstrate that even though these three BRS-3 selective agonists were developed

  18. Brain endogenous liver X receptor ligands selectively promote midbrain neurogenesis.

    PubMed

    Theofilopoulos, Spyridon; Wang, Yuqin; Kitambi, Satish Srinivas; Sacchetti, Paola; Sousa, Kyle M; Bodin, Karl; Kirk, Jayne; Saltó, Carmen; Gustafsson, Magnus; Toledo, Enrique M; Karu, Kersti; Gustafsson, Jan-Åke; Steffensen, Knut R; Ernfors, Patrik; Sjövall, Jan; Griffiths, William J; Arenas, Ernest

    2013-02-01

    Liver X receptors (Lxrα and Lxrβ) are ligand-dependent nuclear receptors critical for ventral midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand selectively regulated the development of distinct midbrain neuronal populations. Whereas cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons, 24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class of highly selective and cell type-specific regulators of neurogenesis and neuronal survival. Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells, suggesting that Lxr ligands may thus contribute to the development of cell replacement and regenerative therapies for Parkinson's disease.

  19. Role of the extracellular loops of G protein-coupled receptors in ligand recognition: a molecular modeling study of the human P2Y1 receptor.

    PubMed

    Moro, S; Hoffmann, C; Jacobson, K A

    1999-03-23

    The P2Y1 receptor is a G protein-coupled receptor (GPCR) and is stimulated by extracellular ADP and ATP. Site-directed mutagenesis of the three extracellular loops (ELs) of the human P2Y1 receptor indicates the existence of two essential disulfide bridges (Cys124 in EL1 and Cys202 in EL2; Cys42 in the N-terminal segment and Cys296 in EL3) and several specific ionic and H-bonding interactions (involving Glu209 and Arg287). Through molecular modeling and molecular dynamics simulations, an energetically sound conformational hypothesis for the receptor has been calculated that includes transmembrane (TM) domains (using the electron density map of rhodopsin as a template), extracellular loops, and a truncated N-terminal region. ATP may be docked in the receptor, both within the previously defined TM cleft and within two other regions of the receptor, termed meta-binding sites, defined by the extracellular loops. The first meta-binding site is located outside of the TM bundle, between EL2 and EL3, and the second higher energy site is positioned immediately underneath EL2. Binding at both the principal TM binding site and the lower energy meta-binding sites potentially affects the observed ligand potency. In meta-binding site I, the side chain of Glu209 (EL2) is within hydrogen-bonding distance (2.8 A) of the ribose O3', and Arg287 (EL3) coordinates both alpha- and beta-phosphates of the triphosphate chain, consistent with the insensitivity in potency of the 5'-monophosphate agonist, HT-AMP, to mutation of Arg287 to Lys. Moreover, the selective reduction in potency of 3'NH2-ATP in activating the E209R mutant receptor is consistent with the hypothesis of direct contact between EL2 and nucleotide ligands. Our findings support ATP binding to at least two distinct domains of the P2Y1 receptor, both outside and within the TM core. The two disulfide bridges present in the human P2Y1 receptor play a major role in the structure and stability of the receptor, to constrain the

  20. Pattern Selection in Network of Coupled Multi-Scroll Attractors.

    PubMed

    Li, Fan; Ma, Jun

    2016-01-01

    Multi-scroll chaotic attractor makes the oscillator become more complex in dynamic behaviors. The collective behaviors of coupled oscillators with multi-scroll attractors are investigated in the regular network in two-dimensional array, which the local kinetics is described by an improved Chua circuit. A feasible scheme of negative feedback with diversity is imposed on the network to stabilize the spatial patterns. Firstly, the Chua circuit is improved by replacing the nonlinear term with Sine function to generate infinite aquariums so that multi-scroll chaotic attractors could be generated under appropriate parameters, which could be detected by calculating the Lyapunov exponent in the parameter region. Furthermore, negative feedback with different gains (D1, D2) is imposed on the local square center area A2 and outer area A1 of the network, it is found that spiral wave, target wave could be developed in the network under appropriate feedback gain with diversity and size of controlled area. Particularly, homogeneous state could be reached after synchronization by selecting appropriate feedback gain and controlled size in the network. Finally, the distribution for statistical factors of synchronization is calculated in the two-parameter space to understand the transition of pattern region. It is found that developed spiral waves, target waves often are associated with smaller factor of synchronization. These results show that emergence of sustained spiral wave and continuous target wave could be effective for further suppression of spatiotemporal chaos in network by generating stable pacemaker completely.

  1. Pattern Selection in Network of Coupled Multi-Scroll Attractors

    PubMed Central

    Li, Fan

    2016-01-01

    Multi-scroll chaotic attractor makes the oscillator become more complex in dynamic behaviors. The collective behaviors of coupled oscillators with multi-scroll attractors are investigated in the regular network in two-dimensional array, which the local kinetics is described by an improved Chua circuit. A feasible scheme of negative feedback with diversity is imposed on the network to stabilize the spatial patterns. Firstly, the Chua circuit is improved by replacing the nonlinear term with Sine function to generate infinite aquariums so that multi-scroll chaotic attractors could be generated under appropriate parameters, which could be detected by calculating the Lyapunov exponent in the parameter region. Furthermore, negative feedback with different gains (D1, D2) is imposed on the local square center area A2 and outer area A1 of the network, it is found that spiral wave, target wave could be developed in the network under appropriate feedback gain with diversity and size of controlled area. Particularly, homogeneous state could be reached after synchronization by selecting appropriate feedback gain and controlled size in the network. Finally, the distribution for statistical factors of synchronization is calculated in the two-parameter space to understand the transition of pattern region. It is found that developed spiral waves, target waves often are associated with smaller factor of synchronization. These results show that emergence of sustained spiral wave and continuous target wave could be effective for further suppression of spatiotemporal chaos in network by generating stable pacemaker completely. PMID:27119986

  2. Novel selective allosteric and bitopic ligands for the S1P(3) receptor.

    PubMed

    Jo, Euijung; Bhhatarai, Barun; Repetto, Emanuela; Guerrero, Miguel; Riley, Sean; Brown, Steven J; Kohno, Yasushi; Roberts, Edward; Schürer, Stephan C; Rosen, Hugh

    2012-12-21

    Sphingosine 1-phosphate (S1P) is a lysophospholipid signaling molecule that regulates important biological functions, including lymphocyte trafficking and vascular development, by activating G protein-coupled receptors for S1P, namely, S1P(1) through S1P(5). Here, we map the S1P(3) binding pocket with a novel allosteric agonist (CYM-5541), an orthosteric agonist (S1P), and a novel bitopic antagonist (SPM-242). With a combination of site-directed mutagenesis, ligand competition assay, and molecular modeling, we concluded that S1P and CYM-5541 occupy different chemical spaces in the ligand binding pocket of S1P(3). CYM-5541 allowed us to identify an allosteric site where Phe263 is a key gate-keeper residue for its affinity and efficacy. This ligand lacks a polar moiety, and the novel allosteric hydrophobic pocket permits S1P(3) selectivity of CYM-5541 within the highly similar S1P receptor family. However, a novel S1P(3)-selective antagonist, SPM-242, in the S1P(3) pocket occupies the ligand binding spaces of both S1P and CYM-5541, showing its bitopic mode of binding. Therefore, our coordinated approach with biochemical data and molecular modeling, based on our recently published S1P(1) crystal structure data in a highly conserved set of related receptors with a shared ligand, provides a strong basis for the successful optimization of orthosteric, allosteric, and bitopic modulators of S1P(3).

  3. GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

    PubMed Central

    Strittmatter, S M; Cannon, S C; Ross, E M; Higashijima, T; Fishman, M C

    1993-01-01

    The neuronal protein GAP-43 is thought to play a role in determining growth-cone motility, perhaps as an intracellular regulator of signal transduction, but its molecular mechanism of action has remained unclear. We find that GAP-43, when microinjected into Xenopus laevis oocytes, increases the oocyte response to G protein-coupled receptor agonists by 10- to 100-fold. Higher levels of GAP-43 cause a transient current flow, even without receptor stimulation. The GAP-43-induced current, like receptor-stimulated currents, is mediated by a calcium-activated chloride channel and can be desensitized by injection of inositol 1,4,5-trisphosphate. This suggests that neuronal GAP-43 may serve as an intracellular signal to greatly enhance the sensitivity of G protein-coupled receptor transduction. Images Fig. 1 Fig. 2 PMID:7685122

  4. Structure-based drug design for G protein-coupled receptors.

    PubMed

    Congreve, Miles; Dias, João M; Marshall, Fiona H

    2014-01-01

    Our understanding of the structural biology of G protein-coupled receptors has undergone a transformation over the past 5 years. New protein-ligand complexes are described almost monthly in high profile journals. Appreciation of how small molecules and natural ligands bind to their receptors has the potential to impact enormously how medicinal chemists approach this major class of receptor targets. An outline of the key topics in this field and some recent examples of structure- and fragment-based drug design are described. A table is presented with example views of each G protein-coupled receptor for which there is a published X-ray structure, including interactions with small molecule antagonists, partial and full agonists. The possible implications of these new data for drug design are discussed.

  5. Role and therapeutic potential of G-protein coupled receptors in breast cancer progression and metastases.

    PubMed

    Singh, Anukriti; Nunes, Jessica J; Ateeq, Bushra

    2015-09-15

    G-protein-coupled receptors (GPCRs) comprise a large family of cell-surface receptors, which have recently emerged as key players in tumorigenesis, angiogenesis and metastasis. In this review, we discussed our current understanding of the many roles played by GPCRs in general, and particularly Angiotensin II type I receptor (AGTR1), a member of the seven-transmembrane-spanning G-protein coupled receptor superfamily, and its significance in breast cancer progression and metastasis. We have also discussed different strategies for targeting AGTR1, and its ligand Angiotension II (Ang II), which might unravel unique opportunities for breast cancer prevention and treatment. For example, AGTR1 blockers (ARBs) which are already in clinical use for treating hypertension, merit further investigation as a therapeutic strategy for AGTR1-positive cancer patients and may have the potential to prevent Ang II-AGTR1 signalling mediated cancer pathogenesis and metastases.

  6. Role and therapeutic potential of G-protein coupled receptors in breast cancer progression and metastases

    PubMed Central

    Singh, Anukriti; Nunes, Jessica J.; Ateeq, Bushra

    2015-01-01

    G-protein-coupled receptors (GPCRs) comprise a large family of cell-surface receptors, which have recently emerged as key players in tumorigenesis, angiogenesis and metastasis. In this review, we discussed our current understanding of the many roles played by GPCRs in general, and particularly Angiotensin II type I receptor (AGTR1), a member of the seven-transmembrane-spanning G-protein coupled receptor superfamily, and its significance in breast cancer progression and metastasis. We have also discussed different strategies for targeting AGTR1, and its ligand Angiotension II (Ang II), which might unravel unique opportunities for breast cancer prevention and treatment. For example, AGTR1 blockers (ARBs) which are already in clinical use for treating hypertension, merit further investigation as a therapeutic strategy for AGTR1-positive cancer patients and may have the potential to prevent Ang II-AGTR1 signalling mediated cancer pathogenesis and metastases. PMID:25981295

  7. G protein-coupled receptor kinase and beta-arrestin-mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylglycerol dynamics.

    PubMed

    Violin, Jonathan D; Dewire, Scott M; Barnes, William G; Lefkowitz, Robert J

    2006-11-24

    Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

  8. Selective glucocorticoid receptor-activating adjuvant therapy in cancer treatments

    PubMed Central

    Sundahl, Nora; Clarisse, Dorien; Bracke, Marc; Offner, Fritz; Berghe, Wim Vanden; Beck, Ilse M.

    2016-01-01

    Although adverse effects and glucocorticoid resistance cripple their chronic use, glucocorticoids form the mainstay therapy for acute and chronic inflammatory disorders, and play an important role in treatment protocols of both lymphoid malignancies and as adjuvant to stimulate therapy tolerability in various solid tumors. Glucocorticoid binding to their designate glucocorticoid receptor (GR), sets off a plethora of cell-specific events including therapeutically desirable effects, such as cell death, as well as undesirable effects, including chemotherapy resistance, systemic side effects and glucocorticoid resistance. In this context, selective GR agonists and modulators (SEGRAMs) with a more restricted GR activity profile have been developed, holding promise for further clinical development in anti-inflammatory and potentially in cancer therapies. Thus far, the research into the prospective benefits of selective GR modulators in cancer therapy limped behind. Our review discusses how selective GR agonists and modulators could improve the therapy regimens for lymphoid malignancies, prostate or breast cancer. We summarize our current knowledge and look forward to where the field should move to in the future. Altogether, our review clarifies novel therapeutic perspectives in cancer modulation via selective GR targeting. PMID:27713909

  9. Intracellular calcium level is an important factor influencing ion channel modulations by PLC-coupled metabotropic receptors in hippocampal neurons.

    PubMed

    Sugawara, Yuto; Echigo, Ryousuke; Kashima, Kousuke; Minami, Hanae; Watanabe, Megumi; Nishikawa, Yuiko; Muranishi, Miho; Yoneda, Mitsugu; Ohno-Shosaku, Takako

    2013-05-28

    Signaling pathways involving phospholipase C (PLC) are involved in various neural functions. Understanding how these pathways are regulated will lead to a better understanding of their roles in neural functions. Previous studies demonstrated that receptor-driven PLCβ activation depends on intracellular Ca(2+) concentration ([Ca(2+)]i), suggesting the possibility that PLCβ-dependent cellular responses are basically Ca(2+) dependent. To test this possibility, we examined whether modulations of ion channels driven by PLC-coupled metabotropic receptors are sensitive to [Ca(2+)]i using cultured hippocampal neurons. Muscarinic activation triggered an inward current at -100 mV (the equilibrium potential for K(+)) in a subpopulation of neurons. This current response was suppressed by pirenzepine (an M1-preferring antagonist), PLC inhibitor, non-selective cation channel blocker, and lowering [Ca(2+)]i. Using the neurons showing no response at -100 mV, effects of muscarinic activation on K(+) channels were examined at -40 mV. Muscarinic activation induced a transient decrease of the holding outward current. This current response was mimicked and occluded by XE991, an M-current K(+) channel blocker, suppressed by pirenzepine, PLC inhibitor and lowering [Ca(2+)]i, and enhanced by elevating [Ca(2+)]i. Similar results were obtained when group I metabotropic glutamate receptors were activated instead of muscarinic receptors. These results clearly show that ion channel modulations driven by PLC-coupled metabotropic receptors are dependent on [Ca(2+)]i, supporting the hypothesis that cellular responses induced by receptor-driven PLCβ activation are basically Ca(2+) dependent.

  10. Phenotypic Regulation of the Sphingosine 1-Phosphate Receptor Miles Apart by G Protein-Coupled Receptor Kinase 2

    PubMed Central

    2016-01-01

    The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m93 (milm93), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling. PMID:25555130

  11. A calixpyrrole derivative acts as an antagonist to GPER, a G-protein coupled receptor: mechanisms and models

    PubMed Central

    Lappano, Rosamaria; Rosano, Camillo; Pisano, Assunta; Santolla, Maria Francesca; De Francesco, Ernestina Marianna; De Marco, Paola; Dolce, Vincenza; Ponassi, Marco; Felli, Lamberto; Cafeo, Grazia; Kohnke, Franz Heinrich; Abonante, Sergio; Maggiolini, Marcello

    2015-01-01

    ABSTRACT Estrogens regulate numerous pathophysiological processes, mainly by binding to and activating estrogen receptor (ER)α and ERβ. Increasing amounts of evidence have recently demonstrated that G-protein coupled receptor 30 (GPR30; also known as GPER) is also involved in diverse biological responses to estrogens both in normal and cancer cells. The classical ER and GPER share several features, including the ability to bind to identical compounds; nevertheless, some ligands exhibit opposed activity through these receptors. It is worth noting that, owing to the availability of selective agonists and antagonists of GPER for research, certain differential roles elicited by GPER compared with ER have been identified. Here, we provide evidence on the molecular mechanisms through which a calixpyrrole derivative acts as a GPER antagonist in different model systems, such as breast tumor cells and cancer-associated fibroblasts (CAFs) obtained from breast cancer patients. Our data might open new perspectives toward the development of a further class of selective GPER ligands in order to better dissect the role exerted by this receptor in different pathophysiological conditions. Moreover, calixpyrrole derivatives could be considered in future anticancer strategies targeting GPER in cancer cells. PMID:26183213

  12. Third generation antipsychotic drugs: partial agonism or receptor functional selectivity?

    PubMed Central

    Mailman, Richard B.; Murthy, Vishakantha

    2010-01-01

    Functional selectivity is the term that describes drugs that cause markedly different signaling through a single receptor (e.g., full agonist at one pathway and antagonist at a second). It has been widely recognized recently that this phenomenon impacts the understanding of mechanism of action of some drugs, and has relevance to drug discovery. One of the clinical areas where this mechanism has particular importance is in the treatment of schizophrenia. Antipsychotic drugs have been grouped according to both pattern of clinical action and mechanism of action. The original antipsychotic drugs such as chlorpromazine and haloperidol have been called typical or first generation. They cause both antipsychotic actions and many side effects (extrapyramidal and endocrine) that are ascribed to their high affinity dopamine D2 receptor antagonism. Drugs such as clozapine, olanzapine, risperidone and others were then developed that avoided the neurological side effects (atypical or second generation antipsychotics). These compounds are divided mechanistically into those that are high affinity D2 and 5-HT2A antagonists, and those that also bind with modest affinity to D2, 5-HT2A, and many other neuroreceptors. There is one approved third generation drug, aripiprazole, whose actions have been ascribed alternately to either D2 partial agonism or D2 functional selectivity. Although partial agonism has been the more widely accepted mechanism, the available data are inconsistent with this mechanism. Conversely, the D2 functional selectivity hypothesis can accommodate all current data for aripiprazole, and also impacts on discovery compounds that are not pure D2 antagonists. PMID:19909227

  13. Functional Coupling of Ca2+ Channels and Ryanodine Receptors in Cardiac Myocytes

    NASA Astrophysics Data System (ADS)

    Sham, James S. K.; Cleemann, Lars; Morad, Martin

    1995-01-01

    In skeletal muscle, dihydropyridine receptors are functionally coupled to ryanodine receptors of the sarcoplasmic reticulum in triadic or diadic junctional complexes. In cardiac muscle direct physical or functional couplings have not been demonstrated. We have tested the hypothesis of functional coupling of L-type Ca2+ channels and ryanodine receptors in rat cardiac myocytes by comparing the efficacies of Ca2+ in triggering Ca2+ release when the ion enters the cell via the Ca2+ channels or the Na^+/Ca2+ exchanger. Ca2+ transported through the Ca2+ channels was 20-160 times more effective than Ca2+ influx via the Na^+/Ca2+ exchanger in gating Ca2+ release from the sarcoplasmic reticulum, suggesting privileged communication between Ca2+ channels and ryanodine receptors. In support of this hypothesis we found that Ca2+ channels were inactivated by Ca2+ release from the sarcoplasmic reticulum, even though the myoplasmic Ca2+ concentrations were buffered with 10 mM EGTA. The data thus suggest privileged cross signaling between the dihydropyridine and ryanodine receptors such that Ca2+ flux through either the Ca2+ channel or the ryanodine receptor alters the gating kinetics of the other channel.

  14. Desensitization of α7 Nicotinic Receptor Is Governed by Coupling Strength Relative to Gate Tightness*

    PubMed Central

    Zhang, Jianliang; Xue, Fenqin; Whiteaker, Paul; Li, Chaokun; Wu, Wen; Shen, Benchang; Huang, Yao; Lukas, Ronald J.; Chang, Yongchang

    2011-01-01

    Binding of a neurotransmitter to its membrane receptor opens an integral ion conducting pore. However, prolonged exposure to the neurotransmitter drives the receptor to a refractory state termed desensitization, which plays an important role in shaping synaptic transmission. Despite intensive research in the past, the structural mechanism of desensitization is still elusive. Using mutagenesis and voltage clamp in an oocyte expression system, we provide several lines of evidence supporting a novel hypothesis that uncoupling between binding and gating machinery is the underlying mechanism for α7 nicotinic receptor (nAChR) desensitization. First, the decrease in gate tightness was highly correlated to the reduced desensitization. Second, nonfunctional mutants in three important coupling loops (loop 2, loop 7, and the M2-M3 linker) could be rescued by a gating mutant. Furthermore, the decrease in coupling strength in these rescued coupling loop mutants reversed the gating effect on desensitization. Finally, coupling between M1 and hinge region of the M2-M3 linker also influenced the receptor desensitization. Thus, the uncoupling between N-terminal domain and transmembrane domain, governed by the balance of coupling strength and gate tightness, underlies the mechanism of desensitization for the α7 nAChR. PMID:21610071

  15. Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2.

    PubMed

    Whalen, Erin J; Foster, Matthew W; Matsumoto, Akio; Ozawa, Kentaro; Violin, Jonathan D; Que, Loretta G; Nelson, Chris D; Benhar, Moran; Keys, Janelle R; Rockman, Howard A; Koch, Walter J; Daaka, Yehia; Lefkowitz, Robert J; Stamler, Jonathan S

    2007-05-04

    beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.

  16. Novel selective glucocorticoid receptor agonists (SEGRAs) with a covalent warhead for long-lasting inhibition.

    PubMed

    Ryabtsova, Oksana; Joossens, Jurgen; Van Der Veken, Pieter; Vanden Berghe, Wim; Augustyns, Koen; De Winter, Hans

    2016-10-15

    The synthesis and in vitro properties of six analogues of the selective glucocorticoid receptor (GR) agonist GSK866, bearing a warhead for covalent linkage to the glucocorticoid receptor, is described.

  17. Large-scale production and protein engineering of G protein-coupled receptors for structural studies

    PubMed Central

    Milić, Dalibor; Veprintsev, Dmitry B.

    2015-01-01

    Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures. PMID:25873898

  18. Large-scale production and protein engineering of G protein-coupled receptors for structural studies.

    PubMed

    Milić, Dalibor; Veprintsev, Dmitry B

    2015-01-01

    Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures.

  19. Covalent coupling of bovine growth hormone to its receptor in bovine liver membranes.

    PubMed

    Badinga, L; Collier, R J; Thatcher, W W; Quintana, S J; Bazer, F W

    1987-07-01

    The structure of bovine somatotropin receptor was examined following covalent coupling of iodinated recombinant bovine growth hormone ([125I]rbGH) to bovine liver membrane receptors using ethylene glycol bis(succinimidyl succinate). Iodinated rbGH was incorporated into a complex of estimated Mr of 140,000 under reducing conditions. Excess unlabeled rbGH, but not bovine prolactin (bPRL), inhibited completely the incorporation of [125I]rbGH into the Mr = 140,000 species. In dairy bulls, the Mr = 140,000 complex was undetectable soon after birth but became predominant at 6 months of age. No evidence was found to support presence of bPRL receptors in steer liver membranes. Assuming a 1:1 stoichiometry of hormone binding to receptor, it appears that bGH binds to a major receptor subunit of Mr = 119,000 which does not recognize bPRL.

  20. Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization

    PubMed Central

    Franco, Rafael; Martínez-Pinilla, Eva; Lanciego, José L.; Navarro, Gemma

    2016-01-01

    Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs) seem an exception to the general rule of receptor–receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backward instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery. PMID:27065866

  1. Vertebrate extracellular calcium-sensing receptor evolution: selection in relation to life history and habitat.

    PubMed

    Herberger, Amanda L; Loretz, Christopher A

    2013-03-01

    Ionic calcium (Ca(2+)) supports essential functions within physiological systems, and consequently its concentration is homeostatically regulated within narrow bounds in the body fluids of animals through endocrine effects at ion-transporting osmoregulatory tissues. In vertebrates, extracellular Ca(2+) is detected at the cell surface by the extracellular calcium-sensing receptor (CaSR), a member of the G protein-coupled receptor (GPCR) superfamily. Interestingly, the taxonomic distribution of CaSRs is restricted to vertebrates, with some CaSR-like receptors apparently present in non-vertebrate chordates. Since bone is a known Ca(2+) storage site and is characteristically restricted to the vertebrate lineage, we hypothesized a functional association of CaSR with vertebrate skeleton that may have an ancient origin. Protein sequence alignment and phylogenetic analysis of vertebrate CaSRs and related GPCRs of the glutamate receptor-like family expose similarities and indel differences among these receptors, and reveal the evolutionary history of CaSRs. Evolutionary selection was tested statistically by evaluating the relationship between non-synonymous (replacement, dN) versus synonymous (silent, dS) amino acid substitution rates (as dN/dS) of protein-coding DNA sequences among branches of the estimated protein phylogeny. On a background of strong purifying selection (dN/dS<1) in the CaSR phylogeny, statistical evidence for adaptive evolution (dN/dS>1) was detected on some branches to major clades in the CaSR phylogeny, especially to the tetrapod vertebrate CaSRs and chordate CaSR-like branches. Testing also revealed overall purifying selection at the codon level. At some sites relaxation from strong purifying selection was seen, but evidence for adaptive evolution was not detected for individual sites. The results suggest purifying selection of CaSRs, and of adaptive evolution among some major vertebrate clades, reflecting clade specific differences in natural history

  2. Structure of the Human Dopamine D3 Receptor in Complex with a D2/D3 Selective Antagonist

    SciTech Connect

    Chien, Ellen Y.T.; Liu, Wei; Zhao, Qiang; Katritch, Vsevolod; Han, Gye Won; Hanson, Michael A.; Shi, Lei; Newman, Amy Hauck; Javitch, Jonathan A.; Cherezov, Vadim; Stevens, Raymond C.

    2010-11-30

    Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.

  3. Amphipols in G protein-coupled receptor pharmacology: what are they good for?

    PubMed

    Mary, Sophie; Damian, Marjorie; Rahmeh, Rita; Mouillac, Bernard; Marie, Jacky; Granier, Sébastien; Banères, Jean-Louis

    2014-10-01

    G protein-coupled receptors are at a central node of all cell communications. Investigating their molecular functioning is therefore crucial for both academic purposes and drug design. However, getting the receptors as isolated, stable and purified proteins for such studies still stumbles over their instability out of the membrane environment. Different membrane-mimicking environments have been developed so far to increase the stability of purified receptors. Among them are amphipols. These polymers not only preserve the native fold of receptors purified from membrane fractions but they also allow specific applications such as folding receptors purified from inclusion bodies back to their native state. Of importance, amphipol-trapped G protein-coupled receptors essentially maintain their pharmacological properties so that they are perfectly adapted to further investigate the molecular mechanisms underlying signaling processes. We review here how amphipols have been used to refold and stabilize detergent-solubilized purified receptors and what are the main subsequent molecular pharmacology analyses that were performed using this strategy.

  4. Orphan G-protein-coupled receptors: the next generation of drug targets?

    PubMed Central

    Wilson, Shelagh; Bergsma, Derk J; Chambers, Jon K; Muir, Alison I; Fantom, Kenneth G M; Ellis, Catherine; Murdock, Paul R; Herrity, Nicole C; Stadel, Jeffrey M

    1998-01-01

    The pharmaceutical industry has readily embraced genomics to provide it with new targets for drug discovery. Large scale DNA sequencing has allowed the identification of a plethora of DNA sequences distantly related to known G protein-coupled receptors (GPCRs), a superfamily of receptors that have a proven history of being excellent therapeutic targets. In most cases the extent of sequence homology is insufficient to assign these `orphan' receptors to a particular receptor subfamily. Consequently, reverse molecular pharmacological and functional genomic strategies are being employed to identify the activating ligands of the cloned receptors. Briefly, the reverse molecular pharmacological methodology includes cloning and expression of orphan GPCRs in mammalian cells and screening these cells for a functional response to cognate or surrogate agonists present in biological extract preparations, peptide libraries, and complex compound collections. The functional genomics approach involves the use of `humanized yeast cells, where the yeast GPCR transduction system is engineered to permit functional expression and coupling of human GPCRs to the endogenous signalling machinery. Both systems provide an excellent platform for identifying novel receptor ligands. Once activating ligands are identified they can be used as pharmacological tools to explore receptor function and relationship to disease. PMID:9884064

  5. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

    PubMed

    Dror, Ron O; Green, Hillary F; Valant, Celine; Borhani, David W; Valcourt, James R; Pan, Albert C; Arlow, Daniel H; Canals, Meritxell; Lane, J Robert; Rahmani, Raphaël; Baell, Jonathan B; Sexton, Patrick M; Christopoulos, Arthur; Shaw, David E

    2013-11-14

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  6. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    NASA Astrophysics Data System (ADS)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  7. Histamine receptor type coupled to nitric oxide-induced relaxation of guinea-pig nasal mucosa.

    PubMed

    Bockman, C S; Zeng, W

    2002-01-01

    1 The aim of this study was to characterize the histamine receptor type mediating relaxation of the vascular bed of the nasal mucosa from the guinea-pig, and to determine the role of cyclo-oxygenase products and nitric oxide in this relaxant response to histamine. These studies were performed in isolated nasal mucosae examined in vitro to obtain potencies of histamine receptor-type selective agonists in causing vasorelaxation and to determine affinities of histamine receptor antagonists for inhibiting histamine-induced relaxation. 2 After contraction of nasal mucosae with noradrenaline, histamine caused a maximal relaxation response that was 75 +/- 6% of the contraction caused by noradrenaline with a mean EC50 value of 4.3 +/- 0.5 microM. Neither dimaprit (H2-receptor selective) nor R-alpha-methylhistamine (H3-receptor selective) caused significant relaxation of nasal mucosae. In contrast, betahistine (H1-receptor selective) caused an 81 +/- 7% relaxation of noradrenaline-induced tone with an EC50 value of 15 +/- 1 microM. 3 pA2 experiments were performed to obtain KB values of chlorpheniramine (H1-receptor selective) and diphenhydramine (H1-receptor selective) for blocking histamine-stimulated relaxation of nasal mucosae. KB values for chlorpheniramine (0.87 nM) and diphenhydramine (7.4 nM) were consistent with their interaction at the H1-receptor type. Additionally, neither 10 microM cimetidine (H2-receptor selective) nor 1 microM thioperamide (H3-receptor selective) had any effect on the relaxation curve for histamine. 4 In the presence of 10 microM indomethacin (cyclo-oxygenase inhibitor), histamine caused a maximal relaxation response of 73 +/- 5% of the noradrenaline-induced tone with an EC50 value of 2.9 +/- 0.2 microM, which was not different from control values (EC50 = 5.0 +/- 0.4 microM; maximal relaxation = 71 +/- 6%). In contrast, 200 microM NG-nitro-L-arginine (nitric oxide synthase inhibitor) completely inhibited histamine-induced relaxation of nasal

  8. Progestin, estrogen and androgen G-protein coupled receptors in fish gonads.

    PubMed

    Thomas, Peter; Dressing, Gwen; Pang, Yefei; Berg, Hakan; Tubbs, Christopher; Benninghoff, Abby; Doughty, Kelly

    2006-04-01

    The identities of the membrane receptors mediating the majority of rapid, cell surface-initiated, nongenomic (i.e. nonclassical) steroid actions described to date are unclear. Two novel 7-transmembrane spanning proteins, representing two distinct classes of steroid membrane receptors, membrane progestin receptor alpha (mPRalpha) and a membrane estrogen receptor (mER), GPR30, have recently been identified in several vertebrate species. Evidence that both receptors activate G-proteins and function as G-protein coupled receptors (GPCRs) is briefly reviewed. New data on progestin actions on fish gametes suggest a widespread involvement of mPRalpha in oocyte maturation and sperm hyperactivity in this vertebrate group. Information on the second messenger pathways activated upon estrogen binding to a membrane estrogen receptor in croaker gonads and preliminary evidence for the presence of a GPR30-like protein in fish gonads are discussed. Finally, initial characterization of the ligand binding, G-protein activation and molecular size of a membrane androgen receptor (mAR) in croaker ovaries suggests the presence of a third unique steroid receptor in fish gonads that also may function as a GPCR.

  9. Topology of Class A G Protein-Coupled Receptors: Insights Gained from Crystal Structures of Rhodopsins, Adrenergic and Adenosine Receptors

    PubMed Central

    Mustafi, Debarshi; Palczewski, Krzysztof

    2009-01-01

    Biological membranes are densely packed with membrane proteins that occupy approximately half of their volume. In almost all cases, membrane proteins in the native state lack the higher-order symmetry required for their direct study by diffraction methods. Despite many technical difficulties, numerous crystal structures of detergent solubilized membrane proteins have been determined that illustrate their internal organization. Among such proteins, class A G protein-coupled receptors have become amenable to crystallization and high resolution X-ray diffraction analyses. The derived structures of native and engineered receptors not only provide insights into their molecular arrangements but also furnish a framework for designing and testing potential models of transformation from inactive to active receptor signaling states and for initiating rational drug design. PMID:18945819

  10. Transactivation of Epidermal Growth Factor Receptor by G Protein-Coupled Receptors: Recent Progress, Challenges and Future Research

    PubMed Central

    Wang, Zhixiang

    2016-01-01

    Both G protein-coupled receptors (GPCRs) and receptor-tyrosine kinases (RTKs) regulate large signaling networks, control multiple cell functions and are implicated in many diseases including various cancers. Both of them are also the top therapeutic targets for disease treatment. The discovery of the cross-talk between GPCRs and RTKs connects these two vast signaling networks and complicates the already complicated signaling networks that regulate cell signaling and function. In this review, we focus on the transactivation of epidermal growth factor receptor (EGFR), a subfamily of RTKs, by GPCRs. Since the first report of EGFR transactivation by GPCR, significant progress has been made including the elucidation of the mechanisms underlying the transactivation. Here, we first provide a basic picture for GPCR, EGFR and EGFR transactivation by GPCR. We then discuss the progress made in the last five years and finally provided our view of the future challenge and future researches needed to overcome these challenges. PMID:26771606

  11. Transactivation of Epidermal Growth Factor Receptor by G Protein-Coupled Receptors: Recent Progress, Challenges and Future Research.

    PubMed

    Wang, Zhixiang

    2016-01-12

    Both G protein-coupled receptors (GPCRs) and receptor-tyrosine kinases (RTKs) regulate large signaling networks, control multiple cell functions and are implicated in many diseases including various cancers. Both of them are also the top therapeutic targets for disease treatment. The discovery of the cross-talk between GPCRs and RTKs connects these two vast signaling networks and complicates the already complicated signaling networks that regulate cell signaling and function. In this review, we focus on the transactivation of epidermal growth factor receptor (EGFR), a subfamily of RTKs, by GPCRs. Since the first report of EGFR transactivation by GPCR, significant progress has been made including the elucidation of the mechanisms underlying the transactivation. Here, we first provide a basic picture for GPCR, EGFR and EGFR transactivation by GPCR. We then discuss the progress made in the last five years and finally provided our view of the future challenge and future researches needed to overcome these challenges.

  12. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

    PubMed Central

    Hudson, Brian D.; Christiansen, Elisabeth; Tikhonova, Irina G.; Grundmann, Manuel; Kostenis, Evi; Adams, David R.; Ulven, Trond; Milligan, Graeme

    2012-01-01

    When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC50 values for inhibition of cAMP, 5.83 ± 0.11; Ca2+ mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.—Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs. PMID:22919070

  13. Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

    SciTech Connect

    Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

    1988-07-12

    The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity (/sup 3/H)fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity (/sup 3/H)fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils.

  14. A PET study comparing receptor occupancy by five selective cannabinoid 1 receptor antagonists in non-human primates

    PubMed Central

    Hjorth, Stephan; Karlsson, Cecilia; Jucaite, Aurelija; Varnäs, Katarina; Hamrén, Ulrika Wählby; Johnström, Peter; Gulyás, Balázs; Donohue, Sean R; Pike, Victor W; Halldin, Christer; Farde, Lars

    2015-01-01

    There is a medical need for safe and efficacious anti-obesity drugs with acceptable side effect profiles. To mitigate the challenge posed by translating target interaction across species and balancing beneficial vs. adverse effects, a positron emission tomography (PET) approach could help guide clinical dose optimization. Thus, as part of a compound differentiation effort, three novel selective CB1 receptor (CB1R) antagonists, developed by AstraZeneca (AZ) for the treatment of obesity, were compared with two clinically tested reference compounds, rimonabant and taranabant, with regard to receptor occupancy relative to dose and exposure. A total of 42 PET measurements were performed in 6 non-human primates using the novel CB1R antagonist radioligand [11C]SD5024. The AZ CB1R antagonists bound in a saturable manner to brain CB1R with in vivo affinities similar to that of rimonabant and taranabant, compounds with proven weight loss efficacy in clinical trials. Interestingly, it was found that exposures corresponding to those needed for optimal clinical efficacy of rimonabant and taranabant resulted in a CB1R occupancy typically around ~20–30%, thus much lower than what would be expected for classical G-protein coupled receptor (GPCR) antagonists in other therapeutic contexts. These findings are also discussed in relation to emerging literature on the potential usefulness of ‘neutral’ vs. ‘classical’ CB1R (inverse agonist) antagonists. The study additionally highlighted the usefulness of the radioligand [11C]SD5024 as a specific tracer for CB1R in the primate brain, though an arterial input function would ideally be required in future studies to further assure accurate quantitative analysis of specific binding. PMID:25791528

  15. A PET study comparing receptor occupancy by five selective cannabinoid 1 receptor antagonists in non-human primates.

    PubMed

    Hjorth, Stephan; Karlsson, Cecilia; Jucaite, Aurelija; Varnäs, Katarina; Wählby Hamrén, Ulrika; Johnström, Peter; Gulyás, Balázs; Donohue, Sean R; Pike, Victor W; Halldin, Christer; Farde, Lars

    2016-02-01

    There is a medical need for safe and efficacious anti-obesity drugs with acceptable side effect profiles. To mitigate the challenge posed by translating target interaction across species and balancing beneficial vs. adverse effects, a positron emission tomography (PET) approach could help guide clinical dose optimization. Thus, as part of a compound differentiation effort, three novel selective CB1 receptor (CB1R) antagonists, developed by AstraZeneca (AZ) for the treatment of obesity, were compared with two clinically tested reference compounds, rimonabant and taranabant, with regard to receptor occupancy relative to dose and exposure. A total of 42 PET measurements were performed in 6 non-human primates using the novel CB1R antagonist radioligand [(11)C]SD5024. The AZ CB1R antagonists bound in a saturable manner to brain CB1R with in vivo affinities similar to that of rimonabant and taranabant, compounds with proven weight loss efficacy in clinical trials. Interestingly, it was found that exposures corresponding to those needed for optimal clinical efficacy of rimonabant and taranabant resulted in a CB1R occupancy typically around ∼20-30%, thus much lower than what would be expected for classical G-protein coupled receptor (GPCR) antagonists in other therapeutic contexts. These findings are also discussed in relation to emerging literature on the potential usefulness of 'neutral' vs. 'classical' CB1R (inverse agonist) antagonists. The study additionally highlighted the usefulness of the radioligand [(11)C]SD5024 as a specific tracer for CB1R in the primate brain, though an arterial input function would ideally be required in future studies to further assure accurate quantitative analysis of specific binding.

  16. Selective antagonism of muscarinic receptors is neuroprotective in peripheral neuropathy.

    PubMed

    Calcutt, Nigel A; Smith, Darrell R; Frizzi, Katie; Sabbir, Mohammad Golam; Chowdhury, Subir K Roy; Mixcoatl-Zecuatl, Teresa; Saleh, Ali; Muttalib, Nabeel; Van der Ploeg, Randy; Ochoa, Joseline; Gopaul, Allison; Tessler, Lori; Wess, Jürgen; Jolivalt, Corinne G; Fernyhough, Paul

    2017-02-01

    Sensory neurons have the capacity to produce, release, and respond to acetylcholine (ACh), but the functional role of cholinergic systems in adult mammalian peripheral sensory nerves has not been established. Here, we have reported that neurite outgrowth from adult sensory neurons that were maintained under subsaturating neurotrophic factor conditions operates under cholinergic constraint that is mediated by muscarinic receptor-dependent regulation of mitochondrial function via AMPK. Sensory neurons from mice lacking the muscarinic ACh type 1 receptor (M1R) exhibited enhanced neurite outgrowth, confirming the role of M1R in tonic suppression of axonal plasticity. M1R-deficient mice made diabetic with streptozotocin were protected from physiological and structural indices of sensory neuropathy. Pharmacological blockade of M1R using specific or selective antagonists, pirenzepine, VU0255035, or muscarinic toxin 7 (MT7) activated AMPK and overcame diabetes-induced mitochondrial dysfunction in vitro and in vivo. These antimuscarinic drugs prevented or reversed indices of peripheral neuropathy, such as depletion of sensory nerve terminals, thermal hypoalgesia, and nerve conduction slowing in diverse rodent models of diabetes. Pirenzepine and MT7 also prevented peripheral neuropathy induced by the chemotherapeutic agents dichloroacetate and paclitaxel or HIV envelope protein gp120. As a variety of antimuscarinic drugs are approved for clinical use against other conditions, prompt translation of this therapeutic approach to clinical trials is feasible.

  17. Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization.

    PubMed

    Tena-Campos, Mercè; Ramon, Eva; Lupala, Cecylia S; Pérez, Juan J; Koch, Karl-W; Garriga, Pere

    2016-04-01

    5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

  18. Coupling of G Proteins to Reconstituted Monomers and Tetramers of the M2 Muscarinic Receptor*

    PubMed Central

    Redka, Dar'ya S.; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V.; Ellis, John; Ernst, Oliver P.; Wells, James W.

    2014-01-01

    G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5′-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[3H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the “ternary complex model”). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. PMID:25023280

  19. Rapid effects of dorsal hippocampal G-protein coupled estrogen receptor on learning in female mice.

    PubMed

    Lymer, Jennifer; Robinson, Alana; Winters, Boyer D; Choleris, Elena

    2017-03-01

    Through rapid mechanisms of action, estrogens affect learning and memory processes. It has been shown that 17β-estradiol and an Estrogen Receptor (ER) α agonist enhances performance in social recognition, object recognition, and object placement tasks when administered systemically or infused in the dorsal hippocampus. In contrast, systemic and dorsal hippocampal ERβ activation only promote spatial learning. In addition, 17β-estradiol, the ERα and the G-protein coupled estrogen receptor (GPER) agonists increase dendritic spine density in the CA1 hippocampus. Recently, we have shown that selective systemic activation of the GPER also rapidly facilitated social recognition, object recognition, and object placement learning in female mice. Whether activation the GPER specifically in the dorsal hippocampus can also rapidly improve learning and memory prior to acquisition is unknown. Here, we investigated the rapid effects of infusion of the GPER agonist, G-1 (dose: 50nM, 100nM, 200nM), in the dorsal hippocampus on social recognition, object recognition, and object placement learning tasks in home cage. These paradigms were completed within 40min, which is within the range of rapid estrogenic effects. Dorsal hippocampal administration of G-1 improved social (doses: 50nM, 200nM G-1) and object (dose: 200nM G-1) recognition with no effect on object placement. Additionally, when spatial cues were minimized by testing in a Y-apparatus, G-1 administration promoted social (doses: 100nM, 200nM G-1) and object (doses: 50nM, 100nM, 200nM G-1) recognition. Therefore, like ERα, the GPER in the hippocampus appears to be sufficient for the rapid facilitation of social and object recognition in female mice, but not for the rapid facilitation of object placement learning. Thus, the GPER in the dorsal hippocampus is involved in estrogenic mediation of learning and memory and these effects likely occur through rapid signalling mechanisms.

  20. Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

    PubMed

    Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

    2012-12-04

    Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

  1. Evolution of hormone selectivity in glucocorticoid and mineralocorticoid receptors.

    PubMed

    Baker, Michael E; Funder, John W; Kattoula, Stephanie R

    2013-09-01

    Mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) are descended from an ancestral corticoid receptor (CR). To date, the earliest CR have been found in lamprey and hagfish, two jawless fish (cyclostomes) that evolved at the base of the vertebrate line. Lamprey CR has both MR and GR activity. Distinct orthologs of the GR and MR first appear in skates and sharks, which are cartilaginous fishes (Chondrichthyes). Aldosterone, the physiological mineralocorticoid in terrestrial vertebrates, first appears in lobe-finned fish, such as lungfish and coelacanth, forerunners of terrestrial vertebrates, but not in sharks, skates or ray-finned fish. Skate MR are transcriptionally activated by glucocorticoids, such as corticosterone and cortisol, as well as by mineralocorticoids such as deoxycorticosterone and (experimentally) aldosterone; skate GR have low affinity for all human corticosteroids and 1α-OH-corticosterone, which has been proposed to be biologically active glucocorticoid. In fish, cortisol is both physiological mineralocorticoid and glucocorticoid; in terrestrial vertebrates, cortisol or corticosterone are the physiological glucocorticoids acting through GR, and aldosterone via MR as the physiologic mineralocorticoid. MR have equally high affinity for cortisol, corticosterone and progesterone. We review this evolutionary process through an analysis of changes in sequence and structure of vertebrate GR and MR, identifying changes in these receptors in skates and lobe-fined fish important in allowing aldosterone to act as an agonist at epithelial MR and glucocorticoid specificity for GR. hMR and hGR have lost a key contact between helix 3 and helix 5 that was present in their common ancestor. A serine that is diagnostic for vertebrate MR, and absent in terrestrial and fish GR, is present in lamprey CR, skate MR and GR, but not in coelacanth GR, marking the transition of the GR from MR ancestor. Based on the response of the CR and skate MR and GR to

  2. A Selective Nociceptin Receptor Antagonist to Treat Depression: Evidence from Preclinical and Clinical Studies.

    PubMed

    Post, Anke; Smart, Trevor S; Krikke-Workel, Judith; Dawson, Gerard R; Harmer, Catherine J; Browning, Michael; Jackson, Kimberley; Kakar, Rishi; Mohs, Richard; Statnick, Michael; Wafford, Keith; McCarthy, Andrew; Barth, Vanessa; Witkin, Jeffrey M

    2016-06-01

    Nociceptin/Orphanin FQ (N/OFQ) is an endogenous ligand of the N/OFQ peptide (NOP) receptor, which is a G protein-coupled receptor in brain regions associated with mood disorders. We used a novel, potent, and selective orally bioavailable antagonist, LY2940094, to test the hypothesis that blockade of NOP receptors would induce antidepressant effects. In this study we demonstrate that targeting NOP receptors with LY2940094 translates to antidepressant-like effects in rodent models and, importantly, to antidepressant efficacy in patients with major depressive disorder (MDD). The proof-of-concept study (POC) was an 8-week, double-blind, placebo-controlled trial that evaluated LY2940094 as a novel oral medication for the treatment of patients with MDD. Once daily oral dosing of LY2940094 at 40 mg for 8 weeks vs placebo provided some evidence for an antidepressant effect based on the change from baseline to week 8 in the GRID-Hamilton Depression Rating Scale-17 item total score, although the predefined POC efficacy criterion (probability of LY2940094 being better than placebo⩾88%) was not met (82.9%). LY2940094 also had an early effect on the processing of emotional stimuli at Week 1 as shown by an increased recognition of positive relative to negative facial expressions in an emotional test battery. LY2940094 was safe and well tolerated. Overall, these are the first human data providing evidence that the blockade of NOP receptor signaling represents a promising strategy for the treatment of MDD.

  3. Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins

    PubMed Central

    2010-01-01

    Background CC chemokine receptor proteins (CCR1 through CCR10) are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions. In mammals, the majority of chemokine receptor genes are clustered together; in humans, seven of the ten genes are clustered in the 3p21-24 chromosome region. Gene conversion events, or exchange of DNA sequence between genes, have been reported in chemokine receptor paralogs in various mammalian lineages, especially between the cytogenetically closely located pairs CCR2/5 and CCR1/3. Datasets of mammalian orthologs for each gene were analyzed separately to minimize the potential confounding impact of analyzing highly similar sequences resulting from gene conversion events. Molecular evolution approaches and the software package Phylogenetic Analyses by Maximum Likelihood (PAML) were utilized to investigate the signature of selection that has acted on the mammalian CC chemokine receptor (CCR) gene family. The results of neutral vs. adaptive evolution (positive selection) hypothesis testing using Site Models are reported. In general, positive selection is defined by a ratio of nonsynonymous/synonymous nucleotide changes (dN/dS, or ω) >1. Results Of the ten mammalian CC motif chemokine receptor sequence datasets analyzed, only CCR2 and CCR3 contain amino acid codon sites that exhibit evidence of positive selection using site based hypothesis testing in PAML. Nineteen of the twenty codon sites putatively indentified as likely to be under positive selection code for amino acid

  4. Effects of central galanin administration on muscarinic cholinergic and galanin receptor G protein coupling.

    PubMed

    Barreda-Gómez, G; Giralt, M T; Rodríguez-Puertas, R

    2005-06-01

    The neuropeptide galanin is expressed in the mammalian central nervous system and has been implicated in neurotrophic actions. Central galanin administration induces cognitive deficits in rodents and inhibits the release of acetylcholine in the hippocampus. In addition, a galanin hyperinnervation of the basal forebrain cholinergic cells in Alzheimer's disease patients has been reported. To evaluate the effect of galanin treatment on galanin and muscarinic cholinergic receptor G protein coupling, galanin was administered into the lateral ventricle of rats via an implanted cannula. Galanin or muscarinic receptor functional coupling to G proteins was quantified by galanin or carbachol stimulation of guanosine 5'-(gamma-[35S]thio)triphosphate binding in rat brain slices. Guanosine 5'-(gamma-[35S]thio)triphosphate basal binding in nucleus basalis of Meynert and thalamic nuclei was increased in the vehicle treated group. This effect was reverted by galanin treatment and indicates that the surgery increased receptor functional coupling to G proteins, which is restored by a possible neurotrophic action mediated by galanin. In addition, in galanin administered animals, galanin-stimulated binding was increased in the amygdala but decreased in the diagonal band, whilst binding stimulation mediated by carbachol was found to be increased in the amygdala, thalamic nuclei and diagonal band. These findings indicate that galanin treatment modulates the coupling of galanin and muscarinic cholinergic receptors to G proteins in specific regions of the rat central nervous system.

  5. Couples Therapy: Treating Selected Personality-disordered Couples Within a Dynamic Therapy Framework

    PubMed Central

    Foley, Gretchen N.

    2014-01-01

    Personality disordered couples present unique challenges for couples therapy. Novice therapists may feel daunted when taking on such a case, especially given the limited literature available to guide them in this specific area of therapy. Much of what is written on couples therapy is embedded in the larger body of literature on family therapy. While family therapy techniques may apply to couples therapy, this jump requires a level of understanding the novice therapist may not yet have. Additionally, the treatment focus within the body of literature on couples therapy tends to be situation-based (how to treat couples dealing with divorce, an affair, illness), neglecting how to treat couples whose dysfunction is not the product of a crisis, but rather a longstanding pattern escalated to the level of crisis. This is exactly the issue in therapy with personality disordered couples, and it is an important topic, as couples with personality pathology often do present for treatment. This article strives to present practical techniques, modeled in case vignettes, that can be applied directly to couples therapy— specifically therapy with personality disordered couples. PMID:24800131

  6. Couples Therapy: Treating Selected Personality-disordered Couples Within a Dynamic Therapy Framework.

    PubMed

    Landucci, Jennifer; Foley, Gretchen N

    2014-03-01

    Personality disordered couples present unique challenges for couples therapy. Novice therapists may feel daunted when taking on such a case, especially given the limited literature available to guide them in this specific area of therapy. Much of what is written on couples therapy is embedded in the larger body of literature on family therapy. While family therapy techniques may apply to couples therapy, this jump requires a level of understanding the novice therapist may not yet have. Additionally, the treatment focus within the body of literature on couples therapy tends to be situation-based (how to treat couples dealing with divorce, an affair, illness), neglecting how to treat couples whose dysfunction is not the product of a crisis, but rather a longstanding pattern escalated to the level of crisis. This is exactly the issue in therapy with personality disordered couples, and it is an important topic, as couples with personality pathology often do present for treatment. This article strives to present practical techniques, modeled in case vignettes, that can be applied directly to couples therapy- specifically therapy with personality disordered couples.

  7. Pramipexole Derivatives as Potent and Selective Dopamine D3 Receptor Agonists with Improved Human Microsomal Stability

    PubMed Central

    Jiang, Cheng; Levant, Beth; Li, Xiaoqin; Zhao, Ting; Wen, Bo; Luo, Ruijuan; Sun, Duxin

    2014-01-01

    We report herein the synthesis and evaluation of a series of new pramipexole derivatives as highly potent and selective dopamine-3 (D3) receptor agonists. A number of these new compounds bind to the D3 receptor with subnanomolar affinities and show excellent selectivity (>10,000) for the D3 receptor over the D1 and D2 receptors. Compound 23 for example, binds to the D3 receptor with a Ki value of 0.53 nM and shows a selectivity of >20,000 over the D2 receptor and the D1 receptor in the binding assays using a rat brain preparation. It has excellent stability in human liver microsomes and in vitro functional assays showed it to be a full agonist for the human D3 receptor. PMID:25338762

  8. G-Protein Coupled Receptors (GPCRs): A Comprehensive Computational Perspective.

    PubMed

    Ramesh, M; Soliman, Mahmoud E

    2015-01-01

    GPCRs are ubiquitous in most of the organs of the human body. These receptors were found to be the important targets to attenuate inflammation, cancer, cardiac dysfunction, diabetes, etc. The advanced technologies employed on GPCRs provided an opportunity to understand the physiological process of various diseases. Recently, GPCRs were viewed as viable therapeutic targets to deliver safer and more efficacious drug. In the literature, several computational studies were reported to describe the biological mechanism, function and three-dimensional structure of GPCRs. These studies revealed the multiple conserved transmembrane domains of GPCRs which were connected by intra and extracellular loops. In this review, we provide an updated overview on the computational tools and methodologies which were conducted to explore the structural and mechanistic features of GPCRs. The study also demonstrates the most recent computer-aided drug design approaches employed on GPCRs. This review provides the information that can be exploited toward the molecular understanding of GPCRs with an aim to design the novel ligands for GPCRs.

  9. Fluorescent Approaches for Understanding Interactions of Ligands with G Protein Coupled Receptors

    PubMed Central

    Sridharan, Rajashri; Zuber, Jeffrey; Connelly, Sara M.; Mathew, Elizabeth; Dumont, Mark E.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remains unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes, that can be difficult to extract from x-ray structures. We present an overview of the extensive array of fluorescent ligands that have been used in studies of GPCRs and describe spectroscopic approaches for assaying binding and probing the environment of receptor-bound ligands with particular attention to examples involving yeast pheromone receptors. In addition, we discuss the use of fluorescence spectroscopy for detecting and characterizing conformational changes in receptors induced by the binding of ligands. Such studies have provided strong evidence for diversity of receptor conformations elicited by different ligands, consistent with the idea that GPCRs are not simple on and off switches. This diversity of states constitutes an underlying mechanistic basis for biased agonism, the observation that different stimuli can produce different responses from a single receptor. It is likely that continued technical advances will allow fluorescence spectroscopy to play an important role in continued probing of structural transitions in GPCRs. PMID:24055822

  10. Endothelial nitric oxide synthase interactions with G-protein-coupled receptors.

    PubMed Central

    Marrero, M B; Venema, V J; Ju, H; He, H; Liang, H; Caldwell, R B; Venema, R C

    1999-01-01

    The endothelial nitric oxide synthase (eNOS) is activated in response to stimulation of endothelial cells by a number of vasoactive substances including, bradykinin (BK), angiotensin II (Ang II), endothelin-1 (ET-1) and ATP. In the present study we have used in vitro activity assays of purified eNOS and in vitro binding assays with glutathione S-transferase fusion proteins to show that the capacity to bind and inhibit eNOS is a common feature of membrane-proximal regions of intracellular domain 4 of the BK B2, the Ang II AT1 and the ET-1 ETB receptors, but not of the ATP P2Y2 receptor. Phosphorylation of serine or tyrosine residues in the eNOS-interacting region of the B2 receptor results in a loss of eNOS inhibition due to a decrease in the binding affinity of the receptor domain for the eNOS enzyme. Furthermore, the B2 receptor is transiently phosphorylated on tyrosine residues in cultured endothelial cells in response to BK stimulation. Phosphorylation occurs during the time in which eNOS transiently dissociates from the receptor accompanied by a transient increase in nitric oxide production. Taken together, these data support the hypotheses that eNOS is regulated in endothelial cells by reversible and inhibitory interactions with G-protein-coupled receptors and that these interactions can be modulated by receptor phosphorylation. PMID:10510297

  11. Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists

    PubMed Central

    Hiranita, Takato; Kopajtic, Theresa A.; Rice, Kenner C.; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H.; McCurdy, Christopher R.

    2016-01-01

    The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. PMID:27189970

  12. Developmental Expression of Orphan G Protein-Coupled Receptor 50 in the Mouse Brain

    PubMed Central

    2012-01-01

    Mental disorders have a complex etiology resulting from interactions between multiple genetic risk factors and stressful life events. Orphan G protein-coupled receptor 50 (GPR50) has been identified as a genetic risk factor for bipolar disorder and major depression in women, and there is additional genetic and functional evidence linking GPR50 to neurite outgrowth, lipid metabolism, and adaptive thermogenesis and torpor. However, in the absence of a ligand, a specific function has not been identified. Adult GPR50 expression has previously been reported in brain regions controlling the HPA axis, but its developmental expression is unknown. In this study, we performed extensive expression analysis of GPR50 and three protein interactors using rt-PCR and immunohistochemistry in the developing and adult mouse brain. Gpr50 is expressed at embryonic day 13 (E13), peaks at E18, and is predominantly expressed by neurons. Additionally we identified novel regions of Gpr50 expression, including brain stem nuclei involved in neurotransmitter signaling: the locus coeruleus, substantia nigra, and raphe nuclei, as well as nuclei involved in metabolic homeostasis. Gpr50 colocalizes with yeast-two-hybrid interactors Nogo-A, Abca2, and Cdh8 in the hypothalamus, amygdala, cortex, and selected brain stem nuclei at E18 and in the adult. With this study, we identify a link between GPR50 and neurotransmitter signaling and strengthen a likely role in stress response and energy homeostasis. PMID:22860215

  13. Developmental and tumoral vascularization is regulated by G protein–coupled receptor kinase 2

    PubMed Central

    Rivas, Verónica; Carmona, Rita; Muñoz-Chápuli, Ramón; Mendiola, Marta; Nogués, Laura; Reglero, Clara; Miguel-Martín, María; García-Escudero, Ramón; Dorn, Gerald W.; Hardisson, David; Mayor, Federico; Penela, Petronila

    2013-01-01

    Tumor vessel dysfunction is a pivotal event in cancer progression. Using an in vivo neovascularization model, we identified G protein–coupled receptor kinase 2 (GRK2) as a key angiogenesis regulator. An impaired angiogenic response involving immature vessels was observed in mice hemizygous for Grk2 or in animals with endothelium-specific Grk2 silencing. ECs isolated from these animals displayed intrinsic alterations in migration, TGF-β signaling, and formation of tubular networks. Remarkably, an altered pattern of vessel growth and maturation was detected in postnatal retinas from endothelium-specific Grk2 knockout animals. Mouse embryos with systemic or endothelium-selective Grk2 ablation had marked vascular malformations involving impaired recruitment of mural cells. Moreover, decreased endothelial Grk2 dosage accelerated tumor growth in mice, along with reduced pericyte vessel coverage and enhanced macrophage infiltration, and this transformed environment promoted decreased GRK2 in ECs and human breast cancer vessels. Our study suggests that GRK2 downregulation is a relevant event in the tumoral angiogenic switch. PMID:24135140

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

    PubMed

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

    2013-12-05

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

  15. The Concise Guide to Pharmacology 2013/14: G Protein-Coupled Receptors

    PubMed Central

    Alexander, Stephen PH; Benson, Helen E; Faccenda, Elena; Pawson, Adam J; Sharman, Joanna L; Spedding, Michael; Peters, John A; Harmar, Anthony J

    2013-01-01

    The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. G protein-coupled receptors are one of the seven major pharmacological targets into which the Guide is divided, with the others being G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors and Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and the Guide to Receptors and Channels, providing a permanent, citable, point-in-time record that will survive database updates. PMID:24517644

  16. Neurovascular coupling to D2/D3 dopamine receptor occupancy using simultaneous PET/functional MRI

    PubMed Central

    Sander, Christin Y.; Hooker, Jacob M.; Catana, Ciprian; Normandin, Marc D.; Alpert, Nathaniel M.; Knudsen, Gitte M.; Vanduffel, Wim; Rosen, Bruce R.; Mandeville, Joseph B.

    2013-01-01

    This study employed simultaneous neuroimaging with positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) to demonstrate the relationship between changes in receptor occupancy measured by PET and changes in brain activity inferred by fMRI. By administering the D2/D3 dopamine receptor antagonist [11C]raclopride at varying specific activities to anesthetized nonhuman primates, we mapped associations between changes in receptor occupancy and hemodynamics [cerebral blood volume (CBV)] in the domains of space, time, and dose. Mass doses of raclopride above tracer levels caused increases in CBV and reductions in binding potential that were localized to the dopamine-rich striatum. Moreover, similar temporal profiles were observed for specific binding estimates and changes in CBV. Injection of graded raclopride mass doses revealed a monotonic coupling between neurovascular responses and receptor occupancies. The distinct CBV magnitudes between putamen and caudate at matched occupancies approximately matched literature differences in basal dopamine levels, suggesting that the relative fMRI measurements reflect basal D2/D3 dopamine receptor occupancy. These results can provide a basis for models that relate dopaminergic occupancies to hemodynamic changes in the basal ganglia. Overall, these data demonstrate the utility of simultaneous PET/fMRI for investigations of neurovascular coupling that correlate neurochemistry with hemodynamic changes in vivo for any receptor system with an available PET tracer. PMID:23723346

  17. Neurovascular coupling to D2/D3 dopamine receptor occupancy using simultaneous PET/functional MRI.

    PubMed

    Sander, Christin Y; Hooker, Jacob M; Catana, Ciprian; Normandin, Marc D; Alpert, Nathaniel M; Knudsen, Gitte M; Vanduffel, Wim; Rosen, Bruce R; Mandeville, Joseph B

    2013-07-02

    This study employed simultaneous neuroimaging with positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) to demonstrate the relationship between changes in receptor occupancy measured by PET and changes in brain activity inferred by fMRI. By administering the D2/D3 dopamine receptor antagonist [(11)C]raclopride at varying specific activities to anesthetized nonhuman primates, we mapped associations between changes in receptor occupancy and hemodynamics [cerebral blood volume (CBV)] in the domains of space, time, and dose. Mass doses of raclopride above tracer levels caused increases in CBV and reductions in binding potential that were localized to the dopamine-rich striatum. Moreover, similar temporal profiles were observed for specific binding estimates and changes in CBV. Injection of graded raclopride mass doses revealed a monotonic coupling between neurovascular responses and receptor occupancies. The distinct CBV magnitudes between putamen and caudate at matched occupancies approximately matched literature differences in basal dopamine levels, suggesting that the relative fMRI measurements reflect basal D2/D3 dopamine receptor occupancy. These results can provide a basis for models that relate dopaminergic occupancies to hemodynamic changes in the basal ganglia. Overall, these data demonstrate the utility of simultaneous PET/fMRI for investigations of neurovascular coupling that correlate neurochemistry with hemodynamic changes in vivo for any receptor system with an available PET tracer.

  18. Selection of cell lines resistant to anti-transferrin receptor antibody: evidence for a mutation in transferrin receptor.

    PubMed Central

    Lesley, J F; Schulte, R J

    1984-01-01

    Some anti-murine transferrin receptor monoclonal antibodies block iron uptake in mouse cell lines and inhibit cell growth. We report here the selection and characterization of mutant murine lymphoma cell lines which escape this growth inhibition by anti-transferrin receptor antibody. Growth assays and immunoprecipitation of transferrin receptor in hybrids between independently derived mutants or between mutants and antibody-susceptible parental cell lines indicate that all of the selected lines have a similar genetic alteration that is codominantly expressed in hybrids. Anti-transferrin receptor antibodies and transferrin itself still bind to the mutant lines with saturating levels and Kd values very similar to those of the parental lines. However, reciprocal clearing experiments by immunoprecipitation and reciprocal blocking of binding to the cell surface with two anti-transferrin receptor antibodies indicate that the mutant lines have altered a fraction of their transferrin receptors such that the growth-inhibiting antibody no longer binds, whereas another portion of their transferrin receptors is similar to those of the parental lines and binds both antibodies. These results argue that the antibody-selected mutant cell lines are heterozygous in transferrin receptor expression, probably with a mutation in one of the transferrin receptor structural genes. Images PMID:6092931

  19. Overexpression of G protein-coupled receptors in cancer cells: involvement in tumor progression.

    PubMed

    Li, Shuyu; Huang, Shuguang; Peng, Sheng-Bin

    2005-11-01

    G protein-coupled receptors (GPCRs) play important roles in a variety of biological and pathological processes. They are considered among the most desirable targets for drug development. Recent studies have demonstrated that many GPCRs, such as endothelin receptors, chemokine receptors and lysophosphatidic acid receptors have been implicated in the tumorigenesis and metastasis of multiple human cancers. In this study, we conducted an in silico analysis of GPCR gene expression in primary human tumors by analyzing some publicly available gene expression profiling data. Statistical analysis was performed on eight microarray data sets of non-small cell lung cancer, breast cancer, prostate cancer, melanoma, gastric cancer and diffused large B cell lymphoma to identify GPCRs that are up-regulated in primary or metastatic cancer cells. Our analysis has demonstrated overexpression of several GPCRs in primary tumor cells, including chemokine receptors and protease-activated receptors that were shown to be important for tumorigenesis by previous studies. In addition, we have uncovered several GPCRs, such as neuropeptide receptors, adenosine A2B receptor, P2Y purinoceptor, calcium-sensing receptor and metabotropic glutamate receptors, that are expressed at a significantly higher level in some cancer tissue and may play a role in cancer progression. Analysis of cancer samples in different disease stages also suggests that some GPCRs, such as endothelin receptor A, may be involved in early tumor progression and others, such as CXCR4, may play a critical role in tumor invasion and metastasis. The present study demonstrates the value of publicly available microarray data as a resource to gain more understanding of cancer biology, to validate previous findings from in vitro experiments, and to identify potential novel anticancer targets and biomarkers.

  20. Quantitative analysis of receptor tyrosine kinase-effector coupling at functionally relevant stimulus levels.

    PubMed

    Li, Simin; Bhave, Devayani; Chow, Jennifer M; Riera, Thomas V; Schlee, Sandra; Rauch, Simone; Atanasova, Mariya; Cate, Richard L; Whitty, Adrian

    2015-04-17

    A major goal of current signaling research is to develop a quantitative understanding of how receptor activation is coupled to downstream signaling events and to functional cellular responses. Here, we measure how activation of the RET receptor tyrosine kinase on mouse neuroblastoma cells by the neurotrophin artemin (ART) is quantitatively coupled to key downstream effectors. We show that the efficiency of RET coupling to ERK and Akt depends strongly on ART concentration, and it is highest at the low (∼100 pM) ART levels required for neurite outgrowth. Quantitative discrimination between ERK and Akt pathway signaling similarly is highest at this low ART concentration. Stimulation of the cells with 100 pM ART activated RET at the rate of ∼10 molecules/cell/min, leading at 5-10 min to a transient peak of ∼150 phospho-ERK (pERK) molecules and ∼50 pAkt molecules per pRET, after which time the levels of these two signaling effectors fell by 25-50% while the pRET levels continued to slowly rise. Kinetic experiments showed that signaling effectors in different pathways respond to RET activation with different lag times, such that the balance of signal flux among the different pathways evolves over time. Our results illustrate that measurements using high, super-physiological growth factor levels can be misleading about quantitative features of receptor signaling. We propose a quantitative model describing how receptor-effector coupling efficiency links signal amplification to signal sensitization between receptor and effector, thereby providing insight into design principles underlying how receptors and their associated signaling machinery decode an extracellular signal to trigger a functional cellular outcome.

  1. Quantitative Analysis of Receptor Tyrosine Kinase-Effector Coupling at Functionally Relevant Stimulus Levels*♦

    PubMed Central

    Li, Simin; Bhave, Devayani; Chow, Jennifer M.; Riera, Thomas V.; Schlee, Sandra; Rauch, Simone; Atanasova, Mariya; Cate, Richard L.; Whitty, Adrian

    2015-01-01

    A major goal of current signaling research is to develop a quantitative understanding of how receptor activation is coupled to downstream signaling events and to functional cellular responses. Here, we measure how activation of the RET receptor tyrosine kinase on mouse neuroblastoma cells by the neurotrophin artemin (ART) is quantitatively coupled to key downstream effectors. We show that the efficiency of RET coupling to ERK and Akt depends strongly on ART concentration, and it is highest at the low (∼100 pm) ART levels required for neurite outgrowth. Quantitative discrimination between ERK and Akt pathway signaling similarly is highest at this low ART concentration. Stimulation of the cells with 100 pm ART activated RET at the rate of ∼10 molecules/cell/min, leading at 5–10 min to a transient peak of ∼150 phospho-ERK (pERK) molecules and ∼50 pAkt molecules per pRET, after which time the levels of these two signaling effectors fell by 25–50% while the pRET levels continued to slowly rise. Kinetic experiments showed that signaling effectors in different pathways respond to RET activation with different lag times, such that the balance of signal flux among the different pathways evolves over time. Our results illustrate that measurements using high, super-physiological growth factor levels can be misleading about quantitative features of receptor signaling. We propose a quantitative model describing how receptor-effector coupling efficiency links signal amplification to signal sensitization between receptor and effector, thereby providing insight into design principles underlying how receptors and their associated signaling machinery decode an extracellular signal to trigger a functional cellular outcome. PMID:25635057

  2. Selective Androgen Receptor Modulators (SARMs) as Function Promoting Therapies

    PubMed Central

    Bhasin, Shalender; Jasuja, Ravi

    2010-01-01

    Purpose of review The last decade has witnessed unprecedented discovery effort to develop selective androgen receptor modulators (SARMs) that improve physical function and bone health without adversely affecting the prostate and cardiovascular outcomes. This review describes the historical evolution, the rationale for SARM development, and the mechanisms of testosterone action and SARM selectivity. Recent Findings While steroidal SARMs have been around since the 1940s, a number of nonsteroidal SARMs that do not serve as substrates for CYP19 aromatase or 5α-reductase, act as full agonists in muscle and bone and as partial agonists in prostate are in development. The differing interactions of steroidal and nonsteroidal compounds with AR contribute to their unique pharmacologic actions. Ligand binding induces specific conformational changes in the ligand binding domain, which could modulate surface topology and protein-protein interactions between AR and coregulators, resulting in tissue-specific gene regulation. Preclinical studies have demonstrated the ability of SARMs to increase muscle and bone mass in preclinical rodent models with varying degree of prostate sparing. Phase I trials of SARMs in humans have reported modest increments in fat-free mass. Summary SARMs hold promise as a new class of function promoting anabolic therapies for a number of clinical indications, including functional limitations associated with aging and chronic disease, frailty, cancer cachexia, and osteoporosis. PMID:19357508

  3. Glucagon-Like Peptide-1 and Its Class B G Protein–Coupled Receptors: A Long March to Therapeutic Successes

    PubMed Central

    de Graaf, Chris; Donnelly, Dan; Wootten, Denise; Lau, Jesper; Sexton, Patrick M.; Miller, Laurence J.; Ahn, Jung-Mo; Liao, Jiayu; Fletcher, Madeleine M.; Brown, Alastair J. H.; Zhou, Caihong; Deng, Jiejie; Wang, Ming-Wei

    2016-01-01

    The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein–coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain–binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders. PMID:27630114

  4. Characteristics of receptor- and transducer-coupled activation of the intracellular signalling in sensory neuron revealed by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Khalisov, M. M.; Penniyaynen, V. A.; Esikova, N. A.; Ankudinov, A. V.; Krylov, B. V.

    2017-01-01

    The mechanical properties of sensory neurons upon activation of intracellular cascade processes by comenic acid binding to a membrane opioid-like receptor (receptor-coupled), as well as a very low (endogenous) concentration of ouabain (transducer-coupled), have been investigated. Using atomic force microscopy, it is established that exposure to ouabain, in contrast to the impact of comenic acid, leads to a hardening of the neuron soma. This suggests that the receptor-coupled signal transmission to the cell genome is carried out through mechanisms that are different from the transducer-coupled signal pathways.

  5. Function of G-Protein-Coupled Estrogen Receptor-1 in Reproductive System Tumors

    PubMed Central

    Qian, Hongyan; Xuan, Jingxiu; Liu, Yuan; Shi, Guixiu

    2016-01-01

    The G-protein-coupled estrogen receptor-1 (GPER-1), also known as GPR30, is a novel estrogen receptor mediating estrogen receptor signaling in multiple cell types. The progress of estrogen-related cancer is promoted by GPER-1 activation through mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K), and phospholipase C (PLC) signaling pathways. However, this promoting effect of GPER-1 is nonclassic estrogen receptor (ER) dependent manner. In addition, clinical evidences revealed that GPER-1 is associated with estrogen resistance in estrogen-related cancer patients. These give a hint that GPER-1 may be a novel therapeutic target for the estrogen-related cancers. However, preclinical studies also found that GPER-1 activation of its special agonist G-1 inhibits cancer cell proliferation. This review aims to summarize the characteristics and complex functions of GPER-1 in cancers. PMID:27314054

  6. Systematic and quantitative analysis of G protein-coupled receptor trafficking motifs.

    PubMed

    Hurt, Carl M; Ho, Vincent K; Angelotti, Timothy

    2013-01-01

    Plasma membrane expression of G protein-coupled receptors (GPCRs) is a dynamic process balancing anterograde and retrograde trafficking. Multiple interrelated cellular processes determine the final level of cell surface expression, including endoplasmic reticulum (ER) export/retention, receptor internalization, recycling, and degradation. These processes are highly regulated to achieve specific localization to subcellular domains (e.g., dendrites or basolateral membranes) and to affect receptor signaling. Analysis of potential ER trafficking motifs within GPCRs requires careful consideration of intracellular dynamics, such as protein folding, ER export and retention, and glycosylation. This chapter presents an approach and methods for qualitative and quantitative assessment of these processes to aid in accurate identification of GPCR trafficking motifs, utilizing the analysis of a hydrophobic extracellular trafficking motif in α2C adrenergic receptors as a model system.

  7. Bazedoxifene: a novel selective estrogen receptor modulator for postmenopausal osteoporosis.

    PubMed

    de Villiers, T J

    2010-06-01

    Several new selective estrogen receptor modulators (SERMs) are currently under clinical development for the prevention and/or treatment of postmenopausal osteoporosis, with the goal of optimizing the estrogen receptor agonist/antagonist activity in target tissues. Bazedoxifene is a novel SERM under clinical investigation for the prevention and treatment of postmenopausal osteoporosis. Emerging clinical data have shown that bazedoxifene is effective in preventing bone loss and osteoporotic fractures in postmenopausal women, with no evidence of breast or endometrial stimulation. Two large, prospective, international phase 3 studies have been completed. In postmenopausal women at risk for osteoporosis, bazedoxifene has been shown to preserve bone mineral density and to reduce bone turnover. In postmenopausal women with osteoporosis, bazedoxifene has demonstrated significant protection against new vertebral fractures and against non-vertebral fractures in women at higher fracture risk. The treatment effects of bazedoxifene were supported by findings from independent re-analyses using the Fracture Risk Assessment Tool (FRAX), which showed that bazedoxifene significantly reduced the risk of all clinical and morphometric vertebral fracture and of non-vertebral fracture in women at or above a FRAX-based threshold. Bazedoxifene was generally safe and well tolerated in the phase 3 studies and showed neutral effects on the breast and an excellent endometrial safety profile; such attributes allow for the partnering of bazedoxifene with conjugated estrogens for menopausal symptom relief. Collectively, these results suggest that bazedoxifene may be a promising new therapy for the prevention and treatment of postmenopausal osteoporosis as a monotherapy or in combination with conjugated estrogens in menopausal hormone therapy.

  8. Agonistic autoantibodies directed against G-protein-coupled receptors and their relationship to cardiovascular diseases.

    PubMed

    Wallukat, Gerd; Schimke, Ingolf

    2014-05-01

    Agonistic autoantibodies (AABs) against G-protein-coupled receptor (GPCR) are present mainly in diseases of the cardiovascular system or in diseases associated with cardiovascular disturbances. The increasing knowledge about the role of autoantibodies against G-protein-coupled receptor (GPCR-AABs) as pathogenic drivers, the resulting development of strategies aimed at their removal or neutralization, and the evidenced patient benefit associated with such therapies have created the need for a summary of GPCR-AAB-associated diseases. Here, we summarize the present knowledge about GPCR-AABs in cardiovascular diseases. The identity of the GPCR-AABs and their prevalence in each of several specific cardiovascular diseases are documented. The structure of GPCR is also briefly discussed. Using this information, differences between classic agonists and GPCR-AABs in their GPCR binding and activation are presented and the resulting pathogenic consequences are discussed. Furthermore, treatment strategies that are currently under study, most of which are aimed at the removal and in vivo neutralization of GPCR-AABs, are indicated and their patient benefits discussed. In this context, immunoadsorption using peptides/proteins or aptamers as binders are introduced. The use of peptides or aptamers for in vivo neutralization of GPCR-AABs is also described. Particular attention is given to the GPCR-AABs directed against the adrenergic beta1-, beta2-, and α1-receptor as well as the muscarinic receptor M2, angiotensin II-angiotensin receptor type I, endothelin1 receptor type A, angiotensin (1-7) Mas-receptor, and 5-hydroxytryptamine receptor 4. Among the diseases associated with GPCR-AABs, special focus is given to idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, malignant and pulmonary hypertension, and kidney diseases. Relationships of GPCR-AABs are indicated to glaucoma, peripartum cardiomyopathy, myocarditis, pericarditis, preeclampsia, Alzheimer's disease, Sj

  9. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    SciTech Connect

    Lee, Byung-Kwon; Jung, Kyung-Sik; Son, Cagdas D; Kim, Heejung; Verberkmoes, Nathan C; Arshava, Boris; Naider, Fred; Becker, Jeffrey Marvin

    2007-01-01

    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  10. Selectively engaging β-arrestins at the angiotensin II type 1 receptor reduces blood pressure and increases cardiac performance.

    PubMed

    Violin, Jonathan D; DeWire, Scott M; Yamashita, Dennis; Rominger, David H; Nguyen, Lisa; Schiller, Kevin; Whalen, Erin J; Gowen, Maxine; Lark, Michael W

    2010-12-01

    Biased G protein-coupled receptor ligands engage subsets of the receptor signals normally stimulated by unbiased agonists. However, it is unclear whether ligand bias can elicit differentiated pharmacology in vivo. Here, we describe the discovery of a potent, selective β-arrestin biased ligand of the angiotensin II type 1 receptor. TRV120027 (Sar-Arg-Val-Tyr-Ile-His-Pro-D-Ala-OH) competitively antagonizes angiotensin II-stimulated G protein signaling, but stimulates β-arrestin recruitment and activates several kinase pathways, including p42/44 mitogen-activated protein kinase, Src, and endothelial nitric-oxide synthase phosphorylation via β-arrestin coupling. Consistent with β-arrestin efficacy, and unlike unbiased antagonists, TRV120027 increased cardiomyocyte contractility in vitro. In rats, TRV120027 reduced mean arterial pressure, as did the unbiased antagonists losartan and telmisartan. However, unlike the unbiased antagonists, which decreased cardiac performance, TRV120027 increased cardiac performance and preserved cardiac stroke volume. These striking differences in vivo between unbiased and β-arrestin biased ligands validate the use of biased ligands to selectively target specific receptor functions in drug discovery.

  11. Tamoxifen regulation of bone growth and endocrine function in the ovariectomized rat: discrimination of responses involving estrogen receptor α/estrogen receptor β, G protein-coupled estrogen receptor, or estrogen-related receptor γ using fulvestrant (ICI 182780).

    PubMed

    Fitts, James M; Klein, Robert M; Powers, C Andrew

    2011-07-01

    Tamoxifen is a selective estrogen receptor (ER) modulator, but it is also a deactivating ligand for estrogen-related receptor-γ (ERRγ) and a full agonist for the G protein-coupled estrogen receptor (GPER). Fulvestrant is a selective ER down-regulator that lacks agonist effects on ERα/ERβ, is inactive on ERRγ, but acts as a full agonist on GPER. Fulvestrant effects on tamoxifen actions on uterine and somatic growth, bone, the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis, and pituitary prolactin were analyzed to pharmacologically discriminate tamoxifen effects that may be mediated by ERα/ERβ versus ERRγ versus GPER. Ovariectomized rats received tamoxifen (0.6 mg/kg/daily) plus fulvestrant at 0, 3, 6, or 12 mg/kg/daily for 5 weeks; controls received vehicle or 6 mg/kg fulvestrant daily. Tamoxifen effects to increase uterine weight, decrease serum IGF-I, increase pituitary prolactin, and increase bone mineral density could be fully blocked by fulvestrant, indicating mediation by ERα/ERβ. Tamoxifen effects to decrease pituitary GH, tibia length, and body weight were only partially blocked by fulvestrant, indicating involvement of mechanisms unrelated to ERα/ERβ. Fulvestrant did not inhibit tamoxifen actions to reduce total pituitary protein, again indicating effects not mediated by ERα/ERβ. Tamoxifen actions to reduce serum GH were mimicked rather than inhibited by fulvestrant, pharmacological features consistent with GPER involvement. However, fulvestrant alone increased IGF-I and also blocked tamoxifen-evoked IGF-I decreases; thus fulvestrant effects on serum GH might reflect increased IGF-I feedback inhibition. Fulvestrant alone had no effect on the other parameters. The findings indicate that mechanisms unrelated to ERα/ERβ contribute to tamoxifen effects on body weight, bone growth, and pituitary function.

  12. Selective inhibition of Ebola entry with selective estrogen receptor modulators by disrupting the endolysosomal calcium

    PubMed Central

    Fan, Hanlu; Du, Xiaohong; Zhang, Jingyuan; Zheng, Han; Lu, Xiaohui; Wu, Qihui; Li, Haifeng; Wang, Han; Shi, Yi; Gao, George; Zhou, Zhuan; Tan, Dun-Xian; Li, Xiangdong

    2017-01-01

    The Ebola crisis occurred in West-Africa highlights the urgency for its clinical treatments. Currently, no Food and Drug Administration (FDA)-approved therapeutics are available. Several FDA-approved drugs, including selective estrogen receptor modulators (SERMs), possess selective anti-Ebola activities. However, the inhibitory mechanisms of these drugs remain elusive. By analyzing the structures of SERMs and their incidental biological activity (cholesterol accumulation), we hypothesized that this incidental biological activity induced by SERMs could be a plausible mechanism as to their inhibitory effects on Ebola infection. Herein, we demonstrated that the same dosages of SERMs which induced cholesterol accumulation also inhibited Ebola infection. SERMs reduced the cellular sphingosine and subsequently caused endolysosomal calcium accumulation, which in turn led to blocking the Ebola entry. Our study clarified the specific anti-Ebola mechanism of SERMs, even the cationic amphiphilic drugs (CADs), this mechanism led to the endolysosomal calcium as a critical target for development of anti-Ebola drugs. PMID:28117364

  13. Use of Kaede fusions to visualize recycling of G protein-coupled receptors.

    PubMed

    Schmidt, Antje; Wiesner, Burkhard; Weisshart, Klaus; Schulz, Katharina; Furkert, Jens; Lamprecht, Björn; Rosenthal, Walter; Schülein, Ralf

    2009-01-01

    The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere with recycling receptors. In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein. In contrast to the widely used green fluorescent protein, the fluorescence of Kaede can be converted from green to red using ultraviolet irradiation. Our methodology allows to study recycling of GPCRs microscopically in real-time bypassing problems with secretory pathway receptors. Initially, receptors are internalized using an agonist. Fluorescence signals in endosomes are switched, and trafficking of the receptors to the plasma membrane can be easily visualized by monitoring their new fluorescence. Using this methodology, we show that the corticotropin-releasing factor receptor type 1 belongs to the family of recycling GPCRs. Moreover, we demonstrate by fluorescence correlation spectroscopy that Kaede does not oligomerize when fused to membrane proteins, representing an additional advantage of this technique. The Kaede technology may be a powerful tool to study membrane protein trafficking in general.

  14. G protein-coupled receptors as oncogenic signals in glioma: emerging therapeutic avenues

    PubMed Central

    Cherry, Allison E; Stella, Nephi

    2014-01-01

    Gliomas are the most common malignant intracranial tumors. Newly developed targeted therapies for these cancers aim to inhibit oncogenic signals, many of which emanate from receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor receptor (VEGFR). Unfortunately, the first generation treatments targeting these oncogenic signals provide little survival benefit in both mouse xenograft models and human patients. The search for new treatment options has uncovered several G protein-coupled receptor (GPCR) candidates and generated a growing interest in this class of proteins as alternative therapeutic targets for the treatment of various cancers, including GBM. GPCRs constitute a large family of membrane receptors that influence oncogenic pathways through canonical and non-canonical signaling. Accordingly, evidence indicates that GPCRs display a unique ability to crosstalk with receptor tyrosine kinases, making them important molecular components controlling tumorigenesis. This review summarizes the current research on GPCR functionality in gliomas and explores the potential of modulating these receptors to treat this devastating disease. PMID:25158675

  15. The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

    PubMed Central

    Salon, John A.; Lodowski, David T.

    2011-01-01

    Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination. PMID:21969326

  16. G protein-coupled receptors as oncogenic signals in glioma: emerging therapeutic avenues.

    PubMed

    Cherry, A E; Stella, N

    2014-10-10

    Gliomas are the most common malignant intracranial tumors. Newly developed targeted therapies for these cancers aim to inhibit oncogenic signals, many of which emanate from receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor receptor (VEGFR). Unfortunately, the first-generation treatments targeting these oncogenic signals provide little survival benefit in both mouse xenograft models and human patients. The search for new treatment options has uncovered several G protein-coupled receptor (GPCR) candidates and generated a growing interest in this class of proteins as alternative therapeutic targets for the treatment of various cancers, including glioblastoma multiforme (GBM). GPCRs constitute a large family of membrane receptors that influence oncogenic pathways through canonical and non-canonical signaling. Accordingly, evidence indicates that GPCRs display a unique ability to crosstalk with receptor tyrosine kinases, making them important molecular components controlling tumorigenesis. This review summarizes the current research on GPCR functionality in gliomas and explores the potential of modulating these receptors to treat this devastating disease.

  17. Discovery of Novel Potent and Selective Agonists at the Melanocortin-3 Receptor.

    PubMed

    Carotenuto, Alfonso; Merlino, Francesco; Cai, Minying; Brancaccio, Diego; Yousif, Ali Munaim; Novellino, Ettore; Hruby, Victor J; Grieco, Paolo

    2015-12-24

    The melanocortin receptors 3 and 4 control energy homeostasis, food-intake behavior, and correlated pathophysiological conditions. The melanocortin-4 receptor (MC4R) has been broadly investigated. In contrast, the knowledge related to physiological roles of the melanocortin-3 receptor (MC3R) is lacking because of the limited number of known MC3R selective ligands. Here, we report the design, synthesis, biological activity, conformational analysis, and docking with receptors of two potent and selective agonists at the human MC3 receptor.

  18. International Union of Basic and Clinical Pharmacology. XCIII. The parathyroid hormone receptors--family B G protein-coupled receptors.

    PubMed

    Gardella, Thomas J; Vilardaga, Jean-Pierre

    2015-01-01

    The type-1 parathyroid hormone receptor (PTHR1) is a family B G protein-coupled receptor (GPCR) that mediates the actions of two polypeptide ligands; parathyroid hormone (PTH), an endocrine hormone that regulates the levels of calcium and inorganic phosphate in the blood by acting on bone and kidney, and PTH-related protein (PTHrP), a paracrine-factor that regulates cell differentiation and proliferation programs in developing bone and other tissues. The type-2 parathyroid hormone receptor (PTHR2) binds a peptide ligand, called tuberoinfundibular peptide-39 (TIP39), and while the biologic role of the PTHR2/TIP39 system is not as defined as that of the PTHR1, it likely plays a role in the central nervous system as well as in spermatogenesis. Mechanisms of action at these receptors have been explored through a variety of pharmacological and biochemical approaches, and the data obtained support a basic "two-site" mode of ligand binding now thought to be used by each of the family B peptide hormone GPCRs. Recent crystallographic studies on the family B GPCRs are providing new insights that help to further refine the specifics of the overall receptor architecture and modes of ligand docking. One intriguing pharmacological finding for the PTHR1 is that it can form surprisingly stable complexes with certain PTH/PTHrP ligand analogs and thereby mediate markedly prolonged cell signaling responses that persist even when the bulk of the complexes are found in internalized vesicles. The PTHR1 thus appears to be able to activate the Gα(s)/cAMP pathway not only from the plasma membrane but also from the endosomal domain. The cumulative findings could have an impact on efforts to develop new drug therapies for the PTH receptors.

  19. Principal pathway coupling agonist binding to channel gating in nicotinic receptors

    NASA Astrophysics Data System (ADS)

    Lee, Won Yong; Sine, Steven M.

    2005-11-01

    Synaptic receptors respond to neurotransmitters by opening an intrinsic ion channel in the final step in synaptic transmission. How binding of the neurotransmitter is conveyed over the long distance to the channel remains a central question in neurobiology. Here we delineate a principal pathway that links neurotransmitter binding to channel gating by using a structural model of the Torpedo acetylcholine receptor at 4-Å resolution, recordings of currents through single receptor channels and determinations of energetic coupling between pairs of residues. We show that a pair of invariant arginine and glutamate residues in each receptor α-subunit electrostatically links peripheral and inner β-sheets from the binding domain and positions them to engage with the channel. The key glutamate and flanking valine residues energetically couple to conserved proline and serine residues emerging from the top of the channel-forming α-helix, suggesting that this is the point at which the binding domain triggers opening of the channel. The series of interresidue couplings identified here constitutes a primary allosteric pathway that links neurotransmitter binding to channel gating.

  20. Exon capture analysis of G protein-coupled receptors identifies activating mutations in GRM3 in melanoma.

    PubMed

    Prickett, Todd D; Wei, Xiaomu; Cardenas-Navia, Isabel; Teer, Jamie K; Lin, Jimmy C; Walia, Vijay; Gartner, Jared; Jiang, Jiji; Cherukuri, Praveen F; Molinolo, Alfredo; Davies, Michael A; Gershenwald, Jeffrey E; Stemke-Hale, Katherine; Rosenberg, Steven A; Margulies, Elliott H; Samuels, Yardena

    2011-09-25

    G protein-coupled receptors (GPCRs), the largest human gene family, are important regulators of signaling pathways. However, knowledge of their genetic alterations is limited. In this study, we used exon capture and massively parallel sequencing methods to analyze the mutational status of 734 GPCRs in melanoma. This investigation revealed that one family member, GRM3, was frequently mutated and that one of its mutations clustered within one position. Biochemical analysis of GRM3 alterations revealed that mutant GRM3 selectively regulated the phosphorylation of MEK, leading to increased anchorage-independent growth and migration. Melanoma cells expressing mutant GRM3 had reduced cell growth and cellular migration after short hairpin RNA-mediated knockdown of GRM3 or treatment with a selective MEK inhibitor, AZD-6244, which is currently being used in phase 2 clinical trials. Our study yields the most comprehensive map of genetic alterations in the GPCR gene family.

  1. Effects of weightlessness on neurotransmitter receptors in selected brain areas

    NASA Technical Reports Server (NTRS)

    Miller, J. D.; Murakami, D. M.; Mcmillen, B. A.; Mcconnaughey, M. M.; Williams, H. L.

    1985-01-01

    The central nervous system receptor dynamics of rats exposed to 7 days of microgravity are studied. The receptor affinity and receptor number at the hippocampus, lateral frontal cortex, prefrontal cortex, corpus striatum, cerebellum and pons-medulla, and the Na(+)/K(+)ATPase activity are examined. The data reveal that there is no significant change in the receptor affinity and receptor number for the lateral frontal cortex, prefrontal cortex, cerebellum and pons-medulla; however, there is an increase from 81 + or - 11 to 120 + or 5 fmole/mg protein in the receptor number for hippocampal binding, and a decrease in receptor number for the striatum from 172 + or - 14 to 143 + or - 10 fmoles/mg protein. A 9 percent decrease in Mg-dependent Na(+)/K(+)ATPase activity is observed. It is detected that the terminal mechanism may be affected by exposure to microgravity.

  2. Immunochemical strategy for quantification of G-coupled olfactory receptor proteins on natural nanovesicles.

    PubMed

    Sanmartí-Espinal, Marta; Galve, Roger; Iavicoli, Patrizia; Persuy, Marie-Annick; Pajot-Augy, Edith; Marco, M-Pilar; Samitier, Josep

    2016-03-01

    Cell membrane proteins are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays using these membrane protein receptors for drug screening or diagnostic require the construction of well-characterized liposome and lipid bilayer arrays that act as support to prevent protein denaturation during biochip processing. Quantification of the protein receptors in the lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. Herein, we report a novel immunochemical analytical approach for the quantification of membrane proteins (i.e., G-protein-coupled receptor, GPCR) in nanovesicles (NVs). The procedure allows direct determination of tagged receptors (i.e., c-myc tag) without any previous protein purification or extraction steps. The immunochemical method is based on a microplate ELISA format and quantifies this tag on proteins embedded in NVs with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptor (OR, c-myc-OR1740) in the cell membrane NVs. The reported method opens the possibility to develop well-characterized drug-screening platforms based on G-coupled proteins embedded on membranes.

  3. Estrogen receptor transcription and transactivation: Estrogen receptor alpha and estrogen receptor beta - regulation by selective estrogen receptor modulators and importance in breast cancer

    PubMed Central

    S Katzenellenbogen, Benita; A Katzenellenbogen, John

    2000-01-01

    Estrogens display intriguing tissue-selective action that is of great biomedical importance in the development of optimal therapeutics for the prevention and treatment of breast cancer, for menopausal hormone replacement, and for fertility regulation. Certain compounds that act through the estrogen receptor (ER), now referred to as selective estrogen receptor modulators (SERMs), can demonstrate remarkable differences in activity in the various estrogen target tissues, functioning as agonists in some tissues but as antagonists in others. Recent advances elucidating the tripartite nature of the biochemical and molecular actions of estrogens provide a good basis for understanding these tissue-selective actions. As discussed in this thematic review, the development of optimal SERMs should now be viewed in the context of two estrogen receptor subtypes, ERα and ERβ, that have differing affinities and responsiveness to various SERMs, and differing tissue distribution and effectiveness at various gene regulatory sites. Cellular, biochemical, and structural approaches have also shown that the nature of the ligand affects the conformation assumed by the ER-ligand complex, thereby regulating its state of phosphorylation and the recruitment of different coregulator proteins. Growth factors and protein kinases that control the phosphorylation state of the complex also regulate the bioactivity of the ER. These interactions and changes determine the magnitude of the transcriptional response and the potency of different SERMs. As these critical components are becoming increasingly well defined, they provide a sound basis for the development of novel SERMs with optimal profiles of tissue selectivity as medical therapeutic agents. PMID:11250726

  4. Regulation of H1-receptor coupling and H1-receptor mRNA by histamine in bovine tracheal smooth muscle

    PubMed Central

    Pype, J L; Dupont, L J; Mak, J C W; Barnes, P J; Verleden, G M

    1998-01-01

    Pretreatment of bovine tracheal smooth muscle (BTSM) with histamine (1–100 μM, 1 h) induced a concentration-dependent desensitization of the contractile response to subsequently administered histamine, with a reduction of the maximum response of 72±8% (n=5) following pre-exposure to 100 μM histamine. In contrast, concentration-response curves to the muscarinic agonist, methacholine were not affected following histamine pretreatment, indicating a homologous desensitization. Furthermore, concentration-response curves to NaF, a G-protein activator, were not altered following histamine pre-incubation.The histamine H1-receptor (H1R) desensitization could be antagonized by mepyramine (an H1-receptor antagonist, 1 μM) but not by cimetidine (an H2-receptor antagonist, 10 μM), indicating that the desensitization occurred via stimulation of histamine H1-receptors, without evidence for the involvement of histamine H2-receptors.Indomethacin (10 μM) did not block the H1R desensitization, suggesting no involvement of prostaglandins. Furthermore, histamine pre-incubation in calcium free medium still induced a functional uncoupling of H1R.GF 109203X, a protein kinase C (PKC) inhibitor, and H-7, a non-selective kinase inhibitor, did not antagonize the homologous H1R desensitization.The steady-state level of H1R mRNA, assessed by Northern blot analysis, was not affected by prolonged histamine exposure (100 μM, 0.5, 1, 2, 4, 16 and 24 h).These results suggest that histamine induces desensitization of the H1R at the level of the receptor protein, which involves a mechanism independent of PKC, PKA, PKG and calcium influx, suggesting the involvement of a receptor-specific kinase. PMID:9535029

  5. Prothymosin Alpha Selectively Enhances Estrogen Receptor Transcriptional Activity by Interacting with a Repressor of Estrogen Receptor Activity

    PubMed Central

    Martini, Paolo G. V.; Delage-Mourroux, Regis; Kraichely, Dennis M.; Katzenellenbogen, Benita S.

    2000-01-01

    We find that prothymosin alpha (PTα) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors. This selectivity for ER is explained by PTα interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking coactivator binding to ER. We isolated PTα, known to be a chromatin-remodeling protein associated with cell proliferation, using REA as bait in a yeast two-hybrid screen with a cDNA library from MCF-7 human breast cancer cells. PTα increases the magnitude of ERα transcriptional activity three- to fourfold. It shows lesser enhancement of ERβ transcriptional activity and has no influence on the transcriptional activity of other nuclear hormone receptors (progesterone receptor, glucocorticoid receptor, thyroid hormone receptor, or retinoic acid receptor) or on the basal activity of ERs. In contrast, the steroid receptor coactivator SRC-1 increases transcriptional activity of all of these receptors. Cotransfection of PTα or SRC-1 with increasing amounts of REA, as well as competitive glutathione S-transferase pulldown and mammalian two-hybrid studies, show that REA competes with PTα (or SRC-1) for regulation of ER transcriptional activity and suppresses the ER stimulation by PTα or SRC-1, indicating that REA can function as an anticoactivator in cells. Our data support a model in which PTα, which does not interact with ER, selectively enhances the transcriptional activity of the ER but not that of other nuclear receptors by recruiting the repressive REA protein away from ER, thereby allowing effective coactivation of ER with SRC-1 or other coregulators. The ability of PTα to directly interact in vitro and in vivo with REA, a selective coregulator of the ER, thereby enabling the interaction of ER with coactivators, appears to explain its ability to selectively enhance

  6. Cardiovascular selectivity of adenosine receptor agonists in anaesthetized dogs.

    PubMed Central

    Gerencer, R. Z.; Finegan, B. A.; Clanachan, A. S.

    1992-01-01

    1. In order to determine the relevance of adenosine (Ado) receptor classification obtained from in vitro methods to the cardiovascular actions of Ado agonists in vivo, the cardiovascular effects of adenosine 5'-monophosphate (AMP), N6-cyclohexyladenosine (CHA, 400 fold A1-selective), 5'-N-ethyl-carboxamidoadenosine (NECA, A1 approximately A2) and 2-phenylaminoadenosine (PAA, 5 fold A2-selective) were compared in open-chest, fentanyl-pentobarbitone anaesthetized dogs. 2. Graded doses of CHA (10 to 1000 micrograms kg-1), NECA (0.5 to 100 micrograms kg-1) or PAA (0.1 to 20 micrograms kg-1) were administered intravenously and changes in haemodynamics and myocardial contractility were assessed 10 min following each dose. The effects of graded infusions of AMP (200 to 1000 micrograms kg-1 min-1) were also evaluated. 3. AMP and each of the Ado analogues (NECA > PAA > CHA) increased the systemic vascular conductance index (SVCI) in a dose-dependent manner and reduced mean arterial pressure (MAP). At doses causing similar increases in SVCI, these agonists caused (i) similar reflex increases in heart rate (HR) and cardiac index (CI) and decreases in AV conduction interval (AVi) and (ii) similar increases in coronary vascular conductance (CVC). 4. After cardiac autonomic blockade with atropine (0.2 mg kg-1) and propranolol (1 mg kg-1), AMP, CHA and PAA still increased SVCI and CVC and decreased MAP. CHA and PAA had no marked effects on HR, CI or AVi. As in the absence of cardiac autonomic blockade, equieffective vasodilator doses of CHA and PAA had identical effects on CVC, CI and AVi.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1467827

  7. Sequential Co-immunoprecipitation and Immunoblot Approach to Determine Oligomerisation of G-Protein-Coupled Receptors.

    PubMed

    Guest, Paul C

    2017-01-01

    G-protein-coupled receptors (GPCRs) play a major role in psychiatric disorders and are the targets of several current therapeutic approaches in this field. A number of studies have now shown that GPCRs can assemble as high molecular weight homo- and hetero-oligomers, which could affect ligand binding, intracellular signalling or trafficking. This information could be critical in design of new drugs to treat neurological and psychiatric disorders. This chapter describes a sequential co-immunoprecipitation and immunoblot protocol for determining oligomerisation of the 5-hydroxytryptamine (HT)1A receptor with other GPCRs in co-transfected HEK-293 cells.

  8. Understanding the added value of g-protein-coupled receptor heteromers.

    PubMed

    Franco, Nuria; Franco, Rafael

    2014-01-01

    G-protein-coupled receptors (GPCRs) constitute the most populated family of proteins within the human genome. Since the early sixties work on GPCRs and on GPCR-mediated signaling has led to a number of awards, the most recent being the Nobel Prize in Chemistry for 2012. The future of GPCRs research is surely based on their capacity for heteromerization. Receptor heteromers offer a series of challenges that will help in providing success in academic/basic research and translation into more effective and safer drugs.

  9. Sf9 cells: a versatile model system to investigate the pharmacological properties of G protein-coupled receptors.

    PubMed

    Schneider, Erich H; Seifert, Roland

    2010-12-01

    The Sf9 cell/baculovirus expression system is widely used for high-level protein expression, often with the purpose of purification. However, proteins may also be functionally expressed in the defined Sf9 cell environment. According to the literature, the pharmacology of G-protein-coupled receptors (GPCRs) functionally reconstituted in Sf9 cells is similar to the receptor properties in mammalian cells. Sf9 cells express both recombinant GPCRs and G-proteins at much higher levels than mammalian cells. Sf9 cells can be grown in suspension culture, providing an inexpensive way of obtaining large protein amounts. Co-infection with various baculoviruses allows free combination of GPCRs with different G-proteins. The absence of constitutively active receptors in Sf9 cells provides an excellent signal-to background ratio in functional assays, allowing the detection of agonist-independent receptor activity and of small ligand-induced signals including partial agonistic and inverse agonistic effects. Insect cell Gα(i)-like proteins mostly do not couple productively to mammalian GPCRs. Thus, unlike in mammalian cells, Sf9 cells do not require pertussis toxin treatment to obtain a Gα(i)-free environment. Co-expression of GPCRs with Gα(i1), Gα(i2), Gα(i3) or Gα(o) in Sf9 cells allows the generation of a selectivity profile for these Gα(i/o)-isoforms. Additionally, GPCR-G-protein combinations can be compared with defined 1:1 stoichiometry by expressing GPCR-Gα fusion proteins. Sf9 cells can also be employed for ligand screening in medicinal chemistry programs, using radioligand binding assays or functional assays, like the steady-state GTPase- or [(35)S]GTPγS binding assay. This review shows that Sf9 cells are a versatile model system to investigate the pharmacological properties of GPCRs.

  10. Vaginal ring delivery of selective progesterone receptor modulators for contraception

    PubMed Central

    Jensen, Jeffrey T.

    2013-01-01

    Vaginal ring delivery of selective progesterone receptor modulators (SPRMs) are under development to address limitations of current hormonal methods that affect use and effectiveness. This method would be appropriate for use in women with contraindications to, or preferences to avoid, estrogens. A contraceptive vaginal ring (CVR) also eliminates the need for daily dosing, and therefore might improve the effectiveness of contraception. The principle contraceptive effect of SPRMs is the suppression of ovulation. One limiting factor of chronic SPRM administration is the development of benign endometrial thickening characterized as PRM-associated endometrial changes. Ulipristal acetate is approved for use as an emergency contraceptive pill, but no SPRM is approved for regular contraception. The Population Council is developing an ulipristal acetate CVR for regular contraception. The CVR studied is of a matrix design composed of micronized UPA mixed in a silicone rubber matrix The target product is a ring designed for continuous use over 3 months delivering near steady-state drug levels that will suppress ovulation. Results from Phase 1–2 studies demonstrate that suppression of ovulation occurs with UPA levels above 6–7 ng/mL. PMID:23040126

  11. [Cardiovascular effects of selective estrogen receptor modulators. Current perspectives].

    PubMed

    Simoncini, Tommaso; Mannella, Paolo; Genazzani, Andrea R

    2003-02-01

    The use of hormone replacement therapy (HRT) after the menopause for the prevention of the long-term complications of estrogen deprivation has recently been questioned after the publication of large clinical trials that failed to show benefits for postmenopausal women. Although these trials risk to dump the widespread opinion of the cardioprotective effects of long-term estrogen use, they have many pitfalls that prevent a direct clinical application of these negative results. Furthermore, the large amount of epidemiological and experimental evidence indicating estrogens as protective on the vascular system cannot be ignored, and efforts should be devoted to understand the reasons for the discrepancy of results of these recent large trials. In the meanwhile, different molecules should be studied in depth as for the actions on the cardiovascular system, and their specific mechanisms of actions should be elucidated. Selective estrogen receptor modulators (SERM) are a promising family of molecules and some of these compounds have positive effects on cardiovascular risk parameters as well as on vascular cells. Large trials are ongoing to study the impact of these substances on cardiovascular risk, and the near future should provide us with answers on the possible use of SERM as possible safer alternatives to HRT for the long-term prevention of cardiovascular disease in postmenopausal women.

  12. Vaginal ring delivery of selective progesterone receptor modulators for contraception.

    PubMed

    Jensen, Jeffrey T

    2013-03-01

    Vaginal ring delivery of selective progesterone receptor modulators (SPRMs) is under development to address the limitations of current hormonal methods that affect use and effectiveness. This method would be appropriate for use in women with contraindications to, or preferences to avoid, estrogens. A contraceptive vaginal ring (CVR) also eliminates the need for daily dosing and therefore might improve the effectiveness of contraception. The principal contraceptive effect of SPRMs is the suppression of ovulation. One limiting factor of chronic SPRM administration is the development of benign endometrial thickening characterized as PRM-associated endometrial changes. Ulipristal acetate (UPA) is approved for use as an emergency contraceptive pill, but no SPRM is approved for regular contraception. The Population Council is developing an ulipristal acetate CVR for regular contraception. The CVR studied is of a matrix design composed of micronized UPA mixed in a silicone rubber matrix The target product is a ring designed for continuous use over 3 months delivering near steady-state drug levels that will suppress ovulation. Results from Phase 1 and 2 studies demonstrate that suppression of ovulation occurs with UPA levels above 6-7 ng/mL.

  13. Prolactin Receptor Coupling to Jak-Stat Pathways in Breast Cancer

    DTIC Science & Technology

    2007-07-01

    receptors (PRLR) have been considered selective activators of tyrosine kinase Jak2 but not Jak1, Jak3 or Tyk2. We now report marked PRL-induced tyrosine...been considered selective activators of tyrosine kinase Jak2 but not Jak1, Jak3 or Tyk2. We now report marked PRL-induced tyrosine phosphorylation...other members of the Janus kinase family, Jak1, Jak3 or Tyk2 (2-5). Upon PRL-induced receptor aggregation, activated Jak2 in turn phosphorylates

  14. Selective agonists and antagonists of formylpeptide receptors: duplex flow cytometry and mixture-based positional scanning libraries.

    PubMed

    Pinilla, Clemencia; Edwards, Bruce S; Appel, Jon R; Yates-Gibbins, Tina; Giulianotti, Marc A; Medina-Franco, Jose L; Young, Susan M; Santos, Radleigh G; Sklar, Larry A; Houghten, Richard A

    2013-09-01

    The formylpeptide receptor (FPR1) and formylpeptide-like 1 receptor (FPR2) are G protein-coupled receptors that are linked to acute inflammatory responses, malignant glioma stem cell metastasis, and chronic inflammation. Although several N-formyl peptides are known to bind to these receptors, more selective small-molecule, high-affinity ligands are needed for a better understanding of the physiologic roles played by these receptors. High-throughput assays using mixture-based combinatorial libraries represent a unique, highly efficient approach for rapid data acquisition and ligand identification. We report the superiority of this approach in the context of the simultaneous screening of a diverse set of mixture-based small-molecule libraries. We used a single cross-reactive peptide ligand for a duplex flow cytometric screen of FPR1 and FPR2 in color-coded cell lines. Screening 37 different mixture-based combinatorial libraries totaling more than five million small molecules (contained in 5,261 mixture samples) resulted in seven libraries that significantly inhibited activity at the receptors. Using positional scanning deconvolution, selective high-affinity (low nM K(i)) individual compounds were identified from two separate libraries, namely, pyrrolidine bis-diketopiperazine and polyphenyl urea. The most active individual compounds were characterized for their functional activities as agonists or antagonists with the most potent FPR1 agonist and FPR2 antagonist identified to date with an EC₅₀ of 131 nM (4 nM K(i)) and an IC₅₀ of 81 nM (1 nM K(i)), respectively, in intracellular Ca²⁺ response determinations. Comparative analyses of other previous screening approaches clearly illustrate the efficiency of identifying receptor selective, individual compounds from mixture-based combinatorial libraries.

  15. Rapid Computational Prediction of Thermostabilizing Mutations for G Protein-Coupled Receptors

    PubMed Central

    2015-01-01

    G protein-coupled receptors (GPCRs) are highly dynamic and often denature when extracted in detergents. Deriving thermostable mutants has been a successful strategy to stabilize GPCRs in detergents, but this process is experimentally tedious. We have developed a computational method to predict the position of the thermostabilizing mutations for a given GPCR sequence. We have validated the method against experimentally measured thermostability data for single mutants of the β1-adrenergic receptor (β1AR), adenosine A2A receptor (A2AR) and neurotensin receptor 1 (NTSR1). To make these predictions we started from homology models of these receptors of varying accuracies and generated an ensemble of conformations by sampling the rigid body degrees of freedom of transmembrane helices. Then, an all-atom force field function was used to calculate the enthalpy gain, known as the “stability score” upon mutation of every residue, in these receptor structures, to alanine. For all three receptors, β1AR, A2AR, and NTSR1, we observed that mutations of hydrophobic residues in the transmembrane domain to alanine that have high stability scores correlate with high experimental thermostability. The prediction using the stability score improves when using an ensemble of receptor conformations compared to a single structure, showing that receptor flexibility is important. We also find that our previously developed LITiCon method for generating conformation ensembles is similar in performance to predictions using ensembles obtained from microseconds of molecular dynamics simulations (which is computationally hundred times slower than LITiCon). We improved the thermostability prediction by including other properties such as residue-based stress and the extent of allosteric communication by each residue in the stability score. Our method is the first step toward a computational method for rapid prediction of thermostable mutants of GPCRs. PMID:25400524

  16. The recent progress in research on effects of anesthetics and analgesics on G protein-coupled receptors.

    PubMed

    Minami, Kouichiro; Uezono, Yasuhito

    2013-04-01

    The exact mechanisms of action behind anesthetics and analgesics are still unclear. Much attention was focused on ion channels in the central nervous system as targets for anesthetics and analgesics in the 1980s. During the 1990s, major advances were made in our understanding of the physiology and pharmacology of G protein coupled receptor (GPCR) signaling. Thus, several lines of studies have shown that G protein coupled receptors (GPCRs) are one of the targets for anesthetics and analgesics and especially, that some of them inhibit the functions of GPCRs, i.e,, muscarinic receptors and substance P receptors. However, these studies had been focused on only G(q) coupled receptors. There has been little work on G(s)- and G(i)-coupled receptors. In the last decade, a new assay system, using chimera G(i/o)-coupled receptor fused to Gq(i5), has been established and the effects of anesthetics and analgesics on the function of G(i)-coupled receptors is now more easily studied. This review highlights the recent progress of the studies regarding the effects of anesthetics and analgesics on GPCRs.

  17. Allosteric mechanisms of G protein coupled receptor signaling: a structural perspective

    PubMed Central

    Thaker, Tarjani M.; Kaya, Ali I.; Preininger, Anita M.; Hamm, Heidi E.; Iverson, T.M.

    2012-01-01

    G protein-Coupled Receptors (GPCRs) use a complex series of intramolecular conformational changes to couple agonist binding to the binding and activation of cognate heterotrimeric G protein (Gαβγ). The mechanisms underlying this long-range activation have been identified using a variety of biochemical and structural approaches and have primarily used visual signal transduction via the GPCR rhodopsin and cognate heterotrimeric G protein transducin (Gt) as a model system. In this chapter, we will review the methods that have revealed allosteric signaling through rhodopsin and transducin. These methods can be applied to a variety of GPCR-mediated signaling pathways. PMID:22052489

  18. GABA(B2) is essential for g-protein coupling of the GABA(B) receptor heterodimer.

    PubMed

    Robbins, M J; Calver, A R; Filippov, A K; Hirst, W D; Russell, R B; Wood, M D; Nasir, S; Couve, A; Brown, D A; Moss, S J; Pangalos, M N

    2001-10-15

    GABA(B) receptors are unique among G-protein-coupled receptors (GPCRs) in their requirement for heterodimerization between two homologous subunits, GABA(B1) and GABA(B2), for functional expression. Whereas GABA(B1) is capable of binding receptor agonists and antagonists, the role of each GABA(B) subunit in receptor signaling is unknown. Here we identified amino acid residues within the second intracellular domain of GABA(B2) that are critical for the coupling of GABA(B) receptor heterodimers to their downstream effector systems. Our results provide strong evidence for a functional role of the GABA(B2) subunit in G-protein coupling of the GABA(B) receptor heterodimer. In addition, they provide evidence for a novel "sequential" GPCR signaling mechanism in which ligand binding to one heterodimer subunit can induce signal transduction through the second partner of a heteromeric complex.

  19. An algebra of dimerization and its implications for G-protein coupled receptor signaling.

    PubMed

    Woolf, Peter J; Linderman, Jennifer J

    2004-07-21

    Many species of receptors form dimers, but how can we use this information to make predictions about signal transduction? This problem is particularly difficult when receptors dimerize with many different species, leading to a combinatoric increase in the possible number of dimer pairs. As an example system, we focus on receptors in the G-protein coupled receptor (GPCR) family. GPCRs have been shown to reversibly form dimers, but this dimerization does not directly affect signal transduction. Here we present a new theoretical framework called a dimerization algebra. This algebra provides a systematic and rational way to represent, manipulate, and in some cases simplify large and often complicated networks of dimerization interactions. To compliment this algebra, Monte Carlo simulations are used to predict dimerization's effect on receptor organization on the membrane, signal transduction, and internalization. These simulation results are directly comparable to various experimental measures such as fluorescence resonance energy transfer (FRET), and as such provide a link between the dimerization algebra and experimental data. As an example, we show how the algebra and computational results can be used to predict the effects of dimerization on the dopamine D2 and somatastatin SSTR1 receptors. When these predictions were compared to experimental findings from the literature, good agreement was found, demonstrating the utility of our approach. Applications of this work to the development of a novel class of dimerization-modulating drugs are also discussed.

  20. Molecular cloning and characterisation of a novel GABAB-related G-protein coupled receptor.

    PubMed

    Calver, A R; Michalovich, D; Testa, T T; Robbins, M J; Jaillard, C; Hill, J; Szekeres, P G; Charles, K J; Jourdain, S; Holbrook, J D; Boyfield, I; Patel, N; Medhurst, A D; Pangalos, M N

    2003-02-20

    Using a homology-based bioinformatics approach we have analysed human genomic sequence and identified the human and rodent orthologues of a novel putative seven transmembrane G protein coupled receptor, termed GABA(BL). The amino acid sequence homology of these cDNAs compared to GABA(B1) and GABA(B2) led us to postulate that GABA(BL) was a putative novel GABA(B) receptor subunit. The C-terminal sequence of GABA(BL) contained a putative coiled-coil domain, di-leucine and several RXR(R) ER retention motifs, all of which have been shown to be critical in GABA(B) receptor subunit function. In addition, the distribution of GABA(BL) in the central nervous system was reminiscent of that of the other known GABA(B) subunits. However, we were unable to detect receptor function in response to any GABA(B) ligands when GABA(BL) was expressed in isolation or in the presence of either GABA(B1) or GABA(B2). Therefore, if GABA(BL) is indeed a GABA(B) receptor subunit, its partner is a potentially novel receptor subunit or chaperone protein which has yet to be identified.

  1. Microwave Kinetic Inductance Detector with Selective Polarization Coupling

    NASA Technical Reports Server (NTRS)

    Wollack, Edward; U-yen, Kongpop; Stevenson, Thomas; Brown, Ari; Moseley, Samuel; Hsieh, Wen-Ting

    2013-01-01

    A conventional low-noise detector requires a technique to both absorb incident power and convert it to an electrical signal at cryogenic temperatures. This innovation combines low-noise detector and readout functionality into one device while maintaining high absorption, controlled polarization sensitivity, and broadband detection capability. The resulting far-infrared detectors can be read out with a simple approach, which is compact and minimizes thermal loading. The proposed microwave kinetic inductance detector (MKID) consists of three basic elements. The first is the absorptive section in which the incident power is coupled to a superconducting resonator at far-infrared frequency above its superconducting critical frequency (where superconductor becomes normal conductor). This absorber's shape effectively absorbs signals in the desired polarization state and is resonant at the radio frequency (RF) used for readout of the device. Control over the metal film used in the absorber allows realization of structures with either a 50% broadband or 100% resonance absorptance over a 30% fractional bandwidth. The second element is a microwave resonator - which is realized from the thin metal films used to make the absorber as transmission lines - whose resonance frequency changes due to a variation in its kinetic inductance. The resonator's kinetic inductance is a function of the power absorbed by the device. A low-loss dielectric (mono-crystalline silicon) is used in a parallel-plate transmission line structure to realize the desired superconducting resonators. There is negligible coupling among the adjacent elements used to define the polarization sensitivity of each detector. The final component of the device is a microwave transmission line, which is coupled to the resonator, and allows detection of changes in resonance frequency for each detector in the focal plane array. The spiral shape of the detector's absorber allows incident power with two polarizations to

  2. An elevation in physical coupling of type 1 inositol 1,4,5-trisphosphate (IP3) receptors to transient receptor potential 3 (TRPC3) channels constricts mesenteric arteries in genetic hypertension.

    PubMed

    Adebiyi, Adebowale; Thomas-Gatewood, Candice M; Leo, M Dennis; Kidd, Michael W; Neeb, Zachary P; Jaggar, Jonathan H

    2012-11-01

    Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C-coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP(3)) that activates sarcoplasmic reticulum IP(3) receptors. In cerebral artery myocytes, IP(3) receptors release sarcoplasmic reticulum Ca(2+) and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP(3) receptors control vascular contractility in systemic arteries and IP(3)R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ≈2.7- and 2-fold higher in mesenteric arteries of spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP(3)R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-SHRs and WKY rats. Control, IP(3)-induced and endothelin-1 (ET-1)-induced fluorescence resonance energy transfer between IP3R1 and TRPC3 was higher in SHR than WKY myocytes. IP3-induced cation current was ≈3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and calmodulin and IP(3) receptor binding domain peptide, an IP(3)R-TRP physical coupling inhibitor, reduced IP(3)-induced cation current and ET-1-induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a sarcoplasmic reticulum Ca(2+)-ATPase blocker, did not alter ET-1-stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP(3)R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP(3)R1 to TRPC3 channel molecular coupling augments

  3. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    PubMed Central

    Lynch, Jennifer R.; Wang, Jenny Yingzi

    2016-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies. PMID:27187360

  4. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

    PubMed

    Lynch, Jennifer R; Wang, Jenny Yingzi

    2016-05-11

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  5. A Coupled Phase-Temperature Model for Dynamics of Transient Neuronal Signal in Mammals Cold Receptor

    PubMed Central

    Kirana, Firman Ahmad; Husein, Irzaman Sulaiman

    2016-01-01

    We propose a theoretical model consisting of coupled differential equation of membrane potential phase and temperature for describing the neuronal signal in mammals cold receptor. Based on the results from previous work by Roper et al., we modified a nonstochastic phase model for cold receptor neuronal signaling dynamics in mammals. We introduce a new set of temperature adjusted functional parameters which allow saturation characteristic at high and low steady temperatures. The modified model also accommodates the transient neuronal signaling process from high to low temperature by introducing a nonlinear differential equation for the “effective temperature” changes which is coupled to the phase differential equation. This simple model can be considered as a candidate for describing qualitatively the physical mechanism of the corresponding transient process. PMID:27774102

  6. Giα and Gβ subunits both define selectivity of G protein activation by α2-adrenergic receptors

    PubMed Central

    Gibson, Scott K.; Gilman, Alfred G.

    2006-01-01

    Previous studies of the specificity of receptor interactions with G protein subunits in living cells have relied on measurements of second messengers or other downstream responses. We have examined the selectivity of interactions between α2-adrenergic receptors (α2R) and various combinations of Giα and Gβ subunit isoforms by measuring changes in FRET between Giα–yellow fluorescent protein and cyan fluorescent protein–Gβ chimeras in HeLa cells. All combinations of Giα1, -2, or -3 with Gβ1, -2, or -4 were activated to some degree by endogenous α2Rs as judged by agonist-dependent decreases in FRET. The degree of G protein activation is determined by the combination of Giα and Gβ subunits rather than by the identity of an individual subunit. RT-PCR analysis and small interfering RNA knockdown of α2R subtypes, followed by quantification of radiolabeled antagonist binding, demonstrated that HeLa cells express α2a- and α2b-adrenergic receptor isoforms in a 2:1 ratio. Increasing receptor number by overexpression of the α2aR subtype minimized the differences among coupling preferences for Giα and Gβ isoforms. The molecular properties of each Giα, Gβ, and α2-adrenergic receptor subtype influence signaling efficiency for the α2-adrenergic receptor-mediated signaling pathway. PMID:16371464

  7. Computational Approaches for Decoding Select Odorant-Olfactory Receptor Interactions Using Mini-Virtual Screening

    PubMed Central

    Harini, K.; Sowdhamini, Ramanathan

    2015-01-01

    Olfactory receptors (ORs) belong to the class A G-Protein Coupled Receptor superfamily of proteins. Unlike G-Protein Coupled Receptors, ORs exhibit a combinatorial response to odors/ligands. ORs display an affinity towards a range of odor molecules rather than binding to a specific set of ligands and conversely a single odorant molecule may bind to a number of olfactory receptors with varying affinities. The diversity in odor recognition is linked to the highly variable transmembrane domains of these receptors. The purpose of this study is to decode the odor-olfactory receptor interactions using in silico docking studies. In this study, a ligand (odor molecules) dataset of 125 molecules was used to carry out in silico docking using the GLIDE docking tool (SCHRODINGER Inc Pvt LTD). Previous studies, with smaller datasets of ligands, have shown that orthologous olfactory receptors respond to similarly-tuned ligands, but are dramatically different in their efficacy and potency. Ligand docking results were applied on homologous pairs (with varying sequence identity) of ORs from human and mouse genomes and ligand binding residues and the ligand profile differed among such related olfactory receptor sequences. This study revealed that homologous sequences with high sequence identity need not bind to the same/ similar ligand with a given affinity. A ligand profile has been obtained for each of the 20 receptors in this analysis which will be useful for expression and mutation studies on these receptors. PMID:26221959

  8. Consequences of splice variation on Secretin family G protein-coupled receptor function

    PubMed Central

    Furness, Sebastian GB; Wootten, Denise; Christopoulos, Arthur; Sexton, Patrick M

    2012-01-01

    The Secretin family of GPCRs are endocrine peptide hormone receptors that share a common genomic organization and are the subject of a wide variety of alternative splicing. All GPCRs contain a central seven transmembrane domain responsible for transducing signals from the outside of the cell as well as extracellular amino and intracellular carboxyl termini. Members of the Secretin receptor family have a relatively large N-terminus and a variety of lines of evidence support a common mode of ligand binding and a common ligand binding fold. These receptors are best characterized as coupling to intracellular signalling pathways via Gαs and Gαq but are also reported to couple to a multitude of other signalling pathways. The intracellular loops are implicated in regulating the interaction between the receptor and heterotrimeric G protein complexes. Alternative splicing of exons encoding both the extracellular N-terminal domain as well as the extracellular loops of some family members has been reported and as expected these splice variants display altered ligand affinity as well as differential activation by endogenous ligands. Various forms of alternative splicing have also been reported to alter intracellular loops 1 and 3 as well as the C-terminus and as one might expect these display differences in signalling bias towards downstream effectors. These diverse pharmacologies require that the physiological role of these splice variants be addressed but should provide unique opportunities for drug design and development. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1 PMID:21718310

  9. G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor*S⃞

    PubMed Central

    Namkung, Yoon; Dipace, Concetta; Javitch, Jonathan A.; Sibley, David R.

    2009-01-01

    We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D2 dopamine receptor (DAR). Agonist activation of D2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [35S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D2 DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor. PMID:19332542

  10. G Protein-Coupled Receptors in cancer: biochemical interactions and drug design.

    PubMed

    Audigier, Yves; Picault, François-Xavier; Chaves-Almagro, Carline; Masri, Bernard

    2013-01-01

    G Protein-Coupled Receptors (GPCRs) share the same topology made of seven-transmembrane segments and represent the largest family of membrane receptors. Initially associated with signal transduction in differentiated cells, GPCRs and heterotrimeric G proteins were shown to behave as proto-oncogenes whose overexpression or activating mutations confer transforming properties. The first part of this review focuses on the link between biochemical interactions of a GPCR with other receptors, such as dimerization or multiprotein complexes, and their oncogenic properties. Alteration of these interactions or deregulation of transduction cascades can promote uncontrolled cell proliferation or cell transformation that leads to tumorigenicity and malignancy. The second part concerns the design of drugs specifically targeting these complex interactions and their promise in cancer therapy.

  11. From Atomic Structures to Neuronal Functions of G Protein–Coupled Receptors

    PubMed Central

    Palczewski, Krzysztof; Orban, Tivadar

    2015-01-01

    G protein–coupled receptors (GPCRs) are essential mediators of signal transduction, neurotransmission, ion channel regulation, and other cellular events. GPCRs are activated by diverse stimuli, including light, enzymatic processing of their N-termini, and binding of proteins, peptides, or small molecules such as neurotransmitters. GPCR dysfunction caused by receptor mutations and environmental challenges contributes to many neurological diseases. Moreover, modern genetic technology has helped identify a rich array of mono- and multigenic defects in humans and animal models that connect such receptor dysfunction with disease affecting neuronal function. The visual system is especially suited to investigate GPCR structure and function because advanced imaging techniques permit structural studies of photoreceptor neurons at both macro and molecular levels that, together with biochemical and physiological assessment in animal models, provide a more complete understanding of GPCR signaling. PMID:23682660

  12. G-protein-coupled receptors in control of natural killer cell migration.

    PubMed

    Walzer, Thierry; Vivier, Eric

    2011-10-01

    Natural killer (NK) cells are highly motile cells that patrol lymphoid and non-lymphoid organs, and are poised to react to infectious or other inflammatory situations. Several NK cell subsets equipped with different sets of chemotactic G-protein-coupled receptors, and which display distinct distribution across lymphoid and non-lymphoid organs, have been described. These receptors detect various guidance cues including sphingosine-1-phosphate and chemokines that orchestrate NK cell trafficking. Here, we highlight recent advances regarding the receptors involved in NK cell migration, with a focus on bone marrow egress, entry into activated lymph nodes, extravasation into inflamed tissues, and motility within lymph nodes or tumors. Understanding NK cell migration could provide a rational basis for the design of novel therapies in various clinical conditions.

  13. G protein-coupled receptor 56 and collagen III, a receptor-ligand pair, regulates cortical development and lamination.

    PubMed

    Luo, Rong; Jeong, Sung-Jin; Jin, Zhaohui; Strokes, Natalie; Li, Shihong; Piao, Xianhua

    2011-08-02

    GPR56, an orphan G protein-coupled receptor (GPCR) from the family of adhesion GPCRs, plays an indispensable role in cortical development and lamination. Mutations in the GPR56 gene cause a malformed cerebral cortex in both humans and mice that resembles cobblestone lissencephaly, which is characterized by overmigration of neurons beyond the pial basement membrane. However, the molecular mechanisms through which GPR56 regulates cortical development remain elusive due to the unknown status of its ligand. Here we identify collagen, type III, alpha-1 (gene symbol Col3a1) as the ligand of GPR56 through an in vitro biotinylation/proteomics approach. Further studies demonstrated that Col3a1 null mutant mice exhibit overmigration of neurons beyond the pial basement membrane and a cobblestone-like cortical malformation similar to the phenotype seen in Gpr56 null mutant mice. Functional studies suggest that the interaction of collagen III with its receptor GPR56 inhibits neural migration in vitro. As for intracellular signaling, GPR56 couples to the Gα(12/13) family of G proteins and activates RhoA pathway upon ligand binding. Thus, collagen III regulates the proper lamination of the cerebral cortex by acting as the major ligand of GPR56 in the developing brain.

  14. Quantitative structure-activity relationships of selective antagonists of glucagon receptor using QuaSAR descriptors.

    PubMed

    Manoj Kumar, Palanivelu; Karthikeyan, Chandrabose; Hari Narayana Moorthy, Narayana Subbiah; Trivedi, Piyush

    2006-11-01

    In the present paper, quantitative structure activity relationship (QSAR) approach was applied to understand the affinity and selectivity of a novel series of triaryl imidazole derivatives towards glucagon receptor. Statistically significant and highly predictive QSARs were derived for glucagon receptor inhibition by triaryl imidazoles using QuaSAR descriptors of molecular operating environment (MOE) employing computer-assisted multiple regression procedure. The generated QSAR models revealed that factors related to hydrophobicity, molecular shape and geometry predominantly influences glucagon receptor binding affinity of the triaryl imidazoles indicating the relevance of shape specific steric interactions between the molecule and the receptor. Further, QSAR models formulated for selective inhibition of glucagon receptor over p38 mitogen activated protein (MAP) kinase of the compounds in the series highlights that the same structural features, which influence the glucagon receptor affinity, also contribute to their selective inhibition.

  15. Synthesis and Characterization of Tricarbonyl-Re/Tc(I) Chelate Probes Targeting the G Protein-Coupled Estrogen Receptor GPER/GPR30

    PubMed Central

    Burai, Ritwik; Ramesh, Chinnasamy; Nayak, Tapan K.; Dennis, Megan K.; Bryant, Bj K.; Prossnitz, Eric R.; Arterburn, Jeffrey B.

    2012-01-01

    The discovery of the G protein-coupled estrogen receptor GPER (also GPR30) and the resulting development of selective chemical probes have revealed new aspects of estrogen receptor biology. The potential clinical relevance of this receptor has been suggested from numerous studies that have identified GPER expression in breast, endometrial, ovarian and other cancers. Thus GPER can be considered a candidate biomarker and target for non-invasive imaging and therapy. We have designed and synthesized a series of organometallic tricarbonyl-rhenium complexes conjugated to a GPER-selective small molecule derived from tetrahydro-3H-cyclopenta[c]quinoline. The activity and selectivity of these chelates in GPER-mediated signaling pathways were evaluated. These results demonstrate that GPER targeting characteristics depend strongly on the structure of the chelate and linkage. Ethanone conjugates functioned as agonists, a 1,2,3-triazole spacer yielded an antagonist, and derivatives with increased steric volume exhibited decreased activities. Promising GPER selectivity was observed, as none of the complexes interacted with the nuclear estrogen receptors. Radiolabeling with technetium-99m in aqueous media was efficient and gave radioligands with high radiochemical yields and purity. These chelates have favorable physicochemical properties, show excellent stability in biologically relevant media, exhibit receptor specificity and are promising candidates for continuing development as diagnostic imaging agents targeting GPER expression in cancer. PMID:23077529

  16. Dynamics of the β2-adrenergic G-protein coupled receptor revealed by hydrogen-deuterium exchange

    PubMed Central

    Zhang, Xi; Chien, Ellen Y.T.; Chalmers, Michael J.; Pascal, Bruce D.; Gatchalian, Jovylyn; Stevens, Raymond C.; Griffin, Patrick R.

    2010-01-01

    To examine the molecular details of ligand activation of G-protein coupled receptor (GPCRs), emphasis has been placed on structure determination of these receptors with stabilizing ligands. Here we present the methodology for receptor dynamics characterization of the GPCR human β2 adrenergic receptor bound to the inverse agonist carazolol using the technique of amide hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS). The HDX MS profile of receptor bound to carazolol is consistent with thermal parameter observations in the crystal structure and provides additional information in highly dynamic regions of the receptor and chemical modifications demonstrating the highly complementary nature of the techniques. Following optimization of HDX experimental conditions for this membrane protein, better than 89% sequence coverage was obtained for the receptor. The methodology presented paves the way for future analysis of β2AR bound to pharmacologically distinct ligands as well as analysis of other GPCR family members. PMID:20058880

  17. G-protein-coupled receptors in aldosterone-producing adenomas: a potential cause of hyperaldosteronism.

    PubMed

    Ye, Ping; Mariniello, Barbara; Mantero, Franco; Shibata, Hirotaka; Rainey, William E

    2007-10-01

    The source of aldosterone in 30-40% of patients with primary hyperaldosteronism (PA) is unilateral aldosterone-producing adenoma (APA). The mechanisms causing elevated aldosterone production in APA are unknown. Herein, we examined the expression of G-protein-coupled receptors (GPCRs) in APA and demonstrated that when compared with normal adrenals, there is a general elevation of certain GPCR in many APA and/or ectopic expression of GPCR in others. RNA samples from normal adrenals (n = 5), APAs (n = 10), and cortisol-producing adenomas (CPAs; n = 13) were used on 15 genomic expression arrays, each of which included 223 GPCR transcripts presented in at least 1 out of 15 of the independent microarrays. The array results were confirmed using real-time RT-PCR (qPCR). Four GPCR transcripts exhibited a statistically significant increase that was greater than threefold when compared with normal adrenals, suggesting a general increase in expression when compared with normal adrenal glands. Four GPCR transcripts exhibited a > 15-fold increase of expression in one or more of the APA samples when compared with normal adrenals. qPCR analysis confirmed array data and found the receptors with the highest fold increase in APA expression to be LH receptor, serotonin receptor 4, GnRH receptor, glutamate receptor metabotropic 3, endothelin receptor type B-like protein, and ACTH receptor. There are also sporadic increased expressions of these genes in the CPAs. Together, these findings suggest a potential role of altered GPCR expression in many cases of PA and provide candidate GPCR for further study.

  18. G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane

    SciTech Connect

    Funakoshi, Takeshi; Yanai, Akie; Shinoda, Koh; Kawano, Michio M.; Mizukami, Yoichi . E-mail: mizukami@yamaguchi-u.ac.jp

    2006-08-04

    Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17{beta}-estradiol or E2) causes an elevation in the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain.

  19. GPR133 (ADGRD1), an adhesion G-protein-coupled receptor, is necessary for glioblastoma growth

    PubMed Central

    Bayin, N S; Frenster, J D; Kane, J R; Rubenstein, J; Modrek, A S; Baitalmal, R; Dolgalev, I; Rudzenski, K; Scarabottolo, L; Crespi, D; Redaelli, L; Snuderl, M; Golfinos, J G; Doyle, W; Pacione, D; Parker, E C; Chi, A S; Heguy, A; MacNeil, D J; Shohdy, N; Zagzag, D; Placantonakis, D G

    2016-01-01

    Glioblastoma (GBM) is a deadly primary brain malignancy with extensive intratumoral hypoxia. Hypoxic regions of GBM contain stem-like cells and are associated with tumor growth and angiogenesis. The molecular mechanisms that regulate tumor growth in hypoxic conditions are incompletely understood. Here, we use primary human tumor biospecimens and cultures to identify GPR133 (ADGRD1), an orphan member of the adhesion family of G-protein-coupled receptors, as a critical regulator of the response to hypoxia and tumor growth in GBM. GPR133 is selectively expressed in CD133+ GBM stem cells (GSCs) and within the hypoxic areas of PPN in human biospecimens. GPR133 mRNA is transcriptionally upregulated by hypoxia in hypoxia-inducible factor 1α (Hif1α)-dependent manner. Genetic inhibition of GPR133 with short hairpin RNA reduces the prevalence of CD133+ GSCs, tumor cell proliferation and tumorsphere formation in vitro. Forskolin rescues the GPR133 knockdown phenotype, suggesting that GPR133 signaling is mediated by cAMP. Implantation of GBM cells with short hairpin RNA-mediated knockdown of GPR133 in the mouse brain markedly reduces tumor xenograft formation and increases host survival. Analysis of the TCGA data shows that GPR133 expression levels are inversely correlated with patient survival. These findings indicate that GPR133 is an important mediator of the hypoxic response in GBM and has significant protumorigenic functions. We propose that GPR133 represents a novel molecular target in GBM and possibly other malignancies where hypoxia is fundamental to pathogenesis. PMID:27775701

  20. Fulfilling the Promise of "Biased" G Protein–Coupled Receptor Agonism

    PubMed Central

    Maudsley, Stuart; Bohn, Laura M.

    2015-01-01

    The fact that over 30% of current pharmaceuticals target heptahelical G protein–coupled receptors (GPCRs) attests to their tractability as drug targets. Although GPCR drug development has traditionally focused on conventional agonists and antagonists, the growing appreciation that GPCRs mediate physiologically relevant effects via both G protein and non–G protein effectors has prompted the search for ligands that can "bias" downstream signaling in favor of one or the other process. Biased ligands are novel entities with distinct signaling profiles dictated by ligand structure, and the potential prospect of biased ligands as better drugs has been pleonastically proclaimed. Indeed, preclinical proof-of-concept studies have demonstrated that both G protein and arrestin pathway-selective ligands can promote beneficial effects in vivo while simultaneously antagonizing deleterious ones. But along with opportunity comes added complexity and new challenges for drug discovery. If ligands can be biased, then ligand classification becomes assay dependent, and more nuanced screening approaches are needed to capture ligand efficacy across several dimensions of signaling. Moreover, because the signaling repertoire of biased ligands differs from that of the native agonist, unpredicted responses may arise in vivo as these unbalanced signals propagate. For any given GPCR target, establishing a framework relating in vitro efficacy to in vivo biologic response is crucial to biased drug discovery. This review discusses approaches to describing ligand efficacy in vitro, translating ligand bias into biologic response, and developing a systems-level understanding of biased agonism in vivo, with the overall goal of overcoming current barriers to developing biased GPCR therapeutics. PMID:26134495

  1. Membrane-Mediated Oligomerization of G Protein Coupled Receptors and Its Implications for GPCR Function

    PubMed Central

    Gahbauer, Stefan; Böckmann, Rainer A.

    2016-01-01

    The dimerization or even oligomerization of G protein coupled receptors (GPCRs) causes ongoing, controversial debates about its functional role and the coupled biophysical, biochemical or biomedical implications. A continously growing number of studies hints to a relation between oligomerization and function of GPCRs and strengthens the assumption that receptor assembly plays a key role in the regulation of protein function. Additionally, progress in the structural analysis of GPCR-G protein and GPCR-ligand interactions allows to distinguish between actively functional and non-signaling complexes. Recent findings further suggest that the surrounding membrane, i.e., its lipid composition may modulate the preferred dimerization interface and as a result the abundance of distinct dimeric conformations. In this review, the association of GPCRs and the role of the membrane in oligomerization will be discussed. An overview of the different reported oligomeric interfaces is provided and their capability for signaling discussed. The currently available data is summarized with regard to the formation of GPCR oligomers, their structures and dependency on the membrane microenvironment as well as the coupling of oligomerization to receptor function. PMID:27826255

  2. G protein-coupled receptors: bridging the gap from the extracellular signals to the Hippo pathway.

    PubMed

    Zhou, Xin; Wang, Zhen; Huang, Wei; Lei, Qun-Ying

    2015-01-01

    The Hippo pathway is crucial in organ size control, whereas its dysregulation contributes to organ degeneration or tumorigenesis. The kinase cascade of MST1/2 and LATS1/2 and the coupling transcription co-activators YAP/TAZ represent the core components of the Hippo pathway. Extensive studies have identified a number of upstream regulators of the Hippo pathway, including contact inhibition, mechanic stress, extracellular matrix stiffness, cytoskeletal rearrangement, and some molecules of cell polarity and cell junction. However, how the diffuse extracellular signals regulate the Hippo pathway puzzles the researchers for a long time. Unexpectedly, recent elegant studies demonstrated that stimulation of some G protein-coupled receptors (GPCRs), such as lysophosphatidic acid receptor, sphingosine-1-phosphate receptor, and the protease activated receptor PAR1, causes potent YAP/TAZ dephosphorylation and activation by promoting actin cytoskeleton assemble. In this review, we briefly describe the components of the Hippo pathway and focus on the recent progress with respect to the regulation of the Hippo pathway by GPCRs and G proteins in cancer cells. In addition, we also discuss the potential therapeutic roles targeting the Hippo pathway in human cancers.

  3. Recent Advances on the Role of G Protein-Coupled Receptors in Hypoxia-Mediated Signaling.

    PubMed

    Lappano, Rosamaria; Rigiracciolo, Damiano; De Marco, Paola; Avino, Silvia; Cappello, Anna Rita; Rosano, Camillo; Maggiolini, Marcello; De Francesco, Ernestina Marianna

    2016-03-01

    G protein-coupled receptors (GPCRs) are cell surface proteins mainly involved in signal transmission; however, they play a role also in several pathophysiological conditions. Chemically heterogeneous molecules like peptides, hormones, lipids, and neurotransmitters activate second messengers and induce several biological responses by binding to these seven transmembrane receptors, which are coupled to heterotrimeric G proteins. Recently, additional molecular mechanisms have been involved in GPCR-mediated signaling, leading to an intricate network of transduction pathways. In this regard, it should be mentioned that diverse GPCR family members contribute to the adaptive cell responses to low oxygen tension, which is a distinguishing feature of several illnesses like neoplastic and cardiovascular diseases. For instance, the G protein estrogen receptor, namely G protein estrogen receptor (GPER)/GPR30, has been shown to contribute to relevant biological effects induced by hypoxia via the hypoxia-inducible factor (HIF)-1α in diverse cell contexts, including cancer. Likewise, GPER has been found to modulate the biological outcome of hypoxic/ischemic stress in both cardiovascular and central nervous systems. Here, we describe the role exerted by GPCR-mediated signaling in low oxygen conditions, discussing, in particular, the involvement of GPER by a hypoxic microenvironment.

  4. Quantitative Signaling and Structure-Activity Analyses Demonstrate Functional Selectivity at the Nociceptin/Orphanin FQ Opioid Receptor

    PubMed Central

    Chang, Steven D.; Mascarella, S. Wayne; Spangler, Skylar M.; Gurevich, Vsevolod V.; Navarro, Hernan A.; Carroll, F. Ivy

    2015-01-01

    Comprehensive studies that consolidate selective ligands, quantitative comparisons of G protein versus arrestin-2/3 coupling, together with structure-activity relationship models for G protein–coupled receptor (GPCR) systems are less commonly employed. Here we examine biased signaling at the nociceptin/orphanin FQ opioid receptor (NOPR), the most recently identified member of the opioid receptor family. Using real-time, live-cell assays, we identified the signaling profiles of several NOPR-selective ligands in upstream GPCR signaling (G protein and arrestin pathways) to determine their relative transduction coefficients and signaling bias. Complementing this analysis, we designed novel ligands on the basis of NOPR antagonist J-113,397 [(±)-1-[(3R*,4R*)-1-(cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] to explore structure-activity relationships. Our study shows that NOPR is capable of biased signaling, and further, the NOPR selective ligands MCOPPB [1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-(3R)-3-piperidinyl-1H-benzimidazole trihydrochloride] and NNC 63-0532 [8-(1-naphthalenylmethyl)-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decane-3-acetic acid, methyl ester] are G protein–biased agonists. Additionally, minor structural modification of J-113,397 can dramatically shift signaling from antagonist to partial agonist activity. We explore these findings with in silico modeling of binding poses. This work is the first to demonstrate functional selectivity and identification of biased ligands at the nociceptin opioid receptor. PMID:26134494

  5. Graded activation and free energy landscapes of a muscarinic G-protein-coupled receptor.

    PubMed

    Miao, Yinglong; McCammon, J Andrew

    2016-10-25

    G-protein-coupled receptors (GPCRs) recognize ligands of widely different efficacies, from inverse to partial and full agonists, which transduce cellular signals at differentiated levels. However, the mechanism of such graded activation remains unclear. Using the Gaussian accelerated molecular dynamics (GaMD) method that enables both unconstrained enhanced sampling and free energy calculation, we have performed extensive GaMD simulations (∼19 μs in total) to investigate structural dynamics of the M2 muscarinic GPCR that is bound by the full agonist iperoxo (IXO), the partial agonist arecoline (ARC), and the inverse agonist 3-quinuclidinyl-benzilate (QNB), in the presence or absence of the G-protein mimetic nanobody. In the receptor-nanobody complex, IXO binding leads to higher fluctuations in the protein-coupling interface than ARC, especially in the receptor transmembrane helix 5 (TM5), TM6, and TM7 intracellular domains that are essential elements for GPCR activation, but less flexibility in the receptor extracellular region due to stronger binding compared with ARC. Two different binding poses are revealed for ARC in the orthosteric pocket. Removal of the nanobody leads to GPCR deactivation that is characterized by inward movement of the TM6 intracellular end. Distinct low-energy intermediate conformational states are identified for the IXO- and ARC-bound M2 receptor. Both dissociation and binding of an orthosteric ligand are observed in a single all-atom GPCR simulation in the case of partial agonist ARC binding to the M2 receptor. This study demonstrates the applicability of GaMD for exploring free energy landscapes of large biomolecules and the simulations provide important insights into the GPCR functional mechanism.

  6. Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction

    PubMed Central

    Bradley, Sophie J.; Iglesias, Max Maza; Kong, Kok Choi; Butcher, Adrian J.; Plouffe, Bianca; Goupil, Eugénie; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; LeGouill, Christian; Russell, Kirsty; Laporte, Stéphane A.; König, Gabriele M.; Kostenis, Evi; Bouvier, Michel; Chung, Kian Fan; Amrani, Yassine; Tobin, Andrew B.

    2016-01-01

    G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR–biased ligands with important implications for drug discovery. PMID:27071102

  7. Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction.

    PubMed

    Bradley, Sophie J; Wiegman, Coen H; Iglesias, Max Maza; Kong, Kok Choi; Butcher, Adrian J; Plouffe, Bianca; Goupil, Eugénie; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; LeGouill, Christian; Russell, Kirsty; Laporte, Stéphane A; König, Gabriele M; Kostenis, Evi; Bouvier, Michel; Chung, Kian Fan; Amrani, Yassine; Tobin, Andrew B

    2016-04-19

    G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR-biased ligands with important implications for drug discovery.

  8. Use of label-free optical biosensors to detect modulation of potassium channels by G-protein coupled receptors.

    PubMed

    Fleming, Matthew R; Shamah, Steven M; Kaczmarek, Leonard K

    2014-02-10

    Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane(1). To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex(2,3). Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein's behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

  9. FRPR-4 Is a G-Protein Coupled Neuropeptide Receptor That Regulates Behavioral Quiescence and Posture in Caenorhabditis elegans

    PubMed Central

    York, Neil; Lee, Kun He; Schoofs, Liliane; Raizen, David M.

    2015-01-01

    Neuropeptides signal through G-protein coupled receptors (GPCRs) to regulate a broad array of animal behaviors and physiological processes. The Caenorhabditis elegans genome encodes approximately 100 predicted neuropeptide receptor GPCRs, but in vivo roles for only a few have been identified. We describe here a role for the GPCR FRPR-4 in the regulation of behavioral quiescence and locomotive posture. FRPR-4 is activated in cell culture by several neuropeptides with an amidated isoleucine-arginine-phenylalanine (IRF) motif or an amidated valine-arginine-phenylalanine (VRF) motif at their carboxy termini, including those encoded by the gene flp-13. Loss of frpr-4 function results in a minor feeding quiescence defect after heat-induced cellular stress. Overexpression of frpr-4 induces quiescence of locomotion and feeding as well as an exaggerated body bend posture. The exaggerated body bend posture requires the gene flp-13. While frpr-4 is expressed broadly, selective overexpression of frpr-4 in the proprioceptive DVA neurons results in exaggerated body bends that require flp-13 in the ALA neuron. Our results suggest that FLP-13 and other neuropeptides signal through FRPR-4 and other receptors to regulate locomotion posture and behavioral quiescence. PMID:26571132

  10. Environmental coupling of selection and heritability limits evolution.

    PubMed

    Wilson, A J; Pemberton, J M; Pilkington, J G; Coltman, D W; Mifsud, D V; Clutton-Brock, T H; Kruuk, L E B

    2006-07-01

    There has recently been great interest in applying theoretical quantitative genetic models to empirical studies of evolution in wild populations. However, while classical models assume environmental constancy, most natural populations exist in variable environments. Here, we applied a novel analytical technique to a long-term study of birthweight in wild sheep and examined, for the first time, how variation in environmental quality simultaneously influences the strength of natural selection and the genetic basis of trait variability. In addition to demonstrating that selection and genetic variance vary dramatically across environments, our results show that environmental heterogeneity induces a negative correlation between these two parameters. Harsh environmental conditions were associated with strong selection for increased birthweight but low genetic variance, and vice versa. Consequently, the potential for microevolution in this population is constrained by either a lack of heritable variation (in poor environments) or by a reduced strength of selection (in good environments). More generally, environmental dependence of this nature may act to limit rates of evolution, maintain genetic variance, and favour phenotypic stasis in many natural systems. Assumptions of environmental constancy are likely to be violated in natural systems, and failure to acknowledge this may generate highly misleading expectations for phenotypic microevolution.

  11. Purinergic glio-endothelial coupling during neuronal activity: role of P2Y1 receptors and eNOS in functional hyperemia in the mouse somatosensory cortex.

    PubMed

    Toth, Peter; Tarantini, Stefano; Davila, Antonio; Valcarcel-Ares, M Noa; Tucsek, Zsuzsanna; Varamini, Behzad; Ballabh, Praveen; Sonntag, William E; Baur, Joseph A; Csiszar, Anna; Ungvari, Zoltan

    2015-12-01

    Impairment of moment-to-moment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of cognitive impairment associated with aging and pathological conditions associated with accelerated cerebromicrovascular aging (e.g., hypertension, obesity). Although previous studies demonstrate that endothelial dysfunction plays a critical role in neurovascular uncoupling in these conditions, the role of endothelial NO mediation in neurovascular coupling responses is not well understood. To establish the link between endothelial function and functional hyperemia, neurovascular coupling responses were studied in mutant mice overexpressing or deficient in endothelial NO synthase (eNOS), and the role of P2Y1 receptors in purinergic glioendothelial coupling was assessed. We found that genetic depletion of eNOS (eNOS(-/-)) and pharmacological inhibition of NO synthesis significantly decreased the CBF responses in the somatosensory cortex evoked by whisker stimulation and by administration of ATP. Overexpression of eNOS enhanced NO mediation of functional hyperemia. In control mice, the selective and potent P2Y1 receptor antagonist MRS2179 attenuated both whisker stimulation-induced and ATP-mediated CBF responses, whereas, in eNOS(-/-) mice, the inhibitory effects of MRS2179 were blunted. Collectively, our findings provide additional evidence for purinergic glio-endothelial coupling during neuronal activity, highlighting the role of ATP-mediated activation of eNOS via P2Y1 receptors in functional hyperemia.

  12. A modern ionotropic glutamate receptor with a K(+) selectivity signature sequence.

    PubMed

    Janovjak, H; Sandoz, G; Isacoff, E Y

    2011-01-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system and gates non-selective cation channels. The origins of glutamate receptors are not well understood as they differ structurally and functionally from simple bacterial ligand-gated ion channels. Here we report the discovery of an ionotropic glutamate receptor that combines the typical eukaryotic domain architecture with the 'TXVGYG' signature sequence of the selectivity filter found in K(+) channels. This receptor exhibits functional properties intermediate between bacterial and eukaryotic glutamate-gated ion channels, suggesting a link in the evolution of ionotropic glutamate receptors.

  13. Kappa-opioid receptor-selective dicarboxylic ester-derived salvinorin A ligands.

    PubMed

    Polepally, Prabhakar R; White, Kate; Vardy, Eyal; Roth, Bryan L; Ferreira, Daneel; Zjawiony, Jordan K

    2013-05-15

    Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum is the most potent known naturally occurring hallucinogen and is a selective κ-opioid receptor agonist. To better understand the ligand-receptor interactions, a series of dicarboxylic ester-type of salvinorin A derivatives were synthesized and evaluated for their binding affinity at κ-, δ- and μ-opioid receptors. Most of the analogues show high affinity to the κ-opioid receptor. Methyl malonyl derivative 4 shows the highest binding affinity (Ki=2nM), analogues 5, 7, and 14 exhibit significant affinity for the κ-receptor (Ki=21, 36 and 39nM).

  14. Kappa-Opioid Receptor-Selective Dicarboxylic Ester-Derived Salvinorin A Ligands

    PubMed Central

    Polepally, Prabhakar R.; White, Kate; Vardy, Eyal; Roth, Bryan L.; Ferreira, Daneel; Zjawiony, Jordan K.

    2013-01-01

    Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum is the most potent known naturally occurring hallucinogen and is a selective κ-opioid receptor agonist. To better understand the ligand-receptor interactions, a series of dicarboxylic ester-type of salvinorin A derivatives were synthesized and evaluated for their binding affinity at κ, δ, and μ-opioid receptors. Most of the analogues show high affinity to the κ-opioid receptor. Methyl malonyl derivative 4 shows the highest binding affinity (Ki = 2 nM), analogues 5, 7, and 14 exhibit significant affinity for the κ-receptor (Ki = 21, 36 and 39 nM). PMID:23587424

  15. Interacting residues in an activated state of a G protein-coupled receptor.

    PubMed

    Lee, Yong-Hun; Naider, Fred; Becker, Jeffrey M

    2006-01-27

    Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn(205) as a potential interacting partner with the Tyr(266) residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.

  16. Selective protected state preparation of coupled dissipative quantum emitters

    PubMed Central

    Plankensteiner, D.; Ostermann, L.; Ritsch, H.; Genes, C.

    2015-01-01

    Inherent binary or collective interactions in ensembles of quantum emitters induce a spread in the energy and lifetime of their eigenstates. While this typically causes fast decay and dephasing, in many cases certain special entangled collective states with minimal decay can be found, which possess ideal properties for spectroscopy, precision measurements or information storage. We show that for a specific choice of laser frequency, power and geometry or a suitable configuration of control fields one can efficiently prepare these states. We demonstrate this by studying preparation schemes for strongly subradiant entangled states of a chain of dipole-dipole coupled emitters. The prepared state fidelity and its entanglement depth is further improved via spatial excitation phase engineering or tailored magnetic fields. PMID:26549501

  17. Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925.

    PubMed

    Rafehi, Muhammad; Burbiel, Joachim C; Attah, Isaac Y; Abdelrahman, Aliaa; Müller, Christa E

    2017-03-01

    The Gq protein-coupled, ATP- and UTP-activated P2Y2 receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties. We therefore developed an improved procedure for the synthesis of AR-C118925 and two derivatives to allow up-scaling and assessed their potency in calcium mobilization assays on the human and rat P2Y2 receptors recombinantly expressed in 1321N1 astrocytoma cells. The compound was further evaluated for inhibition of P2Y2 receptor-induced β-arrestin translocation. AR-C118925 behaved as a competitive antagonist with pA 2 values of 37.2 nM (calcium assay) and 51.3 nM (β-arrestin assay). Selectivity was assessed vs. related receptors including P2X, P2Y, and adenosine receptor subtypes, as well as ectonucleotidases. AR-C118925 showed at least 50-fold selectivity against the other investigated targets, except for the P2X1 and P2X3 receptors which were blocked by AR-C118925 at concentrations of about 1 μM. AR-C118925 is soluble in buffer at pH 7.4 (124 μM) and was found to be metabolically highly stable in human and mouse liver microsomes. In Caco2 cell experiments, the compound displayed moderate permeability indicating that it may show limited peroral bioavailability. AR-C118925 appears to be a useful pharmacological tool for in vitro and in vivo studies.

  18. Selective suppression of interleukin-12 induction after macrophage receptor ligation.

    PubMed

    Sutterwala, F S; Noel, G J; Clynes, R; Mosser, D M

    1997-06-02

    Interleukin (IL)-12 is a monocyte- and macrophage-derived cytokine that plays a crucial role in both the innate and the acquired immune response. In this study, we examined the effects that ligating specific macrophage receptors had on the induction of IL-12 by lipopolysaccharide (LPS). We report that ligation of the macrophage Fcgamma, complement, or scavenger receptors inhibited the induction of IL-12 by LPS. Both mRNA synthesis and protein secretion were diminished to near-undetectable levels following receptor ligation. Suppression was specific to IL-12 since IL-10 and tumor necrosis factor-alpha (TNF-alpha) production were not inhibited by ligating macrophage receptors. The results of several different experimental approaches suggest that IL-12 downregulation was due to extracellular calcium influxes that resulted from receptor ligation. First, preventing extracellular calcium influxes, by performing the assays in EGTA, abrogated FcgammaR-mediated IL-12(p40) mRNA suppression. Second, exposure of macrophages to the calcium ionophores, ionomycin or A23187, mimicked receptor ligation and inhibited IL-12(p40) mRNA induction by LPS. Finally, bone marrow-derived macrophages from FcR gamma chain-deficient mice, which fail to flux calcium after receptor ligation, failed to inhibit IL-12(p40) mRNA induction. These results indicate that the calcium influxes that occur as a result of receptor ligation are responsible for inhibiting the induction of IL-12 by LPS. Hence, the ligation of phagocytic receptors on macrophages can lead to a dramatic decrease in IL-12 induction. This downregulation may be a way of limiting proinflammatory responses of macrophages to extracellular pathogens, or suppressing the development of cell-mediated immunity to intracellular pathogens.

  19. Functional selectivity in CB(2) cannabinoid receptor signaling and regulation: implications for the therapeutic potential of CB(2) ligands.

    PubMed

    Atwood, Brady K; Wager-Miller, James; Haskins, Christopher; Straiker, Alex; Mackie, Ken

    2012-02-01

    Receptor internalization increases the flexibility and scope of G protein-coupled receptor (GPCR) signaling. CB(1) and CB(2) cannabinoid receptors undergo internalization after sustained exposure to agonists. However, it is not known whether different agonists internalize CB(2) to different extents. Because CB(2) is a promising therapeutic target, understanding its trafficking in response to different agonists is necessary for a complete understanding of its biology. Here we profile a number of cannabinoid receptor ligands and provide evidence for marked functional selectivity of cannabinoid receptor internalization. Classic, aminoalkylindole, bicyclic, cannabilactone, iminothiazole cannabinoid, and endocannabinoid ligands varied greatly in their effects on CB(1) and CB(2) trafficking. Our most striking finding was that (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55,212-2) (and other aminoalkylindoles) failed to promote CB(2) receptor internalization, whereas 5-(1,1-dimethylheptyl)-2-(5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)phenol (CP55,940) robustly internalized CB(2) receptors. Furthermore, WIN55,212-2 competitively antagonized CP55,940-induced CB(2) internalization. Despite these differences in internalization, both compounds activated CB(2) receptors as measured by extracellular signal-regulated kinase 1/2 phosphorylation and recruitment of β-arrestin(2) to the membrane. In contrast, whereas CP55,940 inhibited voltage-gated calcium channels via CB(2) receptor activation, WIN55,212-2 was ineffective on its own and antagonized the effects of CP55,940. On the basis of the differences we found between these two ligands, we also tested the effects of other cannabinoids on these signaling pathways and found additional evidence for functional selectivity of CB(2) ligands. These novel data highlight that WIN55,212-2 and other cannabinoids show strong functional selectivity at CB(2

  20. The effects of sigma (σ1) receptor-selective ligands on muscarinic receptor antagonist-induced cognitive deficits in mice

    PubMed Central

    Malik, Maninder; Rangel-Barajas, Claudia; Sumien, Nathalie; Su, Chang; Singh, Meharvan; Chen, Zhenglan; Huang, Ren-Qi; Meunier, Johann; Maurice, Tangui; Mach, Robert H; Luedtke, Robert R

    2015-01-01

    Background and Purpose Cognitive deficits in patients with Alzheimer's disease, Parkinson's disease, traumatic brain injury and stroke often involve alterations in cholinergic signalling. Currently available therapeutic drugs provide only symptomatic relief. Therefore, novel therapeutic strategies are needed to retard and/or arrest the progressive loss of memory. Experimental Approach Scopolamine-induced memory impairment provides a rapid and reversible phenotypic screening paradigm for cognition enhancement drug discovery. Male C57BL/6J mice given scopolamine (1 mg·kg−1) were used to evaluate the ability of LS-1–137, a novel sigma (σ1) receptor-selective agonist, to improve the cognitive deficits associated with muscarinic antagonist administration. Key Results LS-1–137 is a high-affinity (Ki = 3.2 nM) σ1 receptor agonist that is 80-fold selective for σ1, compared with σ2 receptors. LS-1–137 binds with low affinity at D2-like (D2, D3 and D4) dopamine and muscarinic receptors. LS-1–137 was found to partially reverse the learning deficits associated with scopolamine administration using a water maze test and an active avoidance task. LS-1–137 treatment was also found to trigger the release of brain-derived neurotrophic factor from rat astrocytes. Conclusions and Implications The σ1 receptor-selective compound LS-1–137 may represent a novel candidate cognitive enhancer for the treatment of muscarinic receptor-dependent cognitive deficits. PMID:25573298

  1. Cannabinoid Receptors Couple to NMDA Receptors to Reduce the Production of NO and the Mobilization of Zinc Induced by Glutamate

    PubMed Central

    Sánchez-Blázquez, Pilar; Rodríguez-Muñoz, María; Vicente-Sánchez, Ana

    2013-01-01

    Abstract Aims: Overactivation of glutamate N-methyl-D-aspartate receptor (NMDAR) increases the cytosolic concentrations of calcium and zinc, which significantly contributes to neural death. Since cannabinoids prevent the NMDAR-mediated increase in cytosolic calcium, we investigated whether they also control the rise of potentially toxic free zinc ions, as well as the processes implicated in this phenomenon. Results: The cannabinoid receptors type 1 (CNR1) and NMDARs are cross-regulated in different regions of the nervous system. Cannabinoids abrogated the stimulation of the nitric oxide-zinc pathway by NMDAR, an effect that required the histidine triad nucleotide-binding protein 1 (HINT1). Conversely, NMDAR antagonism reduced the analgesia promoted by the CNR1 agonist WIN55,212-2 and impaired its capacity to internalize CNR1s. At the cell surface, CNR1s co-immunoprecipitated with the NR1 subunits of NMDARs, an association that diminished after the administration of NMDA in vivo or as a consequence of neuropathic overactivation of NMDARs, both situations in which cannabinoids do not control NMDAR activity. Under these circumstances, inhibition of protein kinase A (PKA) restored the association between CNR1s and NR1 subunits, and cannabinoids regained control over NMDAR activity. Notably, CNR1 and NR1 associated poorly in HINT1−/− mice, in which there was little cross-regulation between these receptors. Innovation: The CNR1 can regulate NMDAR function when the receptor is coupled to HINT1. Thus, internalization of CNR1s drives the co-internalization of the NR1 subunits, neutralizing the overactivation of NMDARs. Conclusion: Cannabinoids require the HINT1 protein to counteract the toxic effects of NMDAR-mediated NO production and zinc release. This study situates the HINT1 protein at the forefront of cannabinoid protection against NMDAR-mediated brain damage. Antioxid. Redox Signal. 19, 1766–1782. PMID:23600761

  2. Crosstalk between G protein-coupled receptors (GPCRs) and tyrosine kinase receptor (TXR) in the heart after morphine withdrawal.

    PubMed

    Almela, Pilar; García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa

    2013-01-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK) pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signaling, resulting in high expression of protein kinase (PK) A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK), one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR) can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH) at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

  3. Crosstalk between G protein-coupled receptors (GPCRs) and tyrosine kinase receptor (TXR) in the heart after morphine withdrawal

    PubMed Central

    Almela, Pilar; García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa

    2013-01-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK) pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signaling, resulting in high expression of protein kinase (PK) A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK), one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR) can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH) at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies. PMID:24409147

  4. Molecular recognition of parathyroid hormone by its G protein-coupled receptor

    SciTech Connect

    Pioszak, Augen A.; Xu, H. Eric

    2008-08-07

    Parathyroid hormone (PTH) is central to calcium homeostasis and bone maintenance in vertebrates, and as such it has been used for treating osteoporosis. It acts primarily by binding to its receptor, PTH1R, a member of the class B G protein-coupled receptor (GPCR) family that also includes receptors for glucagon, calcitonin, and other therapeutically important peptide hormones. Despite considerable interest and much research, determining the structure of the receptor-hormone complex has been hindered by difficulties in purifying the receptor and obtaining diffraction-quality crystals. Here, we present a method for expression and purification of the extracellular domain (ECD) of human PTH1R engineered as a maltose-binding protein (MBP) fusion that readily crystallizes. The 1.95-{angstrom} structure of PTH bound to the MBP-PTH1R-ECD fusion reveals that PTH docks as an amphipathic helix into a central hydrophobic groove formed by a three-layer {alpha}-{beta}-{beta}{alpha} fold of the PTH1R ECD, resembling a hot dog in a bun. Conservation in the ECD scaffold and the helical structure of peptide hormones emphasizes this hot dog model as a general mechanism of hormone recognition common to class B GPCRs. Our findings reveal critical insights into PTH actions and provide a rational template for drug design that targets this hormone signaling pathway.

  5. Molecular recognition of ketamine by a subset of olfactory G protein–coupled receptors

    PubMed Central

    Saven, Jeffery G.; Matsunami, Hiroaki; Eckenhoff, Roderic G.

    2015-01-01

    Ketamine elicits various neuropharmacological effects, including sedation, analgesia, general anesthesia, and antidepressant activity. Through an in vitro screen, we identified four mouse olfactory receptors (ORs) that responded to ketamine. In addition to their presence in the olfactory epithelium, these G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) are distributed throughout the central nervous system. To better understand the molecular basis of the interactions between ketamine and ORs, we used sequence comparison and molecular modeling to design mutations that (i) increased, reduced, or abolished ketamine responsiveness in responding receptors, and (ii) rendered non-responding receptors responsive to ketamine. We showed that olfactory sensory neurons (OSNs) that expressed distinct ORs responded to ketamine in vivo, suggesting that ORs may serve as functional targets for ketamine. The ability to both abolish and introduce responsiveness to ketamine in GPCRs enabled us to identify and confirm distinct interaction loci in the binding site, which suggested a signature ketamine-binding pocket that may guide exploration of additional receptors for this general anesthetic drug. PMID:25829447

  6. G protein coupled receptor 18: A potential role for endocannabinoid signaling in metabolic dysfunction.

    PubMed

    Rajaraman, Gayathri; Simcocks, Anna; Hryciw, Deanne H; Hutchinson, Dana S; McAinch, Andrew J

    2016-01-01

    Endocannabinoids are products of dietary fatty acids that are modulated by an alteration in food intake levels. Overweight and obese individuals have substantially higher circulating levels of the arachidonic acid derived endocannabinoids, anandamide and 2-arachidonoyl glycerol, and show an altered pattern of cannabinoid receptor expression. These cannabinoid receptors are part of a large family of G protein coupled receptors (GPCRs). GPCRs are major therapeutic targets for various diseases within the cardiovascular, neurological, gastrointestinal, and endocrine systems, as well as metabolic disorders such as obesity and type 2 diabetes mellitus. Obesity is considered a state of chronic low-grade inflammation elicited by an immunological response. Interestingly, the newly deorphanized GPCR (GPR18), which is considered to be a putative cannabinoid receptor, is proposed to have an immunological function. In this review, the current scientific knowledge on GPR18 is explored including its localization, signaling pathways, and pharmacology. Importantly, the involvement of nutritional factors and potential dietary regulation of GPR18 and its (patho)physiological roles are described. Further research on this receptor and its regulation will enable a better understanding of the complex mechanisms of GPR18 and its potential as a novel therapeutic target for treating metabolic disorders.

  7. Computational approaches for ligand discovery and design in class-A G protein- coupled receptors.

    PubMed

    Rodríguez, David; Gutiérrez-de-Terán, Hugo

    2013-01-01

    Our structural understanding of the superfamily of G-protein coupled receptors, a group of targets of utmost pharmacological importance, has improved dramatically in the last few years. This was directly translated in an increase of both the number and the relevance of computer-assisted drug design efforts devoted to these receptors. The field, which had been greatly influenced by ligand-based methods, has experienced a radical transformation with a number of successful structure-based ligand design and ligand discovery studies. This revolution has been accompanied by the exponential increase of computational resources, and as a result the scenario in GPCR structural and chemical studies is now more complex and richer than ever. Virtual screens, both structure- and ligand-based, co-exist with accurate computational characterizations of the receptor conformational dynamics and of the energy landscapes of receptor-ligand interactions. We here provide an integrated and updated view of the different computational techniques applied to the ligand design of GPCRs. Particular emphasis is put on the studies that take into account the novel structural information of GPCRs, together with those that consider the enormous amount of chemical information accumulated on these receptors in the last decades. Indeed, we propose that proper combinations of the different computational techniques: ligand-based, structure-based and molecular dynamics studies, should be performed to better integrate all available information whenever possible. With this in mind, a major impact of computational technologies in the ligand design on GPCRs is expected in the forthcoming years.

  8. Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin.

    PubMed

    Haga, K; Tsuga, H; Haga, T

    1997-02-11

    Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.

  9. Endocannabinoid-mediated modulation of Gq/11 protein-coupled receptor signaling-induced vasoconstriction and hypertension.

    PubMed

    Szekeres, Mária; Nádasy, György L; Turu, Gábor; Soltész-Katona, Eszter; Benyó, Zoltán; Offermanns, Stefan; Ruisanchez, Éva; Szabó, Eszter; Takáts, Zoltán; Bátkai, Sándor; Tóth, Zsuzsanna E; Hunyady, László

    2015-03-05

    Activation of G protein-coupled receptors (GPCRs) can induce vasoconstriction via calcium signal-mediated and Rho-dependent pathways. Earlier reports have shown that diacylglycerol produced during calcium signal generation can be converted to an endocannabinoid, 2-arachidonoylglycerol (2-AG). Our aim was to provide evidence that GPCR signaling-induced 2-AG production and activation of vascular type1 cannabinoid receptors (CB1R) is capable of reducing agonist-induced vasoconstriction and hypertension. Rat and mouse aortic rings were examined by myography. Vascular expression of CB1R was demonstrated with immunohistochemistry. Rat aortic vascular smooth muscle cells (VSMCs) were cultured for calcium measurements and 2-AG-determination. Inhibition or genetic loss of CB1Rs enhanced vasoconstriction induced by angiotensin II (AngII) or phenylephrine (Phe), but not by prostaglandin(PG)F2α. AngII-induced vasoconstriction was augmented by inhibition of diacylglycerol lipase (tetrahydrolipstatin) and was attenuated by inhibition of monoacylglycerol lipase (JZL184) suggesting a functionally relevant role for endogenously produced 2-AG. In Gαq/11-deficient mice vasoconstriction was absent to AngII or Phe, which activate Gq/11-coupled receptors, but was maintained in response to PGF2α. In VSMCs, AngII-stimulated 2-AG-formation was inhibited by tetrahydrolipstatin and potentiated by JZL184. CB1R inhibition increased the sustained phase of AngII-induced calcium signal. Pharmacological or genetic loss of CB1R function augmented AngII-induced blood pressure rise in mice. These data demonstrate that vasoconstrictor effect of GPCR agonists is attenuated via Gq/11-mediated vascular endocannabinoid formation. Agonist-induced endocannabinoid-mediated CB1R activation is a significant physiological modulator of vascular tone. Thus, the selective modulation of GPCR signaling-induced endocannabinoid release has a therapeutic potential in case of increased vascular tone and hypertension.

  10. Intersubunit Physical Couplings Fostered By The Left Flipper Domain Facilitate Channel Opening Of P2X4 Receptors.

    PubMed

    Wang, Jin; Sun, Liang-Fei; Cui, Wen-Wen; Zhao, Wen-Shan; Ma, Xue-Fei; Li, Bin; Liu, Yan; Yang, Yang; Hu, You-Min; Huang, Li-Dong; Cheng, Xiao-Yang; Li, Lingyong; Lu, Xiang-Yang; Tian, Yun; Yu, Ye

    2017-03-16

    P2X receptors are ATP-gated trimeric channels with important roles in diverse pathophysiological functions. A detailed understanding of the mechanism underlying the gating process of these receptors is thus fundamentally important and may open new therapeutic avenues. The left flipper (LF) domain of P2X receptors is a flexible loop structure and its coordinated motions together with the dorsal fin (DF) domain are crucial for the channel gating of the P2X receptors. However, the mechanism underlying the crucial role of the LF domain in the channel gating remains obscure. Here, we propose that the ATP-induced allosteric changes of the LF domain enable it to foster intersubunit physical couplings among the DF and two lower body domains, which is pivotal for the channel gating of P2X4 receptors. Metadynamics analysis indicated that these newly established intersubunit couplings correlate well with the ATP-bound open state of the receptors. Moreover, weakening or strengthening these physical interactions with engineered intersubunit metal bridges remarkably decreased or increased the open probability of the receptors, respectively. Further disulfide crosslinking and covalent modification confirmed that the intersubunit physical couplings among the DF and two lower body domains fostered by the LF domain at the open state act as an integrated structural element that is stringently required for the channel gating of P2X4 receptors. Our observations provide new mechanistic insights into P2X receptor activation and will stimulate development of new allosteric modulators of P2X receptors.

  11. Selective Androgen Receptor Downregulators (SARDs): A New Prostate Cancer Therapy

    DTIC Science & Technology

    2006-10-01

    used to down-regulate the AR include antisense oligonucleotides (9, 10), ribozyme treatments (11, 12), AR dominant negatives (13) and small...findings suggest that ICI may present a useful treatment option for patients with AR-dependent PCa. Unlike the ribozyme , antisense, siRNA, or dominant...of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol, 12: 1558

  12. Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

    PubMed Central

    Caers, Jelle; Peymen, Katleen; Suetens, Nick; Temmerman, Liesbet; Janssen, Tom; Schoofs, Liliane; Beets, Isabel

    2014-01-01

    For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16 construct is co-transfected. Following ligand binding, activation of the Gα16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their

  13. Novel Agonist Bioisosteres and Common Structure-Activity Relationships for The Orphan G Protein-Coupled Receptor GPR139

    PubMed Central

    Shehata, Mohamed A.; Nøhr, Anne C.; Lissa, Delphine; Bisig, Christoph; Isberg, Vignir; Andersen, Kirsten B.; Harpsøe, Kasper; Björkling, Fredrik; Bräuner-Osborne, Hans; Gloriam, David E.

    2016-01-01

    GPR139 is an orphan class A G protein-coupled receptor found mainly in the central nervous system. It has its highest expression levels in the hypothalamus and striatum, regions regulating metabolism and locomotion, respectively, and has therefore been suggested as a potential target for obesity and Parkinson’s disease. The two aromatic amino acids L-Trp and L-Phe have been proposed as putative endogenous agonists, and three structurally related benzohydrazide, glycine benzamide, and benzotriazine surrogate agonist series have been published. Herein, we assayed 158 new analogues selected from a pharmacophore model, and identified 12 new GPR139 agonists, containing previously untested bioisosteres. Furthermore, we present the first combined structure-activity relationships, and a refined pharmacophore model to serve as a rationale for future ligand identification and optimization. PMID:27830715

  14. Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA

    NASA Astrophysics Data System (ADS)

    Tanabe, Tsutomu; Beam, Kurt G.; Powell, Jeanne A.; Numa, Shosaku

    1988-11-01

    Microinjection of an expression plasmid that carries complementary DNA encoding the receptor for dihydropyridine calcium channel blockers of skeletal muscle restores both excitation-contraction coupling and slow calcium current in cultured skeletal muscle cells from mice with muscular dysgenesis. This suggests that the dihydropyridine receptor in the transverse tubule membrane of skeletal muscle functions both as the voltage sensor for excitation-contraction coupling and as the slow calcium channel.

  15. Coupled gating of skeletal muscle ryanodine receptors is modulated by Ca2+, Mg2+, and ATP

    PubMed Central

    Porta, Maura; Diaz-Sylvester, Paula L.; Neumann, Jake T.; Escobar, Ariel L.; Fleischer, Sidney

    2012-01-01

    Coupled gating (synchronous openings and closures) of groups of skeletal muscle ryanodine receptors (RyR1), which mimics RyR1-mediated Ca2+ release underlying Ca2+ sparks, was first described by Marx et al. (Marx SO, Ondrias K, Marks AR. Science 281: 818–821, 1998). The nature of the RyR1-RyR1 interactions for coupled gating still needs to be characterized. Consequently, we defined planar lipid bilayer conditions where ∼25% of multichannel reconstitutions contain mixtures of coupled and independently gating RyR1. In ∼10% of the cases, all RyRs (2–10 channels; most frequently 3–4) gated in coupled fashion, allowing for quantification. Our results indicated that coupling required cytosolic solutions containing ATP/Mg2+ and high (50 mM) luminal Ca2+ (Calum) or Sr2+ solutions. Bursts of coupled activity (events) started and ended abruptly, with all channels activating/deactivating within ∼300 μs. Coupled RyR1 were heterogeneous, where highly active RyR1 (“drivers”) seemed open during the entire coupled event (Po = 1), while other RyR1s (“followers”) displayed abundant flickering and smaller amplitude. Drivers mean open time increased with cytosolic Ca2+ (Cacyt) or caffeine, whereas followers flicker frequency was Cacyt independent and more sensitive to inhibition by cytosolic Mg2+. Coupled events were insensitive to varying lumen-to-cytosol Ca2+ fluxes from ∼1 to 8 pA, which does not corroborate coupling of neighboring RyR1 by local Ca2+-induced Ca2+ release. However, coupling requires specific Calum sites, as it was lost when Calum was replaced by luminal Ba2+ or Mg2+. In summary, coupled events reveal complex interactions among heterogeneous RyR1, differentially modulated by cytosolic ATP/Mg2+, Cacyt, and Calum, which under cell-like ionic conditions may parallel synchronous RyR1 gating during Ca2+ sparks. PMID:22785120

  16. Polarization-Selective Out-Coupling of Whispering-Gallery Modes

    NASA Astrophysics Data System (ADS)

    Sedlmeir, Florian; Foreman, Matthew R.; Vogl, Ulrich; Zeltner, Richard; Schunk, Gerhard; Strekalov, Dmitry V.; Marquardt, Christoph; Leuchs, Gerd; Schwefel, Harald G. L.

    2017-02-01

    Whispering-gallery mode (WGM) resonators are an important platform for linear, nonlinear, and quantum optical experiments. In such experiments, independent control of in-coupling and out-coupling rates to different modes can lead to higher conversion efficiencies and greater flexibility in the generation of nonclassical states based on parametric down-conversion. In this work, we introduce a scheme that enables selective out-coupling of WGMs belonging to a specific polarization family, while the orthogonally polarized modes remain largely unperturbed. Our technique utilizes material birefringence in both the resonator and the coupler such that a negative (positive) birefringence allows for polarization-selective coupling to TE (TM) WGMs. We formulate a refined coupling condition suitable for describing the case where the refractive indices of the resonator and the coupler are almost the same, from which we derive a criterion for polarization-selective coupling. Finally, we experimentally demonstrate our proposed method using a lithium niobate disk resonator coupled to a lithium niobate prism, where we show a 22-dB suppression of coupling to TM modes relative to TE modes.

  17. Selective estrogen receptor modulators differentially alter the immune response of gilthead seabream juveniles.

    PubMed

    Rodenas, M C; Cabas, I; García-Alcázar, A; Meseguer, J; Mulero, V; García-Ayala, A

    2016-05-01

    17α-ethynylestradiol (EE2), a synthetic estrogen used in oral contraceptives and hormone replacement therapy, tamoxifen (Tmx), a selective estrogen-receptor modulator used in hormone replacement therapy, and G1, a G protein-coupled estrogen receptor (GPER) selective agonist, differentially increased the hepatic vitellogenin (vtg) gene expression and altered the immune response in adult gilthead seabream (Sparus aurata L.) males. However, no information exists on the effects of these compounds on the immune response of juveniles. This study aims, for the first time, to investigate the effects of the dietary intake of EE2, Tmx or G1 on the immune response of gilthead seabream juveniles and the capacity of the immune system of the specimens to recover its functionality after ceasing exposures (recovery period). The specimens were immunized with hemocyanin in the presence of aluminium adjuvant 1 (group A) or 120 (group B) days after the treatments ceased (dpt). The results indicate that EE2 and Tmx, but not G1, differentially promoted a transient alteration in hepatic vtg gene expression. Although all three compounds did not affect the production of reactive oxygen intermediates, they inhibited the induction of interleukin-1β (il1b) gene expression after priming. Interestingly, although Tmx increased the percentage of IgM-positive cells in both head kidney and spleen during the recovery period, the antibody response of vaccinated fish varied depending on the compound used and when the immunization was administered. Taken together, our results suggest that these compounds differentially alter the capacity of fish to respond to infection during ontogeny and, more interestingly, that the adaptive immune response remained altered to an extent that depends on the compound.

  18. Heterocyclic 1,7-disubstituted indole sulfonamides are potent and selective human EP3 receptor antagonists.

    PubMed

    Hategan, Georgeta; Polozov, Alexandre M; Zeller, Wayne; Cao, Hua; Mishra, Rama K; Kiselyov, Alex S; Ramirez, Jose; Halldorsdottir, Gudrún; Andrésson, Thornorkell; Gurney, Mark E; Singh, Jasbir

    2009-12-01

    We have developed a pharmacophore model for the EP(3) receptor antagonists based on its endogenous ligand PGE(2). This ligand-based design yielded a series of novel peri-substituted [4.3.0] bicyclic aromatics featuring 1-alklyaryl 7-heterocyclic sulfonamide substituents. The synthesized molecules are potent antagonists of human EP(3) receptor in vitro and show inhibition of rat platelets aggregation. Optimized derivatives display high selectivity over IP, FP, and other EP receptor panels.

  19. The influence of selective vitamin D receptor activator paricalcitol on cardiovascular system and cardiorenal protection.

    PubMed

    Duplancic, Darko; Cesarik, Marijan; Poljak, Nikola Kolja; Radman, Maja; Kovacic, Vedran; Radic, Josipa; Rogosic, Veljko

    2013-01-01

    The ubiquitous distribution of vitamin D receptors in the human body is responsible for the pleiotropic effects of vitamin D-receptor activation. We discuss the possible beneficial effects of a selective activator of vitamin D receptor, paricalcitol, on the cardiovascular system in chronic heart failure patients and chronic kidney patients, in light of new trials. Paricalcitol should provide additional clinical benefits over the standard treatment for chronic kidney and heart failure, especially in cases of cardiorenal syndrome.

  20. Recent advances in drug action and therapeutics: Relevance of novel concepts in G-protein-coupled receptor and signal transduction pharmacology

    PubMed Central

    Brink, C B; Harvey, B H; Bodenstein, J; Venter, D P; Oliver, D W

    2004-01-01

    Problem statement During especially the past two decades many discoveries in biological sciences, and in particular at the molecular and genetic level, have greatly impacted on our knowledge and understanding of drug action and have helped to develop new drugs and therapeutic strategies. Furthermore, many exciting new drugs acting via novel pharmacological mechanisms are expected to be in clinical use in the not too distant future. Scope and contents of review In this educational review, these concepts are explained and their relevance illustrated by examples of drugs used commonly in the clinical setting, with special reference to the pharmacology of G-protein-coupled receptors. The review also addresses the basic theoretical concepts of full and partial agonism, neutral antagonism, inverse agonism and protean and ligand-selective agonism, and the relevance of these concepts in current rational drug therapy. Moreover, the mechanisms whereby receptor signalling (and eventually response to drugs) is fine-tuned, such as receptor promiscuity, agonist-directed trafficking of receptor signalling, receptor trafficking, receptor ‘cross-talk’ and regulators of G-protein signalling (RGSs) are discussed, from theory to proposed therapeutic implications. Conclusions It is concluded that the understanding of molecular receptor and signal transduction pharmacology enables clinicians to improve their effective implementation of current and future pharmacotherapy, ultimately enhancing the quality of life of their patients. PMID:15025734

  1. A selective sigma-2 receptor ligand antagonizes cocaine-induced hyperlocomotion in mice.

    PubMed

    Lever, John R; Miller, Dennis K; Green, Caroline L; Fergason-Cantrell, Emily A; Watkinson, Lisa D; Carmack, Terry L; Fan, Kuo-Hsien; Lever, Susan Z

    2014-02-01

    Cocaine functions, in part, through agonist actions at sigma-1 (σ1 ) receptors, while roles played by sigma-2 (σ2 ) receptors are less established. Attempts to discriminate σ2 receptor-mediated effects of cocaine in locomotor hyperactivity assays have been hampered by the lack of potent and selective antagonists. Certain tetrahydroisoquinolinyl benzamides display high σ2 receptor affinity, and excellent selectivity for binding to σ2 over σ1 receptors. The behavioral properties of this structural class of σ ligands have not yet been investigated. The present study evaluated 5-bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxy-benzamide, 1, a ligand shown by others to bind preferentially to σ2 over σ1 receptors, as well as dopamine D2 and D3 sites. First, we determined binding to monoamine transporters and opioid receptors, and noted 57-fold selectivity for σ2 receptors over the serotonin transporter, and >800-fold selectivity for σ2 receptors over the other sites tested. We then examined 1 in locomotor activity studies using male CD-1® mice, and saw no alteration of basal activity at doses up to 31.6 µmol/kg. Cocaine produced a fivefold increase in locomotor activity, which was attenuated by 66% upon pretreatment of mice with 1 at 31.6 µmol/kg. In vivo radioligand binding studies also were performed, and showed no occupancy of σ1 receptors or the dopamine transporter by 1, or its possible metabolites, at the 31.6 µmol/kg dose. Thus, ligand 1 profiles behaviorally as a σ2 receptor-selective antagonist that is able to counteract cocaine's motor stimulatory effects.

  2. Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer’s disease

    PubMed Central

    Haas, Laura T.; Salazar, Santiago V.; Kostylev, Mikhail A.; Um, Ji Won; Kaufman, Adam C.

    2016-01-01

    Alzheimer’s disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer’s disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer’s disease transgenes or by human Alzheimer’s disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp–Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer’s disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer’s disease pathogenesis, and the complex is a potential target for disease-modifying intervention. PMID:26667279

  3. Dynamic Cholesterol-Conditioned Dimerization of the G Protein Coupled Chemokine Receptor Type 4

    PubMed Central

    Kranz, Franziska

    2016-01-01

    G protein coupled receptors (GPCRs) allow for the transmission of signals across biological membranes. For a number of GPCRs, this signaling was shown to be coupled to prior dimerization of the receptor. The chemokine receptor type 4 (CXCR4) was reported before to form dimers and their functionality was shown to depend on membrane cholesterol. Here, we address the dimerization pattern of CXCR4 in pure phospholipid bilayers and in cholesterol-rich membranes. Using ensembles of molecular dynamics simulations, we show that CXCR4 dimerizes promiscuously in phospholipid membranes. Addition of cholesterol dramatically affects the dimerization pattern: cholesterol binding largely abolishes the preferred dimer motif observed for pure phospholipid bilayers formed mainly by transmembrane helices 1 and 7 (TM1/TM5-7) at the dimer interface. In turn, the symmetric TM3,4/TM3,4 interface is enabled first by intercalating cholesterol molecules. These data provide a molecular basis for the modulation of GPCR activity by its lipid environment. PMID:27812115

  4. The therapeutic potential of G-protein coupled receptors in Huntington's disease.

    PubMed

    Dowie, Megan J; Scotter, Emma L; Molinari, Emanuela; Glass, Michelle

    2010-11-01

    Huntington's disease is a late-onset autosomal dominant inherited neurodegenerative disease characterised by increased symptom severity over time and ultimately premature death. An expanded CAG repeat sequence in the huntingtin gene leads to a polyglutamine expansion in the expressed protein, resulting in complex dysfunctions including cellular excitotoxicity and transcriptional dysregulation. Symptoms include cognitive deficits, psychiatric changes and a movement disorder often referred to as Huntington's chorea, which involves characteristic involuntary dance-like writhing movements. Neuropathologically Huntington's disease is characterised by neuronal dysfunction and death in the striatum and cortex with an overall decrease in cerebral volume (Ho et al., 2001). Neuronal dysfunction begins prior to symptom presentation, and cells of particular vulnerability include the striatal medium spiny neurons. Huntington's is a devastating disease for patients and their families and there is currently no cure, or even an effective therapy for disease symptoms. G-protein coupled receptors are the most abundant receptor type in the central nervous system and are linked to complex downstream pathways, manipulation of which may have therapeutic application in many neurological diseases. This review will highlight the potential of G-protein coupled receptor drug targets as emerging therapies for Huntington's disease.

  5. Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer's disease.

    PubMed

    Haas, Laura T; Salazar, Santiago V; Kostylev, Mikhail A; Um, Ji Won; Kaufman, Adam C; Strittmatter, Stephen M

    2016-02-01

    Alzheimer's disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer's disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer's disease transgenes or by human Alzheimer's disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp-Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer's disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer's disease pathogenesis, and the complex is a potential target for disease-modifying intervention.

  6. Sequestration of human muscarinic acetylcholine receptor hm1-hm5 subtypes: effect of G protein-coupled receptor kinases GRK2, GRK4, GRK5 and GRK6.

    PubMed

    Tsuga, H; Okuno, E; Kameyama, K; Haga, T

    1998-03-01

    Sequestration of porcine muscarinic acetylcholine receptor m2 subtypes (m2 receptors) expressed in COS-7 cells is facilitated by coexpression of G protein-coupled receptor kinases 2 (GRK2). We examined the effect of coexpression of GRK2, GRK4 delta, GRK5 and GRK6 on sequestration of human m1-m5 receptors expressed in COS-7 cells, which was assessed as loss of [3H]N-methylscopolamine binding activity from the cell surface. Sequestration of m4 receptors as well as m2 receptors was facilitated by coexpression of GRK2 and attenuated by coexpression of the dominant negative form of GRK2 (DN-GRK2). Sequestration of m3 and m5 receptors also was facilitated by coexpression of GRK2 but not affected by coexpression of DN-GRK2. On the other hand, proportions of sequestered m1 receptors were not significantly different with coexpression of GRK2 and DN-GRK2. GRK4 delta, GRK5 and GRK6 did not facilitate sequestration of m1-m5 receptors in COS-7 cells, except that the sequestration of m2 receptors tended to be facilitated by coexpression of GRK4 delta, GRK5 and GRK6. However, coexpression of GRK4 delta, GRK5, but not GRK6, in BHK-21 cells facilitated sequestration of m2, but not m3, receptors. These results indicate that the effect of GRK2 to facilitate receptor sequestration is not restricted to m2 receptors but is generalized to other muscarinic receptors except m1 receptors and that other kinases, including GRK4 delta, GRK5 and endogenous kinase(s) in COS-7 cells, also contribute to sequestration of m2 and m4 receptors.

  7. Research Resource: Gene Profiling of G Protein–Coupled Receptors in the Arcuate Nucleus of the Female

    PubMed Central

    Fang, Yuan; Zhang, Chunguang; Nestor, Casey C.; Mao, Peizhong; Kelly, Martin J.

    2014-01-01

    The hypothalamic arcuate nucleus controls many critical homeostatic functions including energy homeostasis, reproduction, and motivated behavior. Although G protein–coupled receptors (GPCRs) are involved in the regulation of these functions, relatively few of the GPCRs have been identified specifically within the arcuate nucleus. Here, using TaqMan low-density arrays we quantified the mRNA expression of nonolfactory GPCRs in mouse arcuate nucleus. An unprecedented number of GPCRs (total of 292) were found to be expressed, of which 183 were known and 109 were orphan GPCRs. The known GPCR genes expressed were classified into several functional clusters including hormone/neurotransmitter, growth factor, angiogenesis and vasoactivity, inflammation and immune system, and lipid messenger receptors. The plethora of orphan genes expressed in the arcuate nucleus were classified into 5 structure-related classes including class A (rhodopsin-like), class B (adhesion), class C (other GPCRs), nonsignaling 7-transmembrane chemokine-binding proteins, and other 7-transmembrane proteins. Therefore, for the first time, we provide a quantitative estimate of the numerous GPCRs expressed in the hypothalamic arcuate nucleus. Finally, as proof of principle, we documented the expression and function of one of these receptor genes, the glucagon-like peptide 1 receptor (Glp1r), which was highly expressed in the arcuate nucleus. Single-cell RT-PCR revealed that Glp1r mRNA was localized in proopiomelanocortin neurons, and using whole-cell recording we found that the glucagon-like peptide 1-selective agonist exendin-4 robustly excited proopiomelanocortin neurons. Thus, the quantitative GPCR data emphasize the complexity of the hypothalamic arcuate nucleus and furthermore provide a valuable resource for future neuroendocrine/endocrine-related experiments. PMID:24933249

  8. Lysophospholipid growth factors and their G protein-coupled receptors in immunity, coronary artery disease, and cancer.

    PubMed

    Goetzl, Edward J; Graeler, Markus; Huang, Mei-Chuan; Shankar, Geetha

    2002-02-06

    The physiological lysophospholipids (LPLs), exemplified by lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), are omnific mediators of normal cellular proliferation, survival, and functions. Although both LPA and S1P attain micromolar concentrations in many biological fluids, numerous aspects of their biosynthesis, transport, and metabolic degradation are unknown. Eight members of a new subfamily of G protein-coupled LPA/S1P receptors, originally termed Edg Rs, bind either LPA or S1P with high affinity and transduce a series of growth-related and/or cytoskeleton-based functional responses. The most critical areas of LPL biology and pathobiology are neural development and neurodegeneration, immunity, atherosclerosis and myocardial injury, and cancer. Data from analyses of T cells established two basic points: (1) the plasticity and adaptability of expression of LPA/S1P Rs by some cells as a function of activation, and (2) the role of opposing signals from two different receptors for the same ligand as a mechanism for fine control of effects of LPLs. In the heart, LPLs may promote coronary atherosclerosis, but are effectively cytoprotective for hypoxic cardiac myocytes and those exposed to oxygen free radicals. The findings of production of LPA by some types of tumor cells, overexpression of selected sets of LPA receptors by the same tumor cells, and augmentation of the effects of protein growth factors by LPA have suggested pathogenetic roles for the LPLs in cancer. The breadth of physiologic and pathologic activities of LPLs emphasizes the importance of developing bioavailable nonlipid agonists and antagonists of the LPA/S1P receptors for diverse therapeutic applications.

  9. Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation.

    PubMed

    Peeters, M C; van Westen, G J P; Li, Q; IJzerman, A P

    2011-01-01

    G protein-coupled receptors (GPCRs) are the major drug target of medicines on the market today. Therefore, much research is and has been devoted to the elucidation of the function and three-dimensional structure of this large family of membrane proteins, which includes multiple conserved transmembrane domains connected by intra- and extracellular loops. In the last few years, the less conserved extracellular loops have garnered increasing interest, particularly after the publication of several GPCR crystal structures that clearly show the extracellular loops to be involved in ligand binding. This review will summarize the recent progress made in the clarification of the ligand binding and activation mechanism of class-A GPCRs and the role of extracellular loops in this process.

  10. Forebrain-selective AMPA-receptor antagonism guided by TARP γ-8 as an antiepileptic mechanism.

    PubMed

    Kato, Akihiko S; Burris, Kevin D; Gardinier, Kevin M; Gernert, Douglas L; Porter, Warren J; Reel, Jon; Ding, Chunjin; Tu, Yuan; Schober, Douglas A; Lee, Matthew R; Heinz, Beverly A; Fitch, Thomas E; Gleason, Scott D; Catlow, John T; Yu, Hong; Fitzjohn, Stephen M; Pasqui, Francesca; Wang, He; Qian, Yuewei; Sher, Emanuele; Zwart, Ruud; Wafford, Keith A; Rasmussen, Kurt; Ornstein, Paul L; Isaac, John T R; Nisenbaum, Eric S; Bredt, David S; Witkin, Jeffrey M

    2016-12-01

    Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.

  11. Utilizing GCaMP transgenic mice to monitor endogenous Gq/11-coupled receptors

    PubMed Central

    Partridge, John G.

    2015-01-01

    The family of GCaMPs are engineered proteins that contain Ca2+ binding motifs within a circularly permutated variant of the Aequorea Victoria green fluorescent protein (cp-GFP). The rapidly advancing field of utilizing GCaMP reporter constructs represents a major step forward in our ability to monitor intracellular Ca2+ dynamics. With the use of these genetically encoded Ca2+ sensors, investigators have studied activation of endogenous Gq types of G protein-coupled receptors (GPCRs) and subsequent rises in intracellular calcium. Escalations in intracellular Ca2+ from GPCR activation can be faithfully monitored in space and time as an increase in fluorescent emission from these proteins. Further, transgenic mice are now commercially available that express GCaMPs in a Cre recombinase dependent fashion. These GCaMP reporter mice can be bred to distinct Cre recombinase driver mice to direct expression of this sensor in unique populations of cells. Concerning the central nervous system (CNS), sources of calcium influx, including those arising from Gq activation can be observed in targeted cell types like neurons or astrocytes. This powerful genetic method allows simultaneous monitoring of the activity of dozens of cells upon activation of endogenous Gq-coupled GPCRs. Therefore, in combination with pharmacological tools, this strategy of monitoring GPCR activation is amenable to analysis of orthosteric and allosteric ligands of Gq-coupled receptors in their endogenous environments. PMID:25805995

  12. Utilizing GCaMP transgenic mice to monitor endogenous Gq/11-coupled receptors.

    PubMed

    Partridge, John G

    2015-01-01

    The family of GCaMPs are engineered proteins that contain Ca(2+) binding motifs within a circularly permutated variant of the Aequorea Victoria green fluorescent protein (cp-GFP). The rapidly advancing field of utilizing GCaMP reporter constructs represents a major step forward in our ability to monitor intracellular Ca(2+) dynamics. With the use of these genetically encoded Ca(2+) sensors, investigators have studied activation of endogenous Gq types of G protein-coupled receptors (GPCRs) and subsequent rises in intracellular calcium. Escalations in intracellular Ca(2+) from GPCR activation can be faithfully monitored in space and time as an increase in fluorescent emission from these proteins. Further, transgenic mice are now commercially available that express GCaMPs in a Cre recombinase dependent fashion. These GCaMP reporter mice can be bred to distinct Cre recombinase driver mice to direct expression of this sensor in unique populations of cells. Concerning the central nervous system (CNS), sources of calcium influx, including those arising from Gq activation can be observed in targeted cell types like neurons or astrocytes. This powerful genetic method allows simultaneous monitoring of the activity of dozens of cells upon activation of endogenous Gq-coupled GPCRs. Therefore, in combination with pharmacological tools, this strategy of monitoring GPCR activation is amenable to analysis of orthosteric and allosteric ligands of Gq-coupled receptors in their endogenous environments.

  13. Visualization of arrestin recruitment by a G Protein-Coupled Receptor

    PubMed Central

    Reis, Rosana I.; Huang, Li-Yin; Tripathi-Shukla, Prachi; Qian, Jiang; Li, Sheng; Blanc, Adi; Oleskie, Austin N.; Dosey, Anne M.; Su, Min; Liang, Cui-Rong; Gu, Ling-Ling; Shan, Jin-Ming; Chen, Xin; Hanna, Rachel; Choi, Minjung; Yao, Xiao Jie; Klink, Bjoern U.; Kahsai, Alem W.; Sidhu, Sachdev S.; Koide, Shohei; Penczek, Pawel A.; Kossiakoff, Anthony A.; Jr, Virgil L. Woods; Kobilka, Brian K.; Skiniotis, Georgios; Lefkowitz, Robert J.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy-terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins. PMID:25043026

  14. Glucocorticoids regulate arrestin gene expression and redirect the signaling profile of G protein-coupled receptors.

    PubMed

    Oakley, Robert H; Revollo, Javier; Cidlowski, John A

    2012-10-23

    G protein-coupled receptors (GPCRs) compose the largest family of cell surface receptors and are the most common target of therapeutic drugs. The nonvisual arrestins, β-arrestin-1 and β-arrestin-2, are multifunctional scaffolding proteins that play critical roles in GPCR signaling. On binding of activated GPCRs at the plasma membrane, β-arrestins terminate G protein-dependent responses (desensitization) and stimulate β-arrestin-dependent signaling pathways. Alterations in the cellular complement of β-arrestin-1 and β-arrestin-2 occur in many human diseases, and their genetic ablation in mice has severe consequences. Surprisingly, however, the factors that control β-arrestin gene expression are poorly understood. We demonstrate that glucocorticoids differentially regulate β-arrestin-1 and β-arrestin-2 gene expression in multiple cell types. Glucocorticoids act via the glucocorticoid receptor (GR) to induce the synthesis of β-arrestin-1 and repress the expression of β-arrestin-2. Glucocorticoid-dependent regulation involves the recruitment of ligand-activated glucocorticoid receptors to conserved and functional glucocorticoid response elements in intron-1 of the β-arrestin-1 gene and intron-11 of the β-arrestin-2 gene. In human lung adenocarcinoma cells, the increased expression of β-arrestin-1 after glucocorticoid treatment impairs G protein-dependent activation of inositol phosphate signaling while enhancing β-arrestin-1-dependent stimulation of the MAPK pathway by protease activated receptor 1. These studies demonstrate that glucocorticoids redirect the signaling profile of GPCRs via alterations in β-arrestin gene expression, revealing a paradigm for cross-talk between nuclear and cell surface receptors and a mechanism by which glucocorticoids alter the clinical efficacy of GPCR-based drugs.

  15. Functional Role of the C-Terminal Amphipathic Helix 8 of Olfactory Receptors and Other G Protein-Coupled Receptors

    PubMed Central

    Sato, Takaaki; Kawasaki, Takashi; Mine, Shouhei; Matsumura, Hiroyoshi

    2016-01-01

    G protein-coupled receptors (GPCRs) transduce various extracellular signals, such as neurotransmitters, hormones, light, and odorous chemicals, into intracellular signals via G protein activation during neurological, cardiovascular, sensory and reproductive signaling. Common and unique features of interactions between GPCRs and specific G proteins are important for structure-based design of drugs in order to treat GPCR-related diseases. Atomic resolution structures of GPCR complexes with G proteins have revealed shared and extensive interactions between the conserved DRY motif and other residues in transmembrane domains 3 (TM3), 5 and 6, and the target G protein C-terminal region. However, the initial interactions formed between GPCRs and their specific G proteins remain unclear. Alanine scanning mutagenesis of the murine olfactory receptor S6 (mOR-S6) indicated that the N-terminal acidic residue of helix 8 of mOR-S6 is responsible for initial transient and specific interactions with chimeric Gα15_olf, resulting in a response that is 2.2-fold more rapid and 1.7-fold more robust than the interaction with Gα15. Our mutagenesis analysis indicates that the hydrophobic core buried between helix 8 and TM1–2 of mOR-S6 is important for the activation of both Gα15_olf and Gα15. This review focuses on the functional role of the C-terminal amphipathic helix 8 based on several recent GPCR studies. PMID:27869740

  16. Expression of striatal D sub 1 dopamine receptors coupled to inositol phosphate production and Ca sup 2+ mobilization in Xenopus oocytes

    SciTech Connect

    Mahan, L.C.; Monsma, F.J. Jr.; Sibley, D.R. ); Burch, R.M. )

    1990-03-01

    Expression of central nervous system receptors for dopamine was examined by injection of poly(A){sup +} RNA (mRNA) from rat striatum into oocytes from Xenopus laevis. Electrophysiological measurements in mRNA-injected oocytes indicated that addition of 100 {mu}M dopamine induced an inward current that was consistent with the activation of endogenous Ca{sup 2+}-dependent Cl{sup {minus}} channels. This current was also elicited by addition of the selective D{sub 1} agonist SKF 38393 but not by the selective D{sub 2} agonist quinpirole. Using {sup 45}Ca{sup 2+} efflux assays, addition of 100 {mu}M dopamine to injected oocytes stimulated efflux 2- to 3-fold. This increase was mimicked by SKF 38393 and was blocked by the D{sub 1}-selective antagonist (+)SCH 23390 but not by the D{sub 2}-selective antagonist domperidone. No increase in {sup 45}Ca{sup 2+} efflux was seen with 100 {mu}M quinpirole. Size fractionation of striatal mRNA yielded a single peak (2.5-3.0 kilobases) of D{sub 1} receptor-mediated {sup 45}Ca{sup 2+} efflux activity in injected oocytes. Thus, in addition to D{sub 1} receptors that stimulate adenylyl cyclase, rat striatum contains D{sub 1} receptors that can couple to InsP{sub 3} formation and mobilization of intracellular Ca{sup 2+}.

  17. Design, Synthesis, and Preclinical Characterization of the Selective Androgen Receptor Modulator (SARM) RAD140.

    PubMed

    Miller, Chris P; Shomali, Maysoun; Lyttle, C Richard; O'Dea, Louis St L; Herendeen, Hillary; Gallacher, Kyla; Paquin, Dottie; Compton, Dennis R; Sahoo, Bishwabhusan; Kerrigan, Sean A; Burge, Matthew S; Nickels, Michael; Green, Jennifer L; Katzenellenbogen, John A; Tchesnokov, Alexei; Hattersley, Gary

    2011-02-10

    This report describes the discovery of RAD140, a potent, orally bioavailable, nonsteroidal selective androgen receptor modulator (SARM). The characterization of RAD140 in several preclinical models of anabolic androgen action is also described.

  18. Adipokinetic hormones and their G protein-coupled receptors emerged in Lophotrochozoa

    PubMed Central

    Li, Shizhong; Hauser, Frank; Skadborg, Signe K.; Nielsen, Stine V.; Kirketerp-Møller, Nikolaj; Grimmelikhuijzen, Cornelis J. P.

    2016-01-01

    Most multicellular animals belong to two evolutionary lineages, the Proto– and Deuterostomia, which diverged 640–760 million years (MYR) ago. Neuropeptide signaling is abundant in animals belonging to both lineages, but it is often unclear whether there exist evolutionary relationships between the neuropeptide systems used by proto- or deuterostomes. An exception, however, are members of the gonadotropin-releasing hormone (GnRH) receptor superfamily, which occur in both evolutionary lineages, where GnRHs are the ligands in Deuterostomia and GnRH-like peptides, adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP) are the ligands in Protostomia. AKH is a well-studied insect neuropeptide that mobilizes lipids and carbohydrates from the insect fat body during flight. In our present paper, we show that AKH is not only widespread in insects, but also in other Ecdysozoa and in Lophotrochozoa. Furthermore, we have cloned and deorphanized two G protein-coupled receptors (GPCRs) from the oyster Crassostrea gigas (Mollusca) that are activated by low nanomolar concentrations of oyster AKH (pQVSFSTNWGSamide). Our discovery of functional AKH receptors in molluscs is especially significant, because it traces the emergence of AKH signaling back to about 550 MYR ago and brings us closer to a more complete understanding of the evolutionary origins of the GnRH receptor superfamily. PMID:27628442

  19. Study of G-protein-coupled receptor-protein interactions by bioluminescence resonance energy transfer.

    PubMed

    Kroeger, Karen M; Eidne, Karin A

    2004-01-01

    Complex networks of protein-protein interactions are key determinants of cellular function, including those regulated by G-protein-coupled receptors (GPCRs). Formation of either stable or transitory complexes are involved in regulating all aspects of receptor function, from ligand binding through to signal transduction, desensitization, resensitization and downregulation. Today, 50% of all recently launched drugs are targeted against GPCRs. This particular class of proteins is extremely useful as a drug target because the receptors are partly located outside the cell, simplifying bioavailability and delivery of drugs directed against them. However, be