THE GRK4 SUBFAMILY OF G PROTEIN-COUPLED RECEPTOR KINASES: ALTERNATIVE SPLICING, GENE ORGANIZATION, AND SEQUENCE CONSERVATION

EPA Science Inventory

The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

Premont RT, Macrae AD, Aparicio SA, Kendall HE, Welch JE, Lefkowitz RJ.

Department of Medicine, Howard Hughes Medical Institute, Duke Univer...

RNA sequencing to determine the contribution of kinase receptor transactivation to G protein coupled receptor signalling in vascular smooth muscle cells.

PubMed

Kamato, Danielle; Bhaskarala, Venkata Vijayanand; Mantri, Nitin; Oh, Tae Gyu; Ling, Dora; Janke, Reearna; Zheng, Wenhua; Little, Peter J; Osman, Narin

2017-01-01

G protein coupled receptor (GPCR) signalling covers three major mechanisms. GPCR agonist engagement allows for the G proteins to bind to the receptor leading to a classical downstream signalling cascade. The second mechanism is via the utilization of the β-arrestin signalling molecule and thirdly via transactivation dependent signalling. GPCRs can transactivate protein tyrosine kinase receptors (PTKR) to activate respective downstream signalling intermediates. In the past decade GPCR transactivation dependent signalling was expanded to show transactivation of serine/threonine kinase receptors (S/TKR). Kinase receptor transactivation enormously broadens the GPCR signalling paradigm. This work utilizes next generation RNA-sequencing to study the contribution of transactivation dependent signalling to total protease activated receptor (PAR)-1 signalling. Transactivation, assessed as gene expression, accounted for 50 percent of the total genes regulated by thrombin acting through PAR-1 in human coronary artery smooth muscle cells. GPCR transactivation of PTKRs is approximately equally important as the transactivation of the S/TKR with 209 and 177 genes regulated respectively, via either signalling pathway. This work shows that genome wide studies can provide powerful insights into GPCR mediated signalling pathways.

Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases.

PubMed

Komolov, Konstantin E; Bhardwaj, Anshul; Benovic, Jeffrey L

2015-08-21

G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5'-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells.

PubMed

Montrose-Rafizadeh, C; Avdonin, P; Garant, M J; Rodgers, B D; Kole, S; Yang, H; Levine, M A; Schwindinger, W; Bernier, M

1999-03-01

Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.

Decreased expression of G-protein coupled receptor kinase 2 in cold thyroid nodules.

PubMed

Voigt, C; Holzapfel, H-P; Paschke, R

2005-02-01

G-protein coupled receptor kinases (GRKs) have been shown to regulate the homologous desensitization of different G-protein coupled receptors. We have previously demonstrated that the expression of GRK 3 and 4 is increased in hyperfunctioning thyroid nodules (HTNs) and that GRKs 2, 3, 5 and 6 are able to desensitize the TSHR in vitro. Since cold thyroid nodules (CTNs) and HTNs show different molecular and functional properties, different expression patterns of GRKs in these nodules can be expected. The comparison of GRK expression between CTNs and HTNs could give additional insight into the regulation mechanisms of these nodules. We therefore examined the expression of GRKs in CTNs and analyzed the differences to HTNs. The expression of the different GRKs in CTNs was measured by Western blot followed by chemiluminescence imaging. We found a decreased expression of GRK 2 in CTNs compared to their surrounding tissues and an increased expression of GRK 3 and 4 in CTNs, which is similar to HTNs. The decreased GRK 2 expression most likely results from reduced cAMP stimulation in CTNs. However, the increased GRK 3 and 4 expression in CTNs remains unclear and requires further investigations.

β2-Adrenergic receptors and G-protein-coupled receptor kinase 2 in rabbit pleural mesothelium.

PubMed

Sironi, Chiara; Bodega, Francesca; Armilli, Marta; Porta, Cristina; Zocchi, Luciano; Agostoni, Emilio

2010-09-30

Former studies on net rate of liquid absorption from small Ringer or 1% albumin-Ringer hydrothoraces in rabbits indicated that Na+ transport and solute-coupled liquid absorption by mesothelium is increased by pleural liquid dilution, and stimulation of β2-adrenoreceptors (β2AR). In this research we tried to provide molecular evidence for β2AR in visceral and parietal mesothelium of rabbit pleura. Moreover, because prolonged stimulation of β2AR may lead to desensitization mediated by G-protein-coupled receptor kinase 2 (GRK2), we also checked whether GRK2 is expressed in pleural mesothelium. To this end we performed immunoblot assays on total protein extracts from scraped visceral and parietal mesothelium, and from cultured pleural mesothelial cells of rabbits. All three samples showed β2AR and GRK2 specific bands. Copyright 2010 Elsevier B.V. All rights reserved.

Induction of Cardiac Fibrosis by β-Blocker in G Protein-independent and G Protein-coupled Receptor Kinase 5/β-Arrestin2-dependent Signaling Pathways*

PubMed Central

Nakaya, Michio; Chikura, Satsuki; Watari, Kenji; Mizuno, Natsumi; Mochinaga, Koji; Mangmool, Supachoke; Koyanagi, Satoru; Ohdo, Shigehiro; Sato, Yoji; Ide, Tomomi; Nishida, Motohiro; Kurose, Hitoshi

2012-01-01

G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called “biased ligands” elicit G protein-independent and β-arrestin-dependent signaling through GPCRs (biased agonism). Several β-blockers are known as biased ligands. All β-blockers inhibit the binding of agonists to the β-adrenergic receptors. In addition to β-blocking action, some β-blockers are reported to induce cellular responses through G protein-independent and β-arrestin-dependent signaling pathways. However, the physiological significance induced by the β-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a β1-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and β-arrestin2-dependent pathway. Metoprolol, a β-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between β1-adrenergic receptor and β-arrestin2, but not β-arrestin1. The interaction between β1-adrenergic receptor and β-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in β-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/β-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and β-arrestin-dependent signaling is a reason for the diversity of the effectiveness of β-blockers. PMID:22888001

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

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

Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian

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} complexmore » 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.« less

Increased expression of G-protein-coupled receptor kinases 3 and 4 in hyperfunctioning thyroid nodules.

PubMed

Voigt, Carsten; Holzapfel, Hans-Peter; Meyer, Silke; Paschke, Ralf

2004-07-01

G-protein-coupled receptor kinases (GRKs) are implicated in the pathophysiology of human diseases such as arterial hypertension, heart failure and rheumatoid arthritis. While G-protein-coupled receptor kinases 2 and 5 have been shown to be involved in the desensitization of the rat thyrotropin receptor (TSHR), their role in the pathophysiology of hyperfunctioning thyroid nodules (HTNs) is unknown. Therefore, we analyzed the expression pattern of the known GRKs in human thyroid tissue and investigated their function in the pathology of HTNs. The expression of different GRKs in human thyroid and HTNs was measured by Western blotting. The influence of GRK expression on TSHR function was analyzed by coexpression experiments in HEK 293 cells. We demonstrate that in addition to GRKs 2, 5 and 6, GRKs 3 and 4 are also expressed in the human thyroid. GRKs 2, 3, 5 and 6 are able to desensitize the TSHR in vitro. This GRK-induced desensitization is amplified by the additional over-expression of beta-arrestin 1 or 2. We did not find any mutations in the GRKs 2, 3 and 5 from 14 HTNs without TSHR mutations and Gsalpha mutations. The expression of GRKs 3 and 4 was increased in HTNs independently from the existence of TSHR mutations or Gsalpha mutations. In conclusion, the increased expression of GRK 3 in HTNs and the ability of GRK 3 to desensitize the TSHR in vitro, suggest a potential role for GRK 3 as a negative feedback regulator for the constitutively activated cAMP pathway in HTNs.

Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein.

PubMed

Liu, Songling; Premont, Richard T; Rockey, Don C

2014-06-27

Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ETB) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser(1177)), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ETB receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

G-protein-coupled receptors signaling pathways in new antiplatelet drug development.

PubMed

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

2015-03-01

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. © 2015 American Heart Association, Inc.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

PubMed Central

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

Regulation of mGlu4 metabotropic glutamate receptor signaling by type-2 G-protein coupled receptor kinase (GRK2).

PubMed

Iacovelli, L; Capobianco, L; Iula, M; Di Giorgi Gerevini, V; Picascia, A; Blahos, J; Melchiorri, D; Nicoletti, F; De Blasi, A

2004-05-01

We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist l-2-amino-4-phosphonobutanoate (l-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. Overexpression of GRK2 (but not GRK4) largely attenuated the stimulation of the MAPK pathway by l-AP4, whereas it slightly potentiated the inhibition of FSK-stimulated cAMP formation. Transfection with a kinase-dead mutant of GRK2 (GRK2-K220R) or with the C-terminal fragment of GRK2 also reduced the mGlu4-mediated stimulation of MAPK, suggesting that GRK2 binds to the Gbetagamma subunits to inhibit signal propagation toward the MAPK pathway. This was confirmed by the evidence that GRK2 coimmunoprecipitated with Gbetagamma subunits in an agonist-dependent manner. Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by l-AP4. We speculate that GRK2 acts as a "switch molecule" by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase.

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment

PubMed Central

Bagley, Elena E.

2014-01-01

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector. PMID

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

PubMed

Bagley, Elena E

2014-01-01

Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than E k . Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector.

Distinct Cellular and Subcellular Distributions of G Protein-Coupled Receptor Kinase and Arrestin Isoforms in the Striatum

PubMed Central

Bychkov, Evgeny; Zurkovsky, Lilia; Garret, Mika B.; Ahmed, Mohamed R.; Gurevich, Eugenia V.

2012-01-01

G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling. PMID:23139825

Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

PubMed

Bychkov, Evgeny; Zurkovsky, Lilia; Garret, Mika B; Ahmed, Mohamed R; Gurevich, Eugenia V

2012-01-01

G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

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

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

Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.

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 bemore » 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.« less

Probing receptor structure/function with chimeric G-protein-coupled receptors.

PubMed

Yin, Dezhong; Gavi, Shai; Wang, Hsien-yu; Malbon, Craig C

2004-06-01

Owing its name to an image borrowed from Greek mythology, a chimera is seen to represent a new entity created as a composite from existing creatures or, in this case, molecules. Making use of various combinations of three basic domains of the receptors (i.e., exofacial, transmembrane, and cytoplasmic segments) that couple agonist binding into activation of effectors through heterotrimeric G-proteins, molecular pharmacology has probed the basic organization, structure/function relationships of this superfamily of heptahelical receptors. Chimeric G-protein-coupled receptors obviate the need for a particular agonist ligand when the ligand is resistant to purification or, in the case of orphan receptors, is not known. Chimeric receptors created from distant members of the heptahelical receptors enable new strategies in understanding how these receptors transduce agonist binding into receptor activation and may be able to offer insights into the evolution of G-protein-coupled receptors from yeast to humans.

Activation of G-protein-coupled receptor 30 increases T-type calcium currents in trigeminal ganglion neurons via the cholera toxin-sensitive protein kinase A pathway.

PubMed

Yue, Jingxia; Zhang, Yi; Li, Xuemin; Gong, Shan; Tao, Jin; Jiang, Xinghong

2014-11-01

G protein-coupled receptor 30 (GPR30) is a seven transmembrane domain G protein coupled receptor. In our study, GPR30 expression was found in trigeminal ganglia (TG) in mice, detected by RT-PCR and western blotting. We examined the effects of GPR30 activation on T-type calcium channels using GPR30-specific compound 1 (G-1), a GPR30-selective agonist, in TG neurons and demonstrated that G-1 induced an increase in T-type calcium channel currents (T-currents) in TGs. Intracellular infusion of GDP-β-S and pre-treatment of the neurons with cholera toxin (CTX) blocked the effects of G-1, suggesting that the G(s)-protein was involved. Intracellular application of the protein kinase A (PKA) inhibitor PKI 6-22 or pretreatment of the neurons with H89 abolished G-1 -induced enhancement of T-currents in TG neurons. However, incubation with PKC inhibitor elicited no such effects. In conclusion, our study shows that activation of GPR30 by G-1 increases T-currents via the CTX-sensitive and PKA-dependent pathway.

The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors.

PubMed

Bondar, Alexey; Lazar, Josef

2017-06-09

The G i/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of G i/o -GPCR coupling and the high rate of G i/o signal transduction have been hypothesized to be enabled by the existence of stable associates between G i/o proteins and their cognate GPCRs in the inactive state (G i/o -GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gα i1 subunits labeled with fluorescent proteins and four GPCRs: the α 2A -adrenergic receptor, GABA B , cannabinoid receptor type 1 (CB 1 R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gα i1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gα i1 subunits and α 2A -adrenergic receptor, GABA B , or dopamine receptor type 2 receptors did not reveal any interaction between the G i1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB 1 R exhibited an interaction with the G i1 protein. Further experiments revealed that this interaction is caused solely by CB 1 R basal activity; no preassembly between CB 1 R and the G i1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active G i1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and G i1 (basal coupling). These findings suggest that G i1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

PubMed Central

El Far, Oussama; Betz, Heinrich

2002-01-01

G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

G protein-coupled receptor 30 in tumor development.

PubMed

Wang, Dengfeng; Hu, Lina; Zhang, Guonan; Zhang, Lin; Chen, Chen

2010-08-01

Estrogen plays several important physiological and pathological functions in not only reproductive system but many other systems as well. Its transcriptional activation has been traditionally described as being mediated by classic nuclear estrogen receptors (ERs). It is however established recently that a novel functional estrogen transmembrane receptor, G protein-coupled receptor 30 (GPR30), modulates both rapid non-genomic events and genomic transcriptional events of estrogen. It has been demonstrated that GPR30 promotes the progress of estrogen-related tumors through mitogen-activated protein kinase (MAPK) signaling pathways. Effects mediated by GPR30 are maintained when classic ERs are absent or blocked. In addition, GPR30 is involved in drug resistance, which is often occurring during cancer treatments. All these new findings strongly imply that GPR30 may be an important therapeutic target for estrogen-related tumors. Simultaneously blocking both GPR30 and classic ERs may be a better strategy for the treatment of estrogen-related tumors.

Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells.

PubMed

Wan, Qingwen; Okashah, Najeah; Inoue, Asuka; Nehmé, Rony; Carpenter, Byron; Tate, Christopher G; Lambert, Nevin A

2018-05-11

G protein-coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of Gα subunits that were developed for structural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organelles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of Gα subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase-mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein subtype-biased ligands. © 2018 Wan et al.

PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

PubMed

Dunn, Henry A; Ferguson, Stephen S G

2015-10-01

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

Lifetime of muscarinic receptor-G-protein complexes determines coupling efficiency and G-protein subtype selectivity.

PubMed

Ilyaskina, Olga S; Lemoine, Horst; Bünemann, Moritz

2018-05-08

G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of G i/o , G q/11 , G s , and G 12/13 proteins to muscarinic M 1 , M 2 , and M 3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M 3 -R interactions but rather the affinities of G q and G o proteins to M 3 -Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

Enhanced Uterine Contractility and Stillbirth in Mice Lacking G Protein-Coupled Receptor Kinase 6 (GRK6): Implications for Oxytocin Receptor Desensitization

PubMed Central

Mao, Lan; Pierce, Stephanie L.; Swamy, Geeta K.; Heine, R. Phillips; Murtha, Amy P.

2016-01-01

Oxytocin is a potent uterotonic agent and is used clinically for induction and augmentation of labor, as well as for prevention and treatment of postpartum hemorrhage. Oxytocin increases uterine contractility by activating the oxytocin receptor (OXTR), a member of the G protein-coupled receptor family, which is prone to molecular desensitization. After oxytocin binding, the OXTR is phosphorylated by a member of the G protein-coupled receptor kinase (GRK) family, which allows for recruitment of β-arrestin, receptor internalization, and desensitization. According to previous in vitro analyses, desensitization of calcium signaling by the OXTR is mediated by GRK6. The objective of this study was to determine the role of GRK6 in mediating uterine contractility. Here, we demonstrate that uterine GRK6 levels increase in pregnancy and using a telemetry device to measure changes in uterine contractility in live mice during labor, show that mice lacking GRK6 produce a phenotype of enhanced uterine contractility during both spontaneous and oxytocin-induced labor compared with wild-type or GRK5 knockout mice. In addition, the observed enhanced contractility was associated with high rates of term stillbirth. Lastly, using a heterologous in vitro model, we show that β-arrestin recruitment to the OXTR, which is necessary for homologous OXTR desensitization, is dependent on GRK6. Our findings suggest that GRK6-mediated OXTR desensitization in labor is necessary for normal uterine contractile patterns and optimal fetal outcome. PMID:26886170

Inhibition of prostatic smooth muscle contraction by the inhibitor of G protein-coupled receptor kinase 2/3, CMPD101.

PubMed

Yu, Qingfeng; Gratzke, Christian; Wang, Yiming; Herlemann, Annika; Strittmatter, Frank; Rutz, Beata; Stief, Christian G; Hennenberg, Martin

2018-07-15

Alpha1-adrenoceptors induce prostate smooth muscle contraction, and hold a prominent role for pathophysiology and therapy of lower urinary tract symptoms in benign prostatic hyperplasia. G protein-coupled receptors are regulated by posttranslational regulation, including phosphorylation by G protein-coupled receptor kinases 2 and 3 (GRK2/3). Although posttranslational adrenoceptor regulation has been recently suggested to occur in the prostate, this is still marginally understood. With the newly developed CMPD101, a small molecule inhibitor with assumed specificity for GRK2/3 is now available. Here, we studied effects of CMPD101 on smooth muscle contraction of human prostate tissue. Electric field stimulation caused frequency-dependent contractions, which were inhibited concentration-dependently by CMPD101 (5 µM, 50 µM). 50 µM of CMPD101 did not affect myosin light chain (MCL) phosphorylation or Rho kinase activity, and did not alter contractions induced by highmolar KCl. Noradrenaline, the α 1 -adrenoceptor agonist phenylephrine, endothelin-1, and the thromboxane A 2 analogue U46619 induced concentration-dependent contractions, which were inhibited by CMPD101 (50 µM). CMPD101 (50 µM) did not change phosphorylation of β 2 -adrenoceptors or β 2 -adrenergic relaxation of prostate strips. Molecular detection by Western blot and peroxidase staining suggested expression of GRK2 and GRK3 in human prostates. Double labeling in fluorescence staining confirmed that immunoreactivity for GRK2 and GRK3 was located to smooth muscle cells in the prostate stroma. In conclusion, CMPD101 inhibits adrenergic, neurogenic, and non-adrenergic smooth muscle contractions in the human prostate. Underlying mechanisms may be independent from GRK inhibition, and from inhibition of MLC kinase and Rho kinase. This may point to unknown properties of CMPD101. Copyright © 2018 Elsevier B.V. All rights reserved.

Thrombin-mediated proteoglycan synthesis utilizes both protein-tyrosine kinase and serine/threonine kinase receptor transactivation in vascular smooth muscle cells.

PubMed

Burch, Micah L; Getachew, Robel; Osman, Narin; Febbraio, Mark A; Little, Peter J

2013-03-08

G protein-coupled receptor signaling is mediated by three main mechanisms of action; these are the classical pathway, β-arrestin scaffold signaling, and the transactivation of protein-tyrosine kinase receptors such as those for EGF and PDGF. Recently, it has been demonstrated that G protein-coupled receptors can also mediate signals via transactivation of serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Atherosclerosis is characterized by the development of lipid-laden plaques in blood vessel walls. Initiation of plaque development occurs via low density lipoprotein retention in the neointima of vessels due to binding with modified proteoglycans secreted by vascular smooth muscle cells. Here we show that transactivation of protein-tyrosine kinase receptors is mediated by matrix metalloproteinase triple membrane bypass signaling. In contrast, serine/threonine kinase receptor transactivation is mediated by a cytoskeletal rearrangement-Rho kinase-integrin system, and both protein-tyrosine kinase and serine/threonine kinase receptor transactivation concomitantly account for the total proteoglycan synthesis stimulated by thrombin in vascular smooth muscle. This work provides evidence of thrombin-mediated proteoglycan synthesis and paves the way for a potential therapeutic target for plaque development and atherosclerosis.

Design and functional characterization of a novel, arrestin-biased designer G protein-coupled receptor.

PubMed

Nakajima, Ken-ichiro; Wess, Jürgen

2012-10-01

Mutational modification of distinct muscarinic receptor subtypes has yielded novel designer G protein-coupled receptors (GPCRs) that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs [alternative name: designer receptors exclusively activated by designer drug (DREADDs)] have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. As is the case with other GPCRs, CNO-activated DREADDs not only couple to heterotrimeric G proteins but can also recruit proteins of the arrestin family (arrestin-2 and -3). Accumulating evidence suggests that arrestins can act as scaffolding proteins to promote signaling through G protein-independent signaling pathways. To explore the physiological relevance of these arrestin-dependent signaling pathways, the availability of an arrestin-biased DREADD would be highly desirable. In this study, we describe the development of an M₃ muscarinic receptor-based DREADD [Rq(R165L)] that is no longer able to couple to G proteins but can recruit arrestins and promote extracellular signal-regulated kinase-1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of mouse insulinoma (MIN6) cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types.

Structure and Function of the Hypertension Variant A486V of G Protein-coupled Receptor Kinase 4

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

Allen, Samantha J.; Parthasarathy, Gopal; Darke, Paul L.

G-protein-coupled receptor (GPCR) kinases (GRKs) bind to and phosphorylate GPCRs, initiating the process of GPCR desensitization and internalization. GRK4 is implicated in the regulation of blood pressure, and three GRK4 polymorphisms (R65L, A142V, and A486V) are associated with hypertension. Here, we describe the 2.6 Å structure of human GRK4α A486V crystallized in the presence of 5'-adenylyl β,γ-imidodiphosphate. The structure of GRK4α is similar to other GRKs, although slight differences exist within the RGS homology (RH) bundle subdomain, substrate-binding site, and kinase C-tail. The RH bundle subdomain and kinase C-terminal lobe form a strikingly acidic surface, whereas the kinase N-terminal lobemore » and RH terminal subdomain surfaces are much more basic. In this respect, GRK4α is more similar to GRK2 than GRK6. A fully ordered kinase C-tail reveals interactions linking the C-tail with important determinants of kinase activity, including the αB helix, αD helix, and the P-loop. Autophosphorylation of wild-type GRK4α is required for full kinase activity, as indicated by a lag in phosphorylation of a peptide from the dopamine D1 receptor without ATP preincubation. In contrast, this lag is not observed in GRK4α A486V. Phosphopeptide mapping by mass spectrometry indicates an increased rate of autophosphorylation of a number of residues in GRK4α A486V relative to wild-type GRK4α, including Ser-485 in the kinase C-tail.« less

Evaluation of the Role of G Protein-Coupled Receptor Kinase 3 in Desensitization of Mouse Odorant Receptors in a Mammalian Cell Line and in Olfactory Sensory Neurons

PubMed Central

Kato, Aya; Reisert, Johannes; Ihara, Sayoko; Yoshikawa, Keiichi

2014-01-01

Thousands of odors are sensed and discriminated by G protein-coupled odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs). G protein-coupled receptor kinases (GRKs) may have a role in desensitization of ORs. However, whether ORs are susceptible to agonist-dependent desensitization and whether GRKs affect odorant responsiveness of OSNs are currently unknown. Here we show that GRK3 attenuated the agonist responsiveness of a specific mouse odorant receptor for eugenol (mOR-EG) upon agonist pretreatment in HEK293 cells, but GRK3 did not affect the response amplitude or the recovery kinetics upon repeated agonist stimulation. We performed electrophysiological recordings of single OSNs which expressed mOR-EG and green fluorescent protein (GFP) in the presence or absence of GRK3. The kinetics and amplitude of agonist responsiveness of individual GFP-labeled mOR-EG neurons were not significantly affected by the absence of GRK3. These results indicate that the role of GRK3 in attenuating ORs responsiveness in OSNs may have been overestimated. PMID:25313015

Design and Functional Characterization of a Novel, Arrestin-Biased Designer G Protein-Coupled Receptor

PubMed Central

Nakajima, Ken-ichiro

2012-01-01

Mutational modification of distinct muscarinic receptor subtypes has yielded novel designer G protein-coupled receptors (GPCRs) that are unable to bind acetylcholine (ACh), the endogenous muscarinic receptor ligand, but can be efficiently activated by clozapine-N-oxide (CNO), an otherwise pharmacologically inert compound. These CNO-sensitive designer GPCRs [alternative name: designer receptors exclusively activated by designer drug (DREADDs)] have emerged as powerful new tools to dissect the in vivo roles of distinct G protein signaling pathways in specific cell types or tissues. As is the case with other GPCRs, CNO-activated DREADDs not only couple to heterotrimeric G proteins but can also recruit proteins of the arrestin family (arrestin-2 and -3). Accumulating evidence suggests that arrestins can act as scaffolding proteins to promote signaling through G protein-independent signaling pathways. To explore the physiological relevance of these arrestin-dependent signaling pathways, the availability of an arrestin-biased DREADD would be highly desirable. In this study, we describe the development of an M3 muscarinic receptor-based DREADD [Rq(R165L)] that is no longer able to couple to G proteins but can recruit arrestins and promote extracellular signal-regulated kinase-1/2 phosphorylation in an arrestin- and CNO-dependent fashion. Moreover, CNO treatment of mouse insulinoma (MIN6) cells expressing the Rq(R165L) construct resulted in a robust, arrestin-dependent stimulation of insulin release, directly implicating arrestin signaling in the regulation of insulin secretion. This newly developed arrestin-biased DREADD represents an excellent novel tool to explore the physiological relevance of arrestin signaling pathways in distinct tissues and cell types. PMID:22821234

Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

PubMed

Ho, Hsiang-Ting; Belevych, Andriy E; Liu, Bin; Bonilla, Ingrid M; Radwański, Przemysław B; Kubasov, Igor V; Valdivia, Héctor H; Schober, Karsten; Carnes, Cynthia A; Györke, Sándor

2016-11-01

Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance. © 2016 American Heart Association, Inc.

Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

PubMed

Stoyanov, B; Volinia, S; Hanck, T; Rubio, I; Loubtchenkov, M; Malek, D; Stoyanova, S; Vanhaesebroeck, B; Dhand, R; Nürnberg, B

1995-08-04

Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility

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

Thal, David M.; Homan, Kristoff T.; Chen, Jun

2012-08-10

G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. In this paper we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine–Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice withmore » paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.« less

Cardiac hyporesponsiveness in severe sepsis is associated with nitric oxide-dependent activation of G protein receptor kinase.

PubMed

Dal-Secco, Daniela; DalBó, Silvia; Lautherbach, Natalia E S; Gava, Fábio N; Celes, Mara R N; Benedet, Patricia O; Souza, Adriana H; Akinaga, Juliana; Lima, Vanessa; Silva, Katiussia P; Kiguti, Luiz Ricardo A; Rossi, Marcos A; Kettelhut, Isis C; Pupo, André S; Cunha, Fernando Q; Assreuy, Jamil

2017-07-01

G protein-coupled receptor kinase isoform 2 (GRK2) has a critical role in physiological and pharmacological responses to endogenous and exogenous substances. Sepsis causes an important cardiovascular dysfunction in which nitric oxide (NO) has a relevant role. The present study aimed to assess the putative effect of inducible NO synthase (NOS2)-derived NO on the activity of GRK2 in the context of septic cardiac dysfunction. C57BL/6 mice were submitted to severe septic injury by cecal ligation and puncture (CLP). Heart function was assessed by isolated and perfused heart, echocardiography, and β-adrenergic receptor binding. GRK2 was determined by immunofluorescence and Western blot analysis in the heart and isolated cardiac myocytes. Sepsis increased NOS2 expression in the heart, increased plasma nitrite + nitrate levels, and reduced isoproterenol-induced isolated ventricle contraction, whole heart tension development, and β-adrenergic receptor density. Treatment with 1400W or with GRK2 inhibitor prevented CLP-induced cardiac hyporesponsiveness 12 and 24 h after CLP. Increased labeling of total and phosphorylated GRK2 was detected in hearts after CLP. With treatment of 1400W or in hearts taken from septic NOS2 knockout mice, the activation of GRK2 was reduced. 1400W or GRK2 inhibitor reduced mortality, improved echocardiographic cardiac parameters, and prevented organ damage. Therefore, during sepsis, NOS2-derived NO increases GRK2, which leads to a reduction in β-adrenergic receptor density, contributing to the heart dysfunction. Isolated cardiac myocyte data indicate that NO acts through the soluble guanylyl cyclase/cGMP/PKG pathway. GRK2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction. NEW & NOTEWORTHY The main novelty presented here is to show that septic shock induces cardiac hyporesponsiveness to isoproterenol by a mechanism dependent on nitric oxide and mediated by G protein-coupled receptor kinase isoform 2. Therefore

G Protein-Coupled Receptor Kinase 2 Promotes Flaviviridae Entry and Replication

PubMed Central

Le Sommer, Caroline; Barrows, Nicholas J.; Bradrick, Shelton S.; Pearson, James L.; Garcia-Blanco, Mariano A.

2012-01-01

Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2×2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK−/− mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases. PMID:23029581

G protein-coupled receptor kinase 2 promotes flaviviridae entry and replication.

PubMed

Le Sommer, Caroline; Barrows, Nicholas J; Bradrick, Shelton S; Pearson, James L; Garcia-Blanco, Mariano A

2012-01-01

Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2 × 2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK(-/-) mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases.

Myocardial pathology induced by aldosterone is dependent on non-canonical activities of G protein-coupled receptor kinases

PubMed Central

Cannavo, Alessandro; Liccardo, Daniela; Eguchi, Akito; Elliott, Katherine J.; Traynham, Christopher J.; Ibetti, Jessica; Eguchi, Satoru; Leosco, Dario; Ferrara, Nicola; Rengo, Giuseppe; Koch, Walter J.

2016-01-01

Hyper-aldosteronism is associated with myocardial dysfunction including induction of cardiac fibrosis and maladaptive hypertrophy. Mechanisms of these cardiotoxicities are not fully understood. Here we show that mineralocorticoid receptor (MR) activation by aldosterone leads to pathological myocardial signalling mediated by mitochondrial G protein-coupled receptor kinase 2 (GRK2) pro-death activity and GRK5 pro-hypertrophic action. Moreover, these MR-dependent GRK2 and GRK5 non-canonical activities appear to involve cross-talk with the angiotensin II type-1 receptor (AT1R). Most importantly, we show that ventricular dysfunction caused by chronic hyper-aldosteronism in vivo is completely prevented in cardiac Grk2 knockout mice (KO) and to a lesser extent in Grk5 KO mice. However, aldosterone-induced cardiac hypertrophy is totally prevented in Grk5 KO mice. We also show human data consistent with MR activation status in heart failure influencing GRK2 levels. Therefore, our study uncovers GRKs as targets for ameliorating pathological cardiac effects associated with high-aldosterone levels. PMID:26932512

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.

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

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

2017-09-01

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 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 signaling 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 ( 19 F-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. Copyright © 2017 by The Author(s).

G protein-coupled estrogen receptor 1/G protein-coupled receptor 30 localizes in the plasma membrane and traffics intracellularly on cytokeratin intermediate filaments.

PubMed

Sandén, Caroline; Broselid, Stefan; Cornmark, Louise; Andersson, Krister; Daszkiewicz-Nilsson, Joanna; Mårtensson, Ulrika E A; Olde, Björn; Leeb-Lundberg, L M Fredrik

2011-03-01

G protein-coupled receptor 30 [G protein-coupled estrogen receptor 1 (GPER1)], has been introduced as a membrane estrogen receptor and a candidate cancer biomarker and therapeutic target. However, several questions surround the subcellular localization and signaling of this receptor. In native cells, including mouse myoblast C(2)C(12) cells, Madin-Darby canine kidney epithelial cells, and human ductal breast epithelial tumor T47-D cells, G-1, a GPER1 agonist, and 17β-estradiol stimulated GPER1-dependent cAMP production, a defined plasma membrane (PM) event, and recruitment of β-arrestin2 to the PM. Staining of fixed and live cells showed that GPER1 was localized both in the PM and on intracellular structures. One such intracellular structure was identified as cytokeratin (CK) intermediate filaments, including those composed of CK7 and CK8, but apparently not endoplasmic reticulum, Golgi, or microtubules. Reciprocal coimmunoprecipitation of GPER1 and CKs confirmed an association of these proteins. Live staining also showed that the PM receptors constitutively internalize apparently to reach CK filaments. Receptor localization was supported using FLAG- and hemagglutinin-tagged GPER1. We conclude that GPER1-mediated stimulation of cAMP production and β-arrestin2 recruitment occur in the PM. Furthermore, the PM receptors constitutively internalize and localize intracellularly on CK. This is the first observation that a G protein-coupled receptor is capable of associating with intermediate filaments, which may be important for GPER1 regulation in epithelial cells and the relationship of this receptor to cancer.

Receptor recruitment: A mechanism for interactions between G protein-coupled receptors

PubMed Central

Holtbäck, Ulla; Brismar, Hjalmar; DiBona, Gerald F.; Fu, Michael; Greengard, Paul; Aperia, Anita

1999-01-01

There is a great deal of evidence for synergistic interactions between G protein-coupled signal transduction pathways in various tissues. As two specific examples, the potent effects of the biogenic amines norepinephrine and dopamine on sodium transporters and natriuresis can be modulated by neuropeptide Y and atrial natriuretic peptide, respectively. Here, we report, using a renal epithelial cell line, that both types of modulation involve recruitment of receptors from the interior of the cell to the plasma membrane. The results indicate that recruitment of G protein-coupled receptors may be a ubiquitous mechanism for receptor sensitization and may play a role in the modulation of signal transduction comparable to that of the well established phenomenon of receptor endocytosis and desensitization. PMID:10377404

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. © 2014 Elsevier B.V. All rights reserved.

The effect of G protein-coupled receptor kinase 2 (GRK2) on lactation and on proliferation of mammary epithelial cells from dairy cows.

PubMed

Hou, Xiaoming; Hu, Hongliu; Lin, Ye; Qu, Bo; Gao, Xuejun; Li, Qingzhang

2016-07-01

Milk protein is an important component of milk and a nutritional source for human consumption. To better understand the molecular events underlying synthesis of milk proteins, the global gene expression patterns in mammary glands of dairy cow with high-quality milk (>3% milk protein; >3.5% milk fat) and low-quality milk (<3% milk protein; <3.5% milk fat) were examined via digital gene expression study. A total of 139 upregulated and 66 downregulated genes were detected in the mammary tissues of lactating cows with high-quality milk compared with the tissues of cows with low-quality milk. A pathway enrichment study of these genes revealed that the top 5 pathways that were differentially affected in the tissues of cows with high- versus low-quality milk involved metabolic pathways, cancer, cytokine-cytokine receptor interactions, regulation of the actin cytoskeleton, and insulin signaling. We also found that the G protein-coupled receptor kinase 2 (GRK2) was one of the most highly upregulated genes in lactating mammary tissue with low-quality milk compared with tissue with high-quality milk. The knockdown of GRK2 in cultured bovine mammary epithelial cells enhanced CSN2 expression and activated signaling molecules related to translation, including protein kinase B, mammalian target of rapamycin, and p70 ribosomal protein S6 kinase 1 (S6K1), whereas overexpression of GRK2 had the opposite effects. However, expression of genes involved in the mitogen-activated protein kinase pathway was positively regulated by GRK2. Therefore, GRK2 seems to act as a negative mediator of milk-protein synthesis via the protein kinase B-mammalian target of rapamycin signaling axis. Furthermore, GRK2 may negatively control milk-protein synthesis by activating the mitogen-activated protein kinase pathway in dairy cow mammary epithelial cells. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

PubMed

Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

2015-03-13

Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

High-fat diet induces protein kinase A and G-protein receptor kinase phosphorylation of β2 -adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts.

PubMed

Fu, Qin; Hu, Yuting; Wang, Qingtong; Liu, Yongming; Li, Ning; Xu, Bing; Kim, Sungjin; Chiamvimonvat, Nipavan; Xiang, Yang K

2017-03-15

Patients with diabetes show a blunted cardiac inotropic response to β-adrenergic stimulation despite normal cardiac contractile reserve. Acute insulin stimulation impairs β-adrenergically induced contractile function in isolated cardiomyocytes and Langendorff-perfused hearts. In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high-fat diet (HFD) feeding on the cardiac β 2 -adrenergic receptor signalling and the impacts on cardiac contractile function. We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β-adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β 2 -adrenergic receptor phosphorylation at protein kinase A and G-protein receptor kinase sites in the myocardium. The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high-fat diet (HFD) on the insulin-adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD-fed mice displayed a significant elevation of phosphorylation of the β 2 -adrenergic receptor (β 2 AR) at both the protein kinase A site serine 261/262 and the G-protein-coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD-fed mice also displayed a reduced contractile response to

c-Src, Insulin-Like Growth Factor I Receptor, G-Protein-Coupled Receptor Kinases and Focal Adhesion Kinase are Enriched Into Prostate Cancer Cell Exosomes.

PubMed

DeRita, Rachel M; Zerlanko, Brad; Singh, Amrita; Lu, Huimin; Iozzo, Renato V; Benovic, Jeffrey L; Languino, Lucia R

2017-01-01

It is well known that Src tyrosine kinase, insulin-like growth factor 1 receptor (IGF-IR), and focal adhesion kinase (FAK) play important roles in prostate cancer (PrCa) development and progression. Src, which signals through FAK in response to integrin activation, has been implicated in many aspects of tumor biology, such as cell proliferation, metastasis, and angiogenesis. Furthermore, Src signaling is known to crosstalk with IGF-IR, which also promotes angiogenesis. In this study, we demonstrate that c-Src, IGF-IR, and FAK are packaged into exosomes (Exo), c-Src in particular being highly enriched in Exo from the androgen receptor (AR)-positive cell line C4-2B and AR-negative cell lines PC3 and DU145. Furthermore, we show that the active phosphorylated form of Src (Src pY416 ) is co-expressed in Exo with phosphorylated FAK (FAK pY861 ), a known target site of Src, which enhances proliferation and migration. We further demonstrate for the first time exosomal enrichment of G-protein-coupled receptor kinase (GRK) 5 and GRK6, both of which regulate Src and IGF-IR signaling and have been implicated in cancer. Finally, Src pY416 and c-Src are both expressed in Exo isolated from the plasma of prostate tumor-bearing TRAMP mice, and those same mice have higher levels of exosomal c-Src than their wild-type counterparts. In summary, we provide new evidence that active signaling molecules relevant to PrCa are enriched in Exo, and this suggests that the Src signaling network may provide useful biomarkers detectable by liquid biopsy, and may contribute to PrCa progression via Exo. J. Cell. Biochem. 118: 66-73, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

PubMed

Miao, Yinglong; McCammon, J Andrew

2018-03-20

Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation.

PubMed

Ferreira, Filipe; Foley, Matthew; Cooke, Alex; Cunningham, Margaret; Smith, Gemma; Woolley, Robert; Henderson, Graeme; Kelly, Eamonn; Mundell, Stuart; Smythe, Elizabeth

2012-08-07

Signaling by transmembrane receptors such as G protein-coupled receptors (GPCRs) occurs at the cell surface and throughout the endocytic pathway, and signaling from the cell surface may differ in magnitude and downstream output from intracellular signaling. As a result, the rate at which signaling molecules traverse the endocytic pathway makes a significant contribution to downstream output. Modulation of the core endocytic machinery facilitates differential uptake of individual cargoes. Clathrin-coated pits are a major entry portal where assembled clathrin forms a lattice around invaginating buds that have captured endocytic cargo. Clathrin assembles into triskelia composed of three clathrin heavy chains and associated clathrin light chains (CLCs). Despite the identification of clathrin-coated pits at the cell surface over 30 years ago, the functions of CLCs in endocytosis have been elusive. In this work, we identify a novel role for CLCs in the regulated endocytosis of specific cargoes. Small interfering RNA-mediated knockdown of either CLCa or CLCb inhibits the uptake of GPCRs. Moreover, we demonstrate that phosphorylation of Ser204 in CLCb is required for efficient endocytosis of a subset of GPCRs and identify G protein-coupled receptor kinase 2 (GRK2) as a kinase that can phosphorylate CLCb on Ser204. Overexpression of CLCb(S204A) specifically inhibits the endocytosis of those GPCRs whose endocytosis is GRK2-dependent. Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs. Copyright © 2012 Elsevier Ltd. All rights reserved.

Ligand screening system using fusion proteins of G protein-coupled receptors with G protein alpha subunits.

PubMed

Suga, Hinako; Haga, Tatsuya

2007-01-01

G protein-coupled receptors (GPCRs) constitute one of the largest families of genes in the human genome, and are the largest targets for drug development. Although a large number of GPCR genes have recently been identified, ligands have not yet been identified for many of them. Various assay systems have been employed to identify ligands for orphan GPCRs, but there is still no simple and general method to screen for ligands of such GPCRs, particularly of G(i)-coupled receptors. We have examined whether fusion proteins of GPCRs with G protein alpha subunit (Galpha) could be utilized for ligand screening and showed that the fusion proteins provide an effective method for the purpose. This article focuses on the followings: (1) characterization of GPCR genes and GPCRs, (2) identification of ligands for orphan GPCRs, (3) characterization of GPCR-Galpha fusion proteins, and (4) identification of ligands for orphan GPCRs using GPCR-Galpha fusion proteins.

Signaling through G protein coupled receptors.

PubMed

Tuteja, Narendra

2009-10-01

Heterotrimeric G proteins (Galpha, Gbeta/Ggamma subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane alpha-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Galpha subunit. This leads to the dissociation of Gbeta/Ggamma dimer from Galpha. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Galpha-GTP is hydrolyzed to GDP and Galpha becomes inactive (Galpha-GDP), which leads to its re-association with the Gbeta/Ggamma dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.

Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons.

PubMed

Zhang, Yanmin; Sheng, Hui; Qi, Jinshun; Ma, Bei; Sun, Jihu; Li, Shaofeng; Ni, Xin

2012-04-01

Glucocorticoids (GCs) have been demonstrated to act through both genomic and nongenomic mechanisms. The present study demonstrated that corticosterone rapidly suppressed the activity of N-methyl-D-aspartate (NMDA) receptors in cultured hippocampal neurons. The effect was maintained with corticosterone conjugated to bovine serum albumin and blocked by inhibition of G protein activity with intracellular GDP-β-S application. Corticosterone increased GTP-bound G(s) protein and cyclic AMP (cAMP) production, activated phospholipase Cβ(3) (PLC-β(3)), and induced inositol-1,4,5-triphosphate (IP(3)) production. Blocking PLC and the downstream cascades with PLC inhibitor, IP(3) receptor antagonist, Ca(2+) chelator, and protein kinase C (PKC) inhibitors prevented the actions of corticosterone. Blocking adenylate cyclase (AC) and protein kinase A (PKA) caused a decrease in NMDA-evoked currents. Application of corticosterone partly reversed the inhibition of NMDA currents caused by blockage of AC and PKA. Intracerebroventricular administration of corticosterone significantly suppressed long-term potentiation (LTP) in the CA1 region of the hippocampus within 30 min in vivo, implicating the possibly physiological significance of rapid effects of GC on NMDA receptors. Taken together, our results indicate that GCs act on a putative G protein-coupled receptor to activate multiple signaling pathways in hippocampal neurons, and the rapid suppression of NMDA activity by GCs is dependent on PLC and downstream signaling.

G-protein-coupled receptor kinase 2 and endothelial dysfunction: molecular insights and pathophysiological mechanisms

PubMed Central

Taguchi, Kumiko; Matsumoto, Takayuki; Kobayashi, Tsuneo

2015-01-01

Smooth muscle cells (SMC) and endothelial cells are the major cell types in blood vessels. The principal function of vascular SMC in the body is to regulate blood flow and pressure through contraction and relaxation. The endothelium performs a crucial role in maintaining vascular integrity by achieving whole-organ metabolic homeostasis via the production of factors associated with vasoconstriction or vasorelaxation. In this review, we have focused on the production of nitric oxide (NO), a vasorelaxation factor. The extent of NO production represents a key marker in vascular health. A decrease in NO is capable of inducing pathological conditions associated with endothelial dysfunction, such as obesity, diabetes, cardiovascular disease, and atherosclerosis. Recent studies have strongly implicated the involvement of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cardiovascular disease. Vasculature which is affected by insulin resistance and type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction by reducing intracellular NO. GRK2 activation also induces changes in the subcellular localization of GRK2 itself and also of β-arrestin 2, a downstream protein. In this review, we describe the pathophysiological mechanisms of insulin resistance and diabetes, focusing on the signal transduction for NO production via GRK2 and β-arrestin 2, providing novel insights into the potential field of translational investigation in the treatment of diabetic complications. PMID:26447102

G-protein-coupled receptor kinase 2 and endothelial dysfunction: molecular insights and pathophysiological mechanisms.

PubMed

Taguchi, Kumiko; Matsumoto, Takayuki; Kobayashi, Tsuneo

2015-01-01

Smooth muscle cells (SMC) and endothelial cells are the major cell types in blood vessels. The principal function of vascular SMC in the body is to regulate blood flow and pressure through contraction and relaxation. The endothelium performs a crucial role in maintaining vascular integrity by achieving whole-organ metabolic homeostasis via the production of factors associated with vasoconstriction or vasorelaxation. In this review, we have focused on the production of nitric oxide (NO), a vasorelaxation factor. The extent of NO production represents a key marker in vascular health. A decrease in NO is capable of inducing pathological conditions associated with endothelial dysfunction, such as obesity, diabetes, cardiovascular disease, and atherosclerosis. Recent studies have strongly implicated the involvement of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cardiovascular disease. Vasculature which is affected by insulin resistance and type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction by reducing intracellular NO. GRK2 activation also induces changes in the subcellular localization of GRK2 itself and also of β-arrestin 2, a downstream protein. In this review, we describe the pathophysiological mechanisms of insulin resistance and diabetes, focusing on the signal transduction for NO production via GRK2 and β-arrestin 2, providing novel insights into the potential field of translational investigation in the treatment of diabetic complications.

Ca(2+)-sensitive tyrosine kinase Pyk2/CAK beta-dependent signaling is essential for G-protein-coupled receptor agonist-induced hypertrophy.

PubMed

Hirotani, Shinichi; Higuchi, Yoshiharu; Nishida, Kazuhiko; Nakayama, Hiroyuki; Yamaguchi, Osamu; Hikoso, Shungo; Takeda, Toshihiro; Kashiwase, Kazunori; Watanabe, Tetsuya; Asahi, Michio; Taniike, Masayuki; Tsujimoto, Ikuko; Matsumura, Yasushi; Sasaki, Terukatsu; Hori, Masatsugu; Otsu, Kinya

2004-06-01

G-protein-coupled receptor agonists including endothelin-1 (ET-1) and phenylephrine (PE) induce hypertrophy in neonatal ventricular cardiomyocytes. Others and we previously reported that Rac1 signaling pathway plays an important role in this agonist-induced cardiomyocyte hypertrophy. In this study reported here, we found that a Ca(2+)-sensitive non-receptor tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2)/cell adhesion kinase beta (CAKbeta), is involved in ET-1- and PE-induced cardiomyocyte hypertrophy medicated through Rac1 activation. ET-1, PE or the Ca(2+) inophore, ionomycin, stimulated a rapid increase in tyrosine phosphorylation of Pyk2. The tyrosine phosphorylation of Pyk2 was suppressed by the Ca(2+) chelator, BAPTA. ET-1- or PE-induced increases in [(3)H]-leucine incorporation and expression of atrial natriuretic factor and the enhancement of sarcomere organization. Infection of cardiomyocytes with an adenovirus expressing a mutant Pyk2 which lacked its kinase domain or its ability to bind to c-Src, eliminated ET-1- and PE-induced hypertrophic responses. Inhibition of Pyk2 activation also suppressed Rac1 activation and reactive oxygen species (ROS) production. These findings suggest that the signal transduction pathway leading to hypertrophy involves Ca(2+)-induced Pyk2 activation followed by Rac1-dependent ROS production.

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.

G protein-coupled receptors: the inside story.

PubMed

Jalink, Kees; Moolenaar, Wouter H

2010-01-01

Recent findings necessitate revision of the traditional view of G protein-coupled receptor (GPCR) signaling and expand the diversity of mechanisms by which receptor signaling influences cell behavior in general. GPCRs elicit signals at the plasma membrane and are then rapidly removed from the cell surface by endocytosis. Internalization of GPCRs has long been thought to serve as a mechanism to terminate the production of second messengers such as cAMP. However, recent studies show that internalized GPCRs can continue to either stimulate or inhibit cAMP production in a sustained manner. They do so by remaining associated with their cognate G protein subunit and adenylyl cyclase at endosomal compartments. Once internalized, the GPCRs produce cellular responses distinct from those elicited at the cell surface.

Arrestin–dependent but G–protein coupled receptor kinase–independent uncoupling of D2–dopamine receptors

PubMed Central

Celver, Jeremy; Sharma, Meenakshi; Thanawala, Vaidehi; Octeau, J. Christopher; Kovoor, Abraham

2016-01-01

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (Kir3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-Kir3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor. PMID:23815307

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

PubMed

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

2017-06-16

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

Prognostic Value of Lymphocyte G Protein-Coupled Receptor Kinase-2 Protein Levels in Patients With Heart Failure

PubMed Central

Rengo, Giuseppe; Pagano, Gennaro; Filardi, Pasquale Perrone; Femminella, Grazia Daniela; Parisi, Valentina; Cannavo, Alessandro; Liccardo, Daniela; Komici, Klara; Gambino, Giuseppina; D’Amico, Maria Loreta; de Lucia, Claudio; Paolillo, Stefania; Trimarco, Bruno; Vitale, Dino Franco; Ferrara, Nicola; Koch, Walter J; Leosco, Dario

2016-01-01

Rationale Sympathetic nervous system (SNS) hyperactivity is associated with poor prognosis in patients with HF, yet routine assessment of SNS activation is not recommended for clinical practice. Myocardial G protein-coupled receptor kinase 2 (GRK2) is up-regulated in heart failure (HF) patients, causing dysfunctional β-adrenergic receptor signaling. Importantly, myocardial GRK2 levels correlate with levels found in peripheral lymphocytes of HF patients. Objective The independent prognostic value of blood GRK2 measurements in HF patients has never been investigated, thus, the purpose of the present study was to evaluate whether lymphocyte GRK2 levels predict clinical outcome in HF patients. Methods and Results We prospectively studied 257 HF patients with mean left ventricular ejection fraction (LVEF) of 31.4±8.5%. At the time of enrollment, plasma norepinephrine, serum NT-proBNP and lymphocyte GRK2 levels, as well as clinical and instrumental variables were measured. The prognostic value of GRK2 to predict cardiovascular (CV) death and all-cause mortality was assessed using the Cox proportional hazard model including demographic, clinical, instrumental and laboratory data. Over a mean follow-up period of 37.5±20.2 months (range: 3–60 months) there were 102 CV deaths. Age, LVEF, NYHA class, Chronic Obstructive Pulmonary Disease, Chronic Kidney Disease, N-terminal-pro Brain Natriuretic Peptide, and lymphocyte GRK2 protein levels were independent predictors of CV mortality in HF patients. GRK2 levels showed an additional prognostic and clinical value over demographic and clinical variables. The independent prognostic value of lymphocyte GRK2 levels was also confirmed for all-cause mortality. Conclusion Lymphocyte GRK2 protein levels can independently predict prognosis in patients with HF. PMID:26884616

The G protein-coupled receptors deorphanization landscape.

PubMed

Laschet, Céline; Dupuis, Nadine; Hanson, Julien

2018-07-01

G protein-coupled receptors (GPCRs) are usually highlighted as being both the largest family of membrane proteins and the most productive source of drug targets. However, most of the GPCRs are understudied and hence cannot be used immediately for innovative therapeutic strategies. Besides, there are still around 100 orphan receptors, with no described endogenous ligand and no clearly defined function. The race to discover new ligands for these elusive receptors seems to be less intense than before. Here, we present an update of the various strategies employed to assign a function to these receptors and to discover new ligands. We focus on the recent advances in the identification of endogenous ligands with a detailed description of newly deorphanized receptors. Replication being a key parameter in these endeavors, we also discuss the latest controversies about problematic ligand-receptor pairings. In this context, we propose several recommendations in order to strengthen the reporting of new ligand-receptor pairs. Copyright © 2018 Elsevier Inc. All rights reserved.

Vasoconstriction induced by G1, a G-protein-coupled oestrogen receptor1 (GPER-1) agonist, in the isolated perfused rat kidney.

PubMed

Kurt, Akif Hakan; Buyukafsar, Kansu

2013-02-28

Vascular effects of the G protein-coupled oestrogen receptor1 (GPER-1) agonist, G1 (10(-7)-5×10(-6) M), the main oestrogenic hormone, 17β-estradiol (10(-9)-10(-4) M), the NR3A1 agonist, PPT (10(-8)-10(-5) M), the NR3A2 agonist DPN (10(-8)-10(-5) M), and the classical oestrogen receptor blocker but also a GPER agonist, ICI-182780 (10(-8)-3×10(-6) M), were investigated on the perfusion pressure in the isolated rat kidney. To seek cellular mechanisms involved in GPER-1-induced signalling we tested several compounds including the inhibitors of Rho-kinase (ROCK) (Y-27632), tyrosine kinase (genistein), p38MAPK (SB203580), p44/42MAPK (PD98059), protein kinase C (PKC) (GF109203X), Jun-kinase (JNK) (SP600125), phosphatidylinositol-3-kinase (PI3K) (LY294002), Ca(2+) channels (nifedipine), GPER-1 (G15) and epidermal growth factor (EGF) receptor kinase (AG-1478). Moreover, the effect of saponin (50mg/ml) that was used for endothelium removal was explored on G1-elicited vascular action. G1, 17β-estradiol and ICI-182780 but not PPT and DPN induced vasoconstrictions in basal renal perfusion pressure. In contrast, G1 promoted vasodilatation when the perfusion pressure was elevated in advance by phenylephrine. G1-elicited vasoconstriction was not modified by endothelial removal; however, it was markedly inhibited by GPER-1 antagonist, G15. The vasoconstrictor response to G1 was also significantly attenuated by Y-27632, PD98059, SB203580, GF109203X, genistein, AG-1478, and nifedipine, but not LY294002 and SP600125. Western blotting indicated the expression of GPER-1 in renal artery, medulla and cortex of rat kidney. In conclusion, GPER-1 could substantially modulate vascular responses through a variety of signalling pathways including ROCK, PKC, p38 MAPK, p42/44 MAPK, tyrosine kinase, EGF receptor kinase and VOCC but not JNK or PI3K in isolated perfused rat kidney. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Estrogen signaling through the G protein-coupled estrogen receptor regulates granulocyte activation in fish.

PubMed

Cabas, Isabel; Rodenas, M Carmen; Abellán, Emilia; Meseguer, José; Mulero, Victoriano; García-Ayala, Alfonsa

2013-11-01

Neutrophils are major participants in innate host responses. It is well known that estrogens have an immune-modulatory role, and some evidence exists that neutrophil physiology can be altered by these molecules. Traditionally, estrogens act via classical nuclear estrogen receptors, but the identification of a G protein-coupled estrogen receptor (GPER), a membrane estrogen receptor that binds estradiol and other estrogens, has opened up the possibility of exploring additional estrogen-mediated effects. However, information on the importance of GPER for immunity, especially, in neutrophils is scant. In this study, we report that gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils, express GPER at both mRNA and protein levels. By using a GPER selective agonist, G1, it was found that GPER activation in vitro slightly reduced the respiratory burst of acidophilic granulocytes and drastically altered the expression profile of several genes encoding major pro- and anti-inflammatory mediators. In addition, GPER signaling in vivo modulated adaptive immunity. Finally, a cAMP analog mimicked the effects of G1 in the induction of the gene coding for PG-endoperoxide synthase 2 and in the induction of CREB phosphorylation, whereas pharmacological inhibition of protein kinase A superinduced PG-endoperoxide synthase 2. Taken together, our results demonstrate for the first time, to our knowledge, that estrogens are able to modulate vertebrate granulocyte functions through a GPER/cAMP/protein kinase A/CREB signaling pathway and could establish therapeutic targets for several immune disorders in which estrogens play a prominent role.

How much do we know about the coupling of G-proteins to serotonin receptors?

PubMed Central

2014-01-01

Serotonin receptors are G-protein-coupled receptors (GPCRs) involved in a variety of psychiatric disorders. G-proteins, heterotrimeric complexes that couple to multiple receptors, are activated when their receptor is bound by the appropriate ligand. Activation triggers a cascade of further signalling events that ultimately result in cell function changes. Each of the several known G-protein types can activate multiple pathways. Interestingly, since several G-proteins can couple to the same serotonin receptor type, receptor activation can result in induction of different pathways. To reach a better understanding of the role, interactions and expression of G-proteins a literature search was performed in order to list all the known heterotrimeric combinations and serotonin receptor complexes. Public databases were analysed to collect transcript and protein expression data relating to G-proteins in neural tissues. Only a very small number of heterotrimeric combinations and G-protein-receptor complexes out of the possible thousands suggested by expression data analysis have been examined experimentally. In addition this has mostly been obtained using insect, hamster, rat and, to a lesser extent, human cell lines. Besides highlighting which interactions have not been explored, our findings suggest additional possible interactions that should be examined based on our expression data analysis. PMID:25011628

How much do we know about the coupling of G-proteins to serotonin receptors?

PubMed

Giulietti, Matteo; Vivenzio, Viviana; Piva, Francesco; Principato, Giovanni; Bellantuono, Cesario; Nardi, Bernardo

2014-07-10

Serotonin receptors are G-protein-coupled receptors (GPCRs) involved in a variety of psychiatric disorders. G-proteins, heterotrimeric complexes that couple to multiple receptors, are activated when their receptor is bound by the appropriate ligand. Activation triggers a cascade of further signalling events that ultimately result in cell function changes. Each of the several known G-protein types can activate multiple pathways. Interestingly, since several G-proteins can couple to the same serotonin receptor type, receptor activation can result in induction of different pathways. To reach a better understanding of the role, interactions and expression of G-proteins a literature search was performed in order to list all the known heterotrimeric combinations and serotonin receptor complexes. Public databases were analysed to collect transcript and protein expression data relating to G-proteins in neural tissues. Only a very small number of heterotrimeric combinations and G-protein-receptor complexes out of the possible thousands suggested by expression data analysis have been examined experimentally. In addition this has mostly been obtained using insect, hamster, rat and, to a lesser extent, human cell lines. Besides highlighting which interactions have not been explored, our findings suggest additional possible interactions that should be examined based on our expression data analysis.

Ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes.

PubMed

Tunc-Ozdemir, Meral; Jones, Alan M

2017-01-01

Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1) modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor-like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction. Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22). These microscopies included Förster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy. The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants. A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP. Finally, the G protein complex becomes

Ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes

DOE PAGES

Tunc-Ozdemir, Meral; Jones, Alan M.

2017-02-10

Background Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1) modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor-like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction. Methods Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKsmore » and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22). These microscopies included FoÈrster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy. Results The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants. Conclusions A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP

Ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes

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

Tunc-Ozdemir, Meral; Jones, Alan M.

Background Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1) modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor-like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction. Methods Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKsmore » and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22). These microscopies included FoÈrster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy. Results The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants. Conclusions A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP

[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.

G-Protein Coupled Receptors: Surface Display and Biosensor Technology

NASA Astrophysics Data System (ADS)

McMurchie, Edward; Leifert, Wayne

Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

G protein-coupled receptor kinase-2 (GRK-2) regulates serotonin metabolism through the monoamine oxidase AMX-2 in Caenorhabditis elegans

PubMed Central

Wang, Jianjun; Luo, Jiansong; Aryal, Dipendra K.; Wetsel, William C.; Nass, Richard; Benovic, Jeffrey L.

2017-01-01

G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans. Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans, and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway. PMID:28213524

Structural studies of G protein-coupled receptors.

PubMed

Lu, Mengjie; Wu, Beili

2016-11-01

G protein-coupled receptors (GPCRs) comprise the largest membrane protein family. These receptors sense a variety of signaling molecules, activate multiple intracellular signal pathways, and act as the targets of over 40% of marketed drugs. Recent progress on GPCR structural studies provides invaluable insights into the structure-function relationship of the GPCR superfamily, deepening our understanding about the molecular mechanisms of GPCR signal transduction. Here, we review recent breakthroughs on GPCR structure determination and the structural features of GPCRs, and take the structures of chemokine receptor CCR5 and purinergic receptors P2Y 1 R and P2Y 12 R as examples to discuss the importance of GPCR structures on functional studies and drug discovery. In addition, we discuss the prospect of GPCR structure-based drug discovery. © 2016 IUBMB Life, 68(11):894-903, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

G protein-coupled odorant receptors: From sequence to structure.

PubMed

de March, Claire A; Kim, Soo-Kyung; Antonczak, Serge; Goddard, William A; Golebiowski, Jérôme

2015-09-01

Odorant receptors (ORs) are the largest subfamily within class A G protein-coupled receptors (GPCRs). No experimental structural data of any OR is available to date and atomic-level insights are likely to be obtained by means of molecular modeling. In this article, we critically align sequences of ORs with those GPCRs for which a structure is available. Here, an alignment consistent with available site-directed mutagenesis data on various ORs is proposed. Using this alignment, the choice of the template is deemed rather minor for identifying residues that constitute the wall of the binding cavity or those involved in G protein recognition. © 2015 The Protein Society.

Highly effective phosphorylation by G protein-coupled receptor kinase 7 of light-activated visual pigment in cones.

PubMed

Tachibanaki, Shuji; Arinobu, Daisuke; Shimauchi-Matsukawa, Yoshie; Tsushima, Sawae; Kawamura, Satoru

2005-06-28

Cone photoreceptors show briefer photoresponses than rod photoreceptors. Our previous study showed that visual pigment phosphorylation, a quenching mechanism of light-activated visual pigment, is much more rapid in cones than in rods. Here, we measured the early time course of this rapid phosphorylation with good time resolution and directly compared it with the photoresponse time course in cones. At the time of photoresponse recovery, almost two phosphates were incorporated into a bleached cone pigment molecule, which indicated that the visual pigment phosphorylation coincides with the photoresponse recovery. The rapid phosphorylation in cones is attributed to very high activity of visual pigment kinase [G protein-coupled receptor kinase (GRK) 7] in cones. Because of this high activity, cone pigment is readily phosphorylated at very high bleach levels, which probably explains why cone photoresponses recover quickly even after a very bright light and do not saturate under intense background light. The high GRK7 activity is brought about by high content of a highly potent enzyme. The expression level of GRK7 was 10 times higher than that of rod kinase (GRK1), and the specific activity of a single GRK7 molecule was approximately 10 times higher than that of GRK1. The specific activity of GRK7 is the highest among the GRKs so far known. Our result seems to explain the response characteristics of cone photoreceptors in many aspects, including the nonsaturation of the cone responses during daylight vision.

Crucial role of neuron-enriched endosomal protein of 21 kDa in sorting between degradation and recycling of internalized G-protein-coupled receptors.

PubMed

Debaigt, Colin; Hirling, Harald; Steiner, Pascal; Vincent, Jean-Pierre; Mazella, Jean

2004-08-20

Recycling of endocytosed G-protein-coupled receptors involves a series of molecular events through early and recycling endosomes. The purpose of this work was to study the role of neuron-enriched endosomal protein of 21 kDa (NEEP21) in the recycling process of neurotensin receptors-1 and -2. Here we showed that suppression of NEEP21 expression does not modify the internalization rate of both receptors but strongly inhibited the recycling of the neurotensin receptor-2. In contrast, overexpression of NEEP21 changes the behavior of the neurotensin receptor-1 from a non-recycling to a recycling state. Recycling of the neurotensin receptor-2 involves both the phosphatidylinositol 3-kinase and the recycling endosome pathways, whereas recycling of the neurotensin receptor-1 induced by overexpression of NEEP21 only occurs by the phosphatidylinositol 3-kinase-dependent pathway. Taken together, these results confirm the essential role of NEEP21 in the recycling mechanism and show that this protein acts at the level of early endosomes to promote sorting of receptors toward a recycling pathway.

Divergent β-Arrestin-dependent Signaling Events Are Dependent upon Sequences within G-protein-coupled Receptor C Termini*

PubMed Central

Pal, Kasturi; Mathur, Maneesh; Kumar, Puneet; DeFea, Kathryn

2013-01-01

β-Arrestins are multifunctional adaptor proteins that, upon recruitment to an activated G-protein-coupled receptor, can promote desensitization of G-protein signaling and receptor internalization while simultaneously eliciting an independent signal. The result of β-arrestin signaling depends upon the activating receptor. For example, activation of two Gαq-coupled receptors, protease-activated receptor-2 (PAR2) and neurokinin-1 receptor (NK1R), results in drastically different signaling events. PAR2 promotes β-arrestin-dependent membrane-sequestered extracellular signal-regulated kinase (ERK1/2) activation, cofilin activation, and cell migration, whereas NK1R promotes nuclear ERK1/2 activation and proliferation. Using bioluminescence resonance energy transfer to monitor receptor/β-arrestin interactions in real time, we observe that PAR2 has a higher apparent affinity for both β-arrestins than does NK1R, recruits them at a faster rate, and exhibits more rapid desensitization of the G-protein signal. Furthermore, recruitment of β-arrestins to PAR2 does not require prior Gαq signaling events, whereas inhibition of Gαq signaling intermediates inhibits recruitment of β-arrestins to NK1R. Using chimeric receptors in which the C terminus of PAR2 is fused to the N terminus of NK1R and vice versa and a critical Ser/Thr mutant of PAR2, we demonstrate that interactions between β-arrestins and specific phosphoresidues in the C termini of each receptor are crucial for determining the rate and magnitude of β-arrestin recruitment as well as the ultimate signaling outcome. PMID:23235155

Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30: a novel signaling pathway with potential significance for breast cancer.

PubMed

Filardo, Edward J

2002-02-01

The biological and biochemical effects of estrogen have been ascribed to its known receptors, which function as ligand-inducible transcription factors. However, estrogen also triggers rapid activation of classical second messengers (cAMP, calcium, and inositol triphosphate) and stimulation of intracellular signaling cascades mitogen-activated protein kinase (MAP K), PI3K and eNOS. These latter events are commonly activated by membrane receptors that either possess intrinsic tyrosine kinase activity or couple to heterotrimeric G-proteins. We have shown that estrogen transactivates the epidermal growth factor receptor (EGFR) to MAP K signaling axis via the G-protein-coupled receptor (GPCR), GPR30, through the release of surface-bound proHB-EGF from estrogen receptor (ER)-negative human breast cancer cells [Molecular Endocrinology 14 (2000) 1649]. This finding is consistent with a growing body of evidence suggesting that transactivation of EGFRs by GPCRs is a recurrent theme in cell signaling. GPCR-mediated transactivation of EGFRs by estrogen provides a previously unappreciated mechanism of cross-talk between estrogen and serum growth factors, and explains prior data reporting the EGF-like effects of estrogen. This novel mechanism by which estrogen activates growth factor-dependent signaling and its implications for breast cancer biology are discussed further in this review.

Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER).

PubMed

Thekkumkara, Thomas; Snyder, Russell; Karamyan, Vardan T

2016-01-01

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

G protein-coupled receptor kinase-2 (GRK-2) regulates serotonin metabolism through the monoamine oxidase AMX-2 in Caenorhabditis elegans.

PubMed

Wang, Jianjun; Luo, Jiansong; Aryal, Dipendra K; Wetsel, William C; Nass, Richard; Benovic, Jeffrey L

2017-04-07

G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans , and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Reduction of lymphocyte G protein-coupled receptor kinase-2 (GRK2) after exercise training predicts survival in patients with heart failure.

PubMed

Rengo, Giuseppe; Galasso, Gennaro; Femminella, Grazia D; Parisi, Valentina; Zincarelli, Carmela; Pagano, Gennaro; De Lucia, Claudio; Cannavo, Alessandro; Liccardo, Daniela; Marciano, Caterina; Vigorito, Carlo; Giallauria, Francesco; Ferrara, Nicola; Furgi, Giuseppe; Filardi, Pasquale Perrone; Koch, Walter J; Leosco, Dario

2014-01-01

Increased cardiac G protein-coupled receptor kinase-2 (GRK2) expression has a pivotal role at inducing heart failure (HF)-related β-adrenergic receptor (βAR) dysfunction. Importantly, abnormalities of βAR signalling in the failing heart, including GRK2 overexpression, are mirrored in circulating lymphocytes and correlate with HF severity. Exercise training has been shown to exert several beneficial effects on the failing heart, including normalization of cardiac βAR function and GRK2 protein levels. In the present study, we evaluated whether lymphocyte GRK2 levels and short-term changes of this kinase after an exercise training programme can predict long-term survival in HF patients. For this purpose, we prospectively studied 193 HF patients who underwent a 3-month exercise training programme. Lymphocyte GRK2 protein levels, plasma N-terminal pro-brain natriuretic peptide, and norepinephrine were measured at baseline and after training along with clinical and functional parameters (left ventricular ejection fraction, NYHA class, and peak-VO2). Cardiac-related mortality was evaluated during a mean follow-up period of 37 ± 20 months. Exercise was associated with a significant reduction of lymphocyte GRK2 protein levels (from 1.29 ± 0.52 to 1.16 ± 0.65 densitometric units, p < 0.0001). Importantly, exercise related changes of GRK2 (delta values) robustly predicted survival in our study population. Interestingly, HF patients who did not show reduced lymphocyte GRK2 protein levels after training presented the poorest outcome. Our data offer the first demonstration that changes of lymphocyte GRK2 after exercise training can strongly predict outcome in advanced HF.

G protein-coupled odorant receptors: From sequence to structure

PubMed Central

de March, Claire A; Kim, Soo-Kyung; Antonczak, Serge; Goddard, William A; Golebiowski, Jérôme

2015-01-01

Odorant receptors (ORs) are the largest subfamily within class A G protein-coupled receptors (GPCRs). No experimental structural data of any OR is available to date and atomic-level insights are likely to be obtained by means of molecular modeling. In this article, we critically align sequences of ORs with those GPCRs for which a structure is available. Here, an alignment consistent with available site-directed mutagenesis data on various ORs is proposed. Using this alignment, the choice of the template is deemed rather minor for identifying residues that constitute the wall of the binding cavity or those involved in G protein recognition. PMID:26044705

The human mu opioid receptor: modulation of functional desensitization by calcium/calmodulin-dependent protein kinase and protein kinase C.

PubMed

Mestek, A; Hurley, J H; Bye, L S; Campbell, A D; Chen, Y; Tian, M; Liu, J; Schulman, H; Yu, L

1995-03-01

Opioids are some of the most efficacious analgesics used in humans. Prolonged administration of opioids, however, often causes the development of drug tolerance, thus limiting their effectiveness. To explore the molecular basis of those mechanisms that may contribute to opioid tolerance, we have isolated a cDNA for the human mu opioid receptor, the target of such opioid narcotics as morphine, codeine, methadone, and fentanyl. The receptor encoded by this cDNA is 400 amino acids long with 94% sequence similarity to the rat mu opioid receptor. Transient expression of this cDNA in COS-7 cells produced high-affinity binding sites to mu-selective agonists and antagonists. This receptor displays functional coupling to a recently cloned G-protein-activated K+ channel. When both proteins were expressed in Xenopus oocytes, functional desensitization developed upon repeated stimulation of the mu opioid receptor, as observed by a reduction in K+ current induced by the second mu receptor activation relative to that induced by the first. The extent of desensitization was potentiated by both the multifunctional calcium/calmodulin-dependent protein kinase and protein kinase C. These results demonstrate that kinase modulation is a molecular mechanism by which the desensitization of mu receptor signaling may be regulated at the cellular level, suggesting that this cellular mechanism may contribute to opioid tolerance in humans.

Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

PubMed

Lucas, Thaís F G; Royer, Carine; Siu, Erica R; Lazari, Maria Fatima M; Porto, Catarina S

2010-08-01

The aim of the present study was to investigate the expression and signaling of the G protein-coupled estrogen receptor 1 (GPER) in cultured immature rat Sertoli cells--in which we have previously described the classical estrogen receptors (ESR1 and ESR2). Expression of GPER in cultured Sertoli cells from 15-day-old rats was detected by RT-PCR and immunoassays. Gper transcripts also were present in testes from 5-, 15-, and 120-day-old rats. Short-term treatment of Sertoli cells with 17beta-estradiol (E2), the GPER agonist G-1, or the ESR antagonist ICI 182,780 (ICI) rapidly activated MAPK3/1 (ERK1/2), even after down-regulation of ESR1 and ESR2, suggesting a role for GPER in the rapid E2 action in these cells. MAPK3/1 phosphorylation induced by ICI or G-1 was blocked by pertussis toxin, selective inhibitor of the SRC family of protein tyrosine kinases, metalloprotease inhibitor, MAP2K1/2 inhibitor, and epidermal growth factor receptor (EGFR) kinase inhibitor. Furthermore, E2, but not G-1, induced up-regulation of cyclin D1 in the Sertoli cells. This effect was blocked by ICI. E2 and G-1 decreased BAX and increased BCL2 expression and these effects were blocked by MAP2K1/2 inhibitor and EGFR kinase inhibitor. The pretreatment with ICI did not block the effect of E2. Taken together, these results indicate that in Sertoli cells 1) GPER-mediated MAPK3/1 activation occurs via EGFR transactivation through G protein beta gamma subunits that promote SRC-mediated metalloprotease-dependent release of EGFR ligands, which bind to EGFR and lead to MAPK3/1 phosphorylation; 2) E2-ESRs play a role in Sertoli cell proliferation; and 3) E2-GPER may regulate gene expression involved with apoptosis. ESR and GPER may mediate actions important for Sertoli cell function and maintenance of normal testis development and homeostasis.

Structural Basis of G Protein-coupled Receptor-Gi Protein Interaction

PubMed Central

Mnpotra, Jagjeet S.; Qiao, Zhuanhong; Cai, Jian; Lynch, Diane L.; Grossfield, Alan; Leioatts, Nicholas; Hurst, Dow P.; Pitman, Michael C.; Song, Zhao-Hui; Reggio, Patricia H.

2014-01-01

In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)- Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins. PMID:24855641

Expression and functional roles of G-protein-coupled estrogen receptor (GPER) in human eosinophils.

PubMed

Tamaki, Mami; Konno, Yasunori; Kobayashi, Yoshiki; Takeda, Masahide; Itoga, Masamichi; Moritoki, Yuki; Oyamada, Hajime; Kayaba, Hiroyuki; Chihara, Junichi; Ueki, Shigeharu

2014-07-01

Sexual dimorphism in asthma links the estrogen and allergic immune responses. The function of estrogen was classically believed to be mediated through its nuclear receptors, i.e., estrogen receptors (ERs). However, recent studies established the important roles of G-protein-coupled estrogen receptor (GPER/GPR30) as a novel membrane receptor for estrogen. To date, the role of GPER in allergic inflammation is poorly understood. The purpose of this study was to examine whether GPER might affect the functions of eosinophils, which play an important role in the pathogenesis of asthma. Here, we demonstrated that GPER was expressed in purified human peripheral blood eosinophils both at the mRNA and protein levels. Although GPER agonist G-1 did not induce eosinophil chemotaxis or chemokinesis, preincubation with G-1 enhanced eotaxin (CCL11)-directed eosinophil chemotaxis. G-1 inhibited eosinophil spontaneous apoptosis and caspase-3 activities. The anti-apoptotic effect was not affected by the cAMP-phospodiesterase inhibitor rolipram or phosphoinositide 3-kinase inhibitors. In contrast to resting eosinophils, G-1 induced apoptosis and increased caspase-3 activities when eosinophils were co-stimulated with IL-5. No effect of G-1 was observed on eosinophil degranulation in terms of release of eosinophil-derived neurotoxin (EDN). The current study indicates the functional capacities of GPER on human eosinophils and also provides the previously unrecognized mechanisms of interaction between estrogen and allergic inflammation. Copyright © 2014 Elsevier B.V. All rights reserved.

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. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

G Protein-Coupled Receptor Rhodopsin: A Prospectus

PubMed Central

Filipek, Sławomir; Stenkamp, Ronald E.; Teller, David C.; Palczewski, Krzysztof

2006-01-01

Rhodopsin is a retinal photoreceptor protein of bipartite structure consisting of the transmembrane protein opsin and a light-sensitive chromophore 11-cis-retinal, linked to opsin via a protonated Schiff base. Studies on rhodopsin have unveiled many structural and functional features that are common to a large and pharmacologically important group of proteins from the G protein-coupled receptor (GPCR) superfamily, of which rhodopsin is the best-studied member. In this work, we focus on structural features of rhodopsin as revealed by many biochemical and structural investigations. In particular, the high-resolution structure of bovine rhodopsin provides a template for understanding how GPCRs work. We describe the sensitivity and complexity of rhodopsin that lead to its important role in vision. PMID:12471166

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.

Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

PubMed

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

2015-01-01

Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.

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

Cardioprotective role of G-Protein Coupled Estrogen Receptor 1 (GPER1).

PubMed

Koganti, Sivaramakrishna

2015-01-01

G-Protein Coupled Estrogen Receptor 1 (GPER1), also known as G-Protein Coupled Receptor 30 (GPR30) and initially considered an orphan receptor, has become one of the most important pharmacological targets in cardiovascular research. Since the gene encoding this putative receptor was cloned nearly 20 years ago, researchers have addressed its role in various aspects of physiology, including cardioprotection. Although extensive research has been carried out to understand the role of GPER1 as a pharmacological target to treat cardiovascular diseases, there are few current reviews addressing the overall cardioprotective benefits of this receptor and the signaling intermediates involved. This review considers the origins of GPER1, its cell biology, its physiological and pharmacological roles as a therapeutic target in cardiovascular disease, and what future research on GPER1 might entail. More specifically, the review focuses on GPER1 regulation of Angiotensin Type I Receptor (AT1R) and the role of estrogen receptors, epidermal growth factor receptor (EGFR) and matrix metalloproteinases (MMPs) in bringing about the cardioprotective effects of GPER1. Areas where improved knowledge of GPER1 biology is still needed to better understand the receptor's cardioprotective effects are also discussed.

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 (Gα q and Gα 11 ) 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 Gα q /Gα 11 -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 G q / 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 G q / 11 signaling because the G q / 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 G q / 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

[G protein-coupled receptors in the spot light].

PubMed

Benleulmi-Chaachoua, Abla; Wojciech, Stefanie; Jockers, Ralf

2013-01-01

G protein-coupled receptors (GPCRs), also known as seven transmembrane domain-spanning proteins (7TM), play an important role in tissue homeostasis and cellular and hormonal communication. GPCRs are targeted by a large panel of natural ligands such as photons, ions, metabolites, lipids and proteins but also by numerous drugs. Research efforts in the GPCR field have been rewarded in 2012 by the Nobel Price in Chemistry. The present article briefly summarizes our current knowledge on GPCRs and discusses future challenges in terms of fundamental aspects and therapeutic applications. © Société de Biologie, 2013.

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

The ubiquitin ligase Mdm2 controls oligodendrocyte maturation by intertwining mTOR with G protein-coupled receptor kinase 2 in the regulation of GPR17 receptor desensitization.

PubMed

Fumagalli, Marta; Bonfanti, Elisabetta; Daniele, Simona; Zappelli, Elisa; Lecca, Davide; Martini, Claudia; Trincavelli, Maria L; Abbracchio, Maria P

2015-12-01

During oligodendrocyte precursor cell (OPC) differentiation, defective control of the membrane receptor GPR17 has been suggested to block cell maturation and impair remyelination under demyelinating conditions. After the immature oligodendrocyte stage, to enable cells to complete maturation, GPR17 is physiologically down-regulated via phosphorylation/desensitization by G protein-coupled receptor kinases (GRKs); conversely, GRKs are regulated by the "mammalian target of rapamycin" mTOR. However, how GRKs and mTOR are connected to each other in modulating GPR17 function and oligodendrogenesis has remained elusive. Here we show, for the first time, a role for Murine double minute 2 (Mdm2), a ligase previously involved in ubiquitination/degradation of the onco-suppressor p53 protein. In maturing OPCs, both rapamycin and Nutlin-3, a small molecule inhibitor of Mdm2-p53 interactions, increased GRK2 sequestration by Mdm2, leading to impaired GPR17 down-regulation and OPC maturation block. Thus, Mdm2 intertwines mTOR with GRK2 in regulating GPR17 and oligodendrogenesis and represents a novel actor in myelination. © 2015 Wiley Periodicals, Inc.

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

PubMed

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

2017-02-01

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

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.

Characterization of the Caenorhabditis elegans G protein-coupled serotonin receptors.

PubMed

Carre-Pierrat, Maïté; Baillie, David; Johnsen, Robert; Hyde, Rhonda; Hart, Anne; Granger, Laure; Ségalat, Laurent

2006-12-01

Serotonin (5-HT) regulates a wide range of behaviors in Caenorhabditis elegans, including egg laying, male mating, locomotion and pharyngeal pumping. So far, four serotonin receptors have been described in the nematode C. elegans, three of which are G protein-coupled receptors (GPCR), (SER-1, SER-4 and SER-7), and one is an ion channel (MOD-1). By searching the C. elegans genome for additional 5-HT GPCR genes, we identified five further genes which encode putative 5-HT receptors, based on sequence similarities to 5-HT receptors from other species. Using loss-of-function mutants and RNAi, we performed a systematic study of the role of the eight GPCR genes in serotonin-modulated behaviors of C. elegans (F59C12.2, Y22D7AR.13, K02F2.6, C09B7.1, M03F4.3, F16D3.7, T02E9.3, C24A8.1). We also examined their expression patterns. Finally, we tested whether the most likely candidate receptors were able to modulate adenylate cyclase activity in transfected cells in a 5-HT-dependent manner. This paper is the first comprehensive study of G protein-coupled serotonin receptors of C. elegans. It provides a direct comparison of the expression patterns and functional roles for 5-HT receptors in C. elegans.

Oligomerization of G protein-coupled receptors: computational methods.

PubMed

Selent, J; Kaczor, A A

2011-01-01

Recent research has unveiled the complexity of mechanisms involved in G protein-coupled receptor (GPCR) functioning in which receptor dimerization/oligomerization may play an important role. Although the first high-resolution X-ray structure for a likely functional chemokine receptor dimer has been deposited in the Protein Data Bank, the interactions and mechanisms of dimer formation are not yet fully understood. In this respect, computational methods play a key role for predicting accurate GPCR complexes. This review outlines computational approaches focusing on sequence- and structure-based methodologies as well as discusses their advantages and limitations. Sequence-based approaches that search for possible protein-protein interfaces in GPCR complexes have been applied with success in several studies, but did not yield always consistent results. Structure-based methodologies are a potent complement to sequence-based approaches. For instance, protein-protein docking is a valuable method especially when guided by experimental constraints. Some disadvantages like limited receptor flexibility and non-consideration of the membrane environment have to be taken into account. Molecular dynamics simulation can overcome these drawbacks giving a detailed description of conformational changes in a native-like membrane. Successful prediction of GPCR complexes using computational approaches combined with experimental efforts may help to understand the role of dimeric/oligomeric GPCR complexes for fine-tuning receptor signaling. Moreover, since such GPCR complexes have attracted interest as potential drug target for diverse diseases, unveiling molecular determinants of dimerization/oligomerization can provide important implications for drug discovery.

GPR30: A G protein-coupled receptor for estrogen.

PubMed

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

2007-02-01

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

Skeletal Muscle-specific G Protein-coupled Receptor Kinase 2 Ablation Alters Isolated Skeletal Muscle Mechanics and Enhances Clenbuterol-stimulated Hypertrophy*

PubMed Central

Woodall, Benjamin P.; Woodall, Meryl C.; Luongo, Timothy S.; Grisanti, Laurel A.; Tilley, Douglas G.; Elrod, John W.; Koch, Walter J.

2016-01-01

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β2-adrenergic receptor (β2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2AR-induced hypertrophy. PMID:27566547

Skeletal Muscle-specific G Protein-coupled Receptor Kinase 2 Ablation Alters Isolated Skeletal Muscle Mechanics and Enhances Clenbuterol-stimulated Hypertrophy.

PubMed

Woodall, Benjamin P; Woodall, Meryl C; Luongo, Timothy S; Grisanti, Laurel A; Tilley, Douglas G; Elrod, John W; Koch, Walter J

2016-10-14

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2 fl/fl ) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2 fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β 2 -adrenergic receptor (β 2 AR) agonist, was significantly enhanced in MLC-Cre:GRK2 fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β 2 AR-induced hypertrophy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) regulates G-protein-coupled receptor kinase 5 (GRK5)-induced cardiac hypertrophy in vitro.

PubMed

Yeh, Szu-Tsen; Zambrano, Cristina M; Koch, Walter J; Purcell, Nicole H

2018-05-25

PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to regulate cell growth and survival through dephosphorylation of several members of the AGC family of kinases. G-protein-coupled receptor kinase 5 (GRK5) is an AGC kinase that regulates phenylephrine (PE)-induced cardiac hypertrophy through its noncanonical function of directly targeting proteins to the nucleus to regulate transcription. Here we investigated the possibility that the PHLPP2 isoform can regulate GRK5-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). We show that removal of PHLPP2 by siRNA induces hypertrophic growth of NRVMs as measured by cell size changes at baseline, potentiated PE-induced cell size changes, and re-expression of fetal genes atrial natriuretic factor and brain natriuretic peptide. Endogenous GRK5 and PHLPP2 were found to interact in NRVMs, and PE-induced nuclear accumulation of GRK5 was enhanced upon down-regulation of PHLPP2. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes, and nuclear accumulation of GRK5, which depended on its phosphatase activity. Finally, using siRNA against GRK5, we found that GRK5 was necessary for the hypertrophic response induced by PHLPP2 knockdown. Our findings demonstrate for the first time a novel regulation of GRK5 by the phosphatase PHLPP2, which modulates hypertrophic growth. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of estradiol-induced insulin secretion by the G protein-coupled estrogen receptor GPR30/GPER in pancreatic beta-cells.

PubMed

Sharma, Geetanjali; Prossnitz, Eric R

2011-08-01

Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes.

Mechanisms of Estradiol-Induced Insulin Secretion by the G Protein-Coupled Estrogen Receptor GPR30/GPER in Pancreatic β-Cells

PubMed Central

Sharma, Geetanjali

2011-01-01

Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes. PMID:21673097

Estrogenic G protein-coupled receptor 30 signaling is involved in regulation of endometrial carcinoma by promoting proliferation, invasion potential, and interleukin-6 secretion via the MEK/ERK mitogen-activated protein kinase pathway.

PubMed

He, Yin-Yan; Cai, Bin; Yang, Yi-Xia; Liu, Xue-Lian; Wan, Xiao-Ping

2009-06-01

The regulatory mechanism of endometrial carcinoma and the signal transduction pathways involved in hormone action are poorly defined. It has become apparent that the G protein-coupled receptor (GPR) 30 mediates the non-genomic signaling of 17beta-estradiol (E2). Here we show that GPR30 is highly expressed in endometrial cancer tissues and cancer cell lines and positively regulates cell proliferation and invasion. GPR30 expression was detected in 50 human endometrial carcinomas. The transcription level of GPR30 was significantly higher in the tissue of endometrial carcinoma than in normal endometrium (P < 0.05). Immunohistochemical assays revealed that the positive expression rate of GPR30 protein in endometrial carcinoma tissue (35/50, 70%) was statistically higher than in normal endometrium tissue (8/30, 26.67%) (chi2 = 14.16, P = 0.0002). GPR30 overexpression was correlated with high-grade endometrial carcinoma. GPR30 expression was also found in two human endometrial cancer cell lines: RL95-2 (estrogen receptor positive) and KLE (estrogen receptor negative). The roles of GPR30 in proliferative and invasive responses to E2 and G1, a non-steroidal GPR30-specific agonist, in RL95-2 and KLE cell lines were then explored. We showed that E2 and G1 could initiate the MAPK/ERK mitogen-activated protein kinase pathway in both cell lines. What's more, E2 and G1 promoted KLE and RL95-2 proliferation and stimulated matrix metalloproteinase production and activity via the GPR30-mediated MEK/ERK mitogen-activated protein kinase pathway, as well as increased interleukin-6 secretion. These findings suggest that GPR30-mediated non-genomic signaling could play an important role in endometrial cancer.

Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

PubMed

Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J

2014-01-07

Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. Copyright © 2014 Elsevier Ltd. All rights reserved.

The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma.

PubMed

Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J H; Okkenhaug, Klaus; Vanhaesebroeck, Bart

2008-06-17

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.

The p110β isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110γ

PubMed Central

Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J. H.; Okkenhaug, Klaus; Vanhaesebroeck, Bart

2008-01-01

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110α, p110β, and p110δ) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110α and p110δ to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110γ class IB PI3K lack SH2 domains and instead couple p110γ to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110β and cells derived from a p110β-deficient mouse line, that p110β is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110β and p110γ contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110β but not p110γ, p110β mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110γ in these cells reduced the contribution of p110β to GPCR signaling. Taken together, these data show that p110β and p110γ can couple redundantly to the same GPCR agonists. p110β, which shows a much broader tissue distribution than the leukocyte-restricted p110γ, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110γ expression is low or absent. PMID:18544649

G protein-coupled estrogen receptor inhibits the P2Y receptor-mediated Ca(2+) signaling pathway in human airway epithelia.

PubMed

Hao, Yuan; Chow, Alison W; Yip, Wallace C; Li, Chi H; Wan, Tai F; Tong, Benjamin C; Cheung, King H; Chan, Wood Y; Chen, Yangchao; Cheng, Christopher H; Ko, Wing H

2016-08-01

P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (E2) receptor, may play an anti-inflammatory role in this process. We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca(2+) signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca(2+)]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5'-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca(2+) were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca(2+) mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.

PubMed

Shi, Mei; Bennett, Teresa A; Cimino, Daniel F; Maestas, Diane C; Foutz, Terry D; Gurevich, Vsevolod V; Sklar, Larry A; Prossnitz, Eric R

2003-06-24

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.

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

PubMed

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

2017-02-01

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

The changing world of G protein-coupled receptors: from monomers to dimers and receptor mosaics with allosteric receptor-receptor interactions.

PubMed

Fuxe, Kjell; Marcellino, Daniel; Borroto-Escuela, Dasiel Oscar; Frankowska, Malgorzata; Ferraro, Luca; Guidolin, Diego; Ciruela, Francisco; Agnati, Luigi F

2010-10-01

Based on indications of direct physical interactions between neuropeptide and monoamine receptors in the early 1980s, the term receptor-receptor interactions was introduced and later on the term receptor heteromerization in the early 1990s. Allosteric mechanisms allow an integrative activity to emerge either intramolecularly in G protein-coupled receptor (GPCR) monomers or intermolecularly via receptor-receptor interactions in GPCR homodimers, heterodimers, and receptor mosaics. Stable heteromers of Class A receptors may be formed that involve strong high energy arginine-phosphate electrostatic interactions. These receptor-receptor interactions markedly increase the repertoire of GPCR recognition, signaling and trafficking in which the minimal signaling unit in the GPCR homomers appears to be one receptor and one G protein. GPCR homomers and GPCR assemblies are not isolated but also directly interact with other proteins to form horizontal molecular networks at the plasma membrane.

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

PubMed

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

2016-10-25

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

Bacterial Dose-Dependent Role of G Protein-Coupled Receptor Kinase 5 in Escherichia coli-Induced Pneumonia.

PubMed

Packiriswamy, Nandakumar; Steury, Michael; McCabe, Ian C; Fitzgerald, Scott D; Parameswaran, Narayanan

2016-05-01

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase previously shown to mediate polymicrobial sepsis-induced inflammation. The goal of the present study was to examine the role of GRK5 in monomicrobial pulmonary infection by using an intratracheal Escherichia coli infection model of pneumonia. We used sublethal and lethal doses of E. coli to examine the mechanistic differences between low-grade and high-grade inflammation induced by E. coli infection. With a sublethal dose of E. coli, GRK5 knockout (KO) mice exhibited higher plasma CXCL1/KC levels and enhanced lung neutrophil recruitment early after infection, and lower bacterial loads, than wild-type (WT) mice. The inflammatory response was also diminished, and resolution of inflammation advanced, in the lungs of GRK5 KO mice. In contrast to the reduced bacterial loads in GRK5 KO mice following a sublethal dose, at a lethal dose of E. coli, the bacterial burdens remained high in GRK5 KO mice relative to those in WT mice. This occurred in spite of enhanced plasma CXCL1 levels as well as neutrophil recruitment in the KO mice. But the recruited neutrophils (following high-dose infection) exhibited decreased CD11b expression and reduced reactive oxygen species production, suggesting decreased neutrophil activation or increased neutrophil exhaustion in the GRK5 KO mice. In agreement with the increased bacterial burden, KO mice showed poorer survival than WT mice following E. coli infection at a lethal dose. Overall, our data suggest that GRK5 negatively regulates CXCL1/KC levels during bacterial pneumonia but that the role of GRK5 in the clinical outcome in this model is dependent on the bacterial dose. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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

PubMed Central

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. PMID:27379162

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.

Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation

PubMed Central

Becker, Elena; Huynh-Do, Uyen; Holland, Sacha; Pawson, Tony; Daniel, Tom O.; Skolnik, Edward Y.

2000-01-01

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62dok, RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs. PMID:10669731

Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs.

PubMed

Navarro, Gemma; Cordomí, Arnau; Zelman-Femiak, Monika; Brugarolas, Marc; Moreno, Estefania; Aguinaga, David; Perez-Benito, Laura; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Pardo, Leonardo; García-Sáez, Ana J; McCormick, Peter J; Franco, Rafael

2016-04-05

G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.

Estrogen-mediated inactivation of FOXO3a by the G protein-coupled estrogen receptor GPER.

PubMed

Zekas, Erin; Prossnitz, Eric R

2015-10-15

Estrogen (17β-estradiol) promotes the survival and proliferation of breast cancer cells and its receptors represent important therapeutic targets. The cellular actions of estrogen are mediated by the nuclear estrogen receptors ERα and ERβ as well as the 7-transmembrane spanning G protein-coupled estrogen receptor (GPER). We previously reported that estrogen activates the phosphoinositide 3-kinase (PI3Kinase) pathway via GPER, resulting in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production within the nucleus of breast cancer cells; however, the mechanisms and consequences of this activity remained unclear. MCF7 breast cancer cells were transfected with GFP-fused Forkhead box O3 (FOXO3) as a reporter to assess localization in response to estrogen stimulation. Inhibitors of PI3Kinases and EGFR were employed to determine the mechanisms of estrogen-mediated FOXO3a inactivation. Receptor knockdown with siRNA and the selective GPER agonist G-1 elucidated the estrogen receptor(s) responsible for estrogen-mediated FOXO3a inactivation. The effects of selective estrogen receptor modulators and downregulators (SERMs and SERDs) on FOXO3a in MCF7 cells were also determined. Cell survival (inhibition of apoptosis) was assessed by caspase activation. In the estrogen-responsive breast cancer cell line MCF7, FOXO3a inactivation occurs on a rapid time scale as a result of GPER, but not ERα, stimulation by estrogen, established by the GPER-selective agonist G-1 and knockdown of GPER and ERα. GPER-mediated inactivation of FOXO3a is effected by the p110α catalytic subunit of PI3Kinase as a result of transactivation of the EGFR. The SERMs tamoxifen and raloxifene, as well as the SERD ICI182,780, were active in mediating FOXO3a inactivation in a GPER-dependent manner. Additionally, estrogen-and G-1-mediated stimulation of MCF7 cells results in a decrease in caspase activation under proapoptotic conditions. Our results suggest that non-genomic signaling by GPER contributes

Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells.

PubMed Central

Graness, A; Hanke, S; Boehmer, F D; Presek, P; Liebmann, C

2000-01-01

Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway

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

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

Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the nativemore » ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.« less

G Protein-Coupled Receptor-G-Protein βγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

PubMed

Kamal, Fadia A; Travers, Joshua G; Schafer, Allison E; Ma, Qing; Devarajan, Prasad; Blaxall, Burns C

2017-01-01

Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein βγ-subunit (Gβγ), GPCR-kinase 2, and β-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gβγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gβγ signaling and ET system expression. Notably, systemic pharmacologic Gβγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gβγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gβγ in AKI and suggest GPCR-Gβγ inhibition as a novel therapeutic approach for treating CRS2 and AKI. Copyright © 2016 by the American Society of Nephrology.

Automated large-scale purification of a G protein-coupled receptor for neurotensin.

PubMed

White, Jim F; Trinh, Loc B; Shiloach, Joseph; Grisshammer, Reinhard

2004-04-30

Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis.

Tonic inhibition by G protein-coupled receptor kinase 2 of Akt/endothelial nitric-oxide synthase signaling in human vascular endothelial cells under conditions of hyperglycemia with high insulin levels.

PubMed

Taguchi, Kumiko; Sakata, Kimimasa; Ohashi, Wakana; Imaizumi, Takahiro; Imura, Joji; Hattori, Yuichi

2014-05-01

G protein-coupled receptor kinase 2 (GRK2) participates together with β-arrestins in the regulation of G protein-coupled receptor signaling, but emerging evidence suggests that GRK2 can interact with a growing number of proteins involved in signaling mediated by other membrane receptor families under various pathologic conditions. We tested the hypothesis that GRK2 may be an important contributor to vascular endothelial dysfunction in diabetes. Human umbilical venous endothelial cells (HUVECs) were exposed to high glucose and high insulin (HG/HI) to mimic insulin-resistant diabetic conditions. GRK2 expression and membrane translocation were up-regulated under HG/HI conditions. HG/HI did not modify activation of Akt or endothelial nitric-oxide synthase (eNOS), but GRK2 inhibitor or small interfering RNA (siRNA) resulted in an increase in Akt and eNOS activation in HUVECs exposed to HG/HI. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation was increased after exposure to HG/HI, which was prevented by GRK2 inhibitor or siRNA. ERK1/2-mediated GRK2 phosphorylation at Ser-670 confirmed that ERK1/2 participated in a negative feedback regulatory loop. In human embryonic kidney 293T cells that overexpressed GRK2, Akt activity was unchanged, whereas ERK1/2 activity was raised. The effect of GRK inhibitor treatment on Akt/eNOS signaling was associated with membrane translocation of β-arrestin 2. The experiments with β-arrestin 2 siRNA showed that β-arrestin 2 may act as a positive modulator of Akt/eNOS signaling. Our studies reveal that GRK2, which is up-regulated by HG/HI, leads to a tonic inhibition of the insulin Akt/eNOS pathway in endothelial cells. We provide new insights into the pathogenesis of diabetes-associated vascular endothelial dysfunction.

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling.

PubMed

Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L; Hille, Bertil; Koh, Duk-Su

2016-03-01

Activated Gq protein-coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein-tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein-coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary

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

Functional relevance of G-protein-coupled-receptor-associated proteins, exemplified by receptor-activity-modifying proteins (RAMPs).

PubMed

Fischer, J A; Muff, R; Born, W

2002-08-01

The calcitonin (CT) receptor (CTR) and the CTR-like receptor (CRLR) are close relatives within the type II family of G-protein-coupled receptors, demonstrating sequence identity of 50%. Unlike the interaction between CT and CTR, receptors for the related hormones and neuropeptides amylin, CT-gene-related peptide (CGRP) and adrenomedullin (AM) require one of three accessory receptor-activity-modifying proteins (RAMPs) for ligand recognition. An amylin/CGRP receptor is revealed when CTR is co-expressed with RAMP1. When complexed with RAMP3, CTR interacts with amylin alone. CRLR, initially classed as an orphan receptor, is a CGRP receptor when co-expressed with RAMP1. The same receptor is specific for AM in the presence of RAMP2. Together with human RAMP3, CRLR defines an AM receptor, and with mouse RAMP3 it is a low-affinity CGRP/AM receptor. CTR-RAMP1, antagonized preferentially by salmon CT-(8-32) and not by CGRP-(8-37), and CRLR-RAMP1, antagonized by CGRP-(8-37), are two CGRP receptor isotypes. Thus amylin and CGRP interact specifically with heterodimeric complexes between CTR and RAMP1 or RAMP3, and CGRP and AM interact with complexes between CRLR and RAMP1, RAMP2 or RAMP3.

G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons

PubMed Central

Connelly, Timothy; Yu, Yiqun; Grosmaitre, Xavier; Wang, Jue; Santarelli, Lindsey C.; Savigner, Agnes; Qiao, Xin; Wang, Zhenshan; Storm, Daniel R.; Ma, Minghong

2015-01-01

Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR–G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction. PMID:25550517

Spectral methods for study of the G-protein-coupled receptor rhodopsin. II. Magnetic resonance methods

NASA Astrophysics Data System (ADS)

Struts, A. V.; Barmasov, A. V.; Brown, M. F.

2016-02-01

This article continues our review of spectroscopic studies of G-protein-coupled receptors. Magnetic resonance methods including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) provide specific structural and dynamical data for the protein in conjunction with optical methods (vibrational, electronic spectroscopy) as discussed in the accompanying article. An additional advantage is the opportunity to explore the receptor proteins in the natural membrane lipid environment. Solid-state 2H and 13C NMR methods yield information about both the local structure and dynamics of the cofactor bound to the protein and its light-induced changes. Complementary site-directed spin-labeling studies monitor the structural alterations over larger distances and correspondingly longer time scales. A multiscale reaction mechanism describes how local changes of the retinal cofactor unlock the receptor to initiate large-scale conformational changes of rhodopsin. Activation of the G-protein-coupled receptor involves an ensemble of conformational substates within the rhodopsin manifold that characterize the dynamically active receptor.

Recombinant G protein-coupled receptor expression in Saccharomyces cerevisiae for protein characterization.

PubMed

Blocker, Kory M; Britton, Zachary T; Naranjo, Andrea N; McNeely, Patrick M; Young, Carissa L; Robinson, Anne S

2015-01-01

G protein-coupled receptors (GPCRs) are membrane proteins that mediate signaling across the cellular membrane and facilitate cellular responses to external stimuli. Due to the critical role that GPCRs play in signal transduction, therapeutics have been developed to influence GPCR function without an extensive understanding of the receptors themselves. Closing this knowledge gap is of paramount importance to improving therapeutic efficacy and specificity, where efforts to achieve this end have focused chiefly on improving our knowledge of the structure-function relationship. The purpose of this chapter is to review methods for the heterologous expression of GPCRs in Saccharomyces cerevisiae, including whole-cell assays that enable quantitation of expression, localization, and function in vivo. In addition, we describe methods for the micellular solubilization of the human adenosine A2a receptor and for reconstitution of the receptor in liposomes that have enabled its biophysical characterization. © 2015 Elsevier Inc. All rights reserved.

Absorption of PCB126 by upper airways impairs G protein-coupled receptor-mediated immune response

NASA Astrophysics Data System (ADS)

Shimada, Ana Lúcia B.; Cruz, Wesley S.; Loiola, Rodrigo A.; Drewes, Carine C.; Dörr, Fabiane; Figueiredo, Natália G.; Pinto, Ernani; Farsky, Sandra H. P.

2015-10-01

PCB126 is a dioxin-like polychlorinated biphenyl (PCB) environmental pollutant with a significant impact on human health, as it bioaccumulates and causes severe toxicity. PCB126-induced immune toxicity has been described, although the mechanisms have not been fully elucidated. In this study, an in vivo protocol of PCB126 intoxication into male Wistar rats by intranasal route was used, which has not yet been described. The intoxication was characterised by PCB126 accumulation in the lungs and liver, and enhanced aryl hydrocarbon receptor expression in the liver, lungs, kidneys, and adipose tissues. Moreover, an innate immune deficiency was characterised by impairment of adhesion receptors on blood leukocytes and by reduced blood neutrophil locomotion and oxidative burst activation elicited by ex vivo G protein-coupled receptor (GPCR) activation. Specificity of PCB126 actions on the GPCR pathway was shown by normal burst oxidative activation evoked by Toll-like receptor 4 and protein kinase C direct activation. Moreover, in vivo PCB180 intoxication did not alter adhesion receptors on blood leukocytes either blood neutrophil locomotion, and only partially reduced the GPCR-induced burst oxidative activation on neutrophils. Therefore, a novel mechanism of in vivo PCB126 toxicity is described which impairs a pivotal inflammatory pathway to the host defence against infections.

Activation of G-protein coupled estrogen receptor inhibits the proliferation of cervical cancer cells via sustained activation of ERK1/2.

PubMed

Zhang, Qiong; Wu, Yuan-Zhe; Zhang, Yan-Mei; Ji, Xiao-Hong; Hao, Qun

2015-04-01

Cervical cancer is one of the most common gynaecological women cancer and suggested to be modulated by estrogenic signals. G protein-coupled receptor (GPER), a seven-transmembrane G protein-coupled receptor, has been reported to regulate the cell proliferation of various cancers. But there is no study investigating the effects of GPER on the progression of cervical cancer. In the present study, we revealed for the first time that GPER was also highly expressed in various human cervical cancer cells. Activation of GPER via its specific agonist G-1 induced G2/M cell cycle arrest and down regulation of cyclin B via a time dependent manner. Furthermore, G-1 treatment induced sustained activation of extracellular-signal-regulated kinases (ERK)1/2 via epidermal growth factor receptor (EGFR) signals. Both inhibitors of ERK1/2 and EGFR significantly abolished G-1-induced suppression of cell proliferation and down regulation of cyclin B. Generally, our study revealed that GPER is highly expressed in human cervical cancer cells and its activation inhibits cell proliferation via EGFR/ERK1/2 signals. It suggested that G-1 can be considered as a potential new pharmacological tool to reduce the growth of cervical cancer. Copyright © 2015 John Wiley & Sons, Ltd.

Molecular Dynamics Simulations of G Protein-Coupled Receptors.

PubMed

Bruno, Agostino; Costantino, Gabriele

2012-04-01

G protein-coupled receptors (GPCRs) constitute the largest family of membrane-bound receptors with more than 800 members encoded by 351 genes in humans. It has been estimated that more than 50 % of clinically available drugs act on GPCRs, with an amount of 400, 50 and 25 druggable proteins for the class A, B and C, respectively. Furthermore, Class A GPCRs with approximately 25 % of marketed small drugs represent the most attractive pharmaceutical class. The recent availability of high-resolution 3-dimensional structures of some GPCRs supports the notion that GPCRs are dynamically versatile, and their functions can be modulated by several factors. In this scenario, molecular dynamics (MD) simulations techniques appear to be crucial when studying GPCR flexibility associated to functioning and ligand recognition. A general overview of biased and unbiased MD techniques is here presented with special emphasis on the recent results obtained in the GPCRs field. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Serial femtosecond crystallography datasets from G protein-coupled receptors.

PubMed

White, Thomas A; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R; Yoon, Chun Hong; Yefanov, Oleksandr M; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

2016-08-01

We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data.

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. Copyright © 2010 Elsevier Inc. All rights reserved.

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

Cellular and molecular biology of orphan G protein-coupled receptors.

PubMed

Oh, Da Young; Kim, Kyungjin; Kwon, Hyuk Bang; Seong, Jae Young

2006-01-01

The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

Modulation of Pain Transmission by G Protein-Coupled Receptors

PubMed Central

Pan, Hui-Lin; Wu, Zi-Zhen; Zhou, Hong-Yi; Chen, Shao-Rui; Zhang, Hong-Mei; Li, De-Pei

2010-01-01

The heterotrimeric G protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors and proteins. GPCRs are widely distributed in the peripheral and central nervous systems and are one of the most important therapeutic targets in pain medicine. GPCRs are present on the plasma membrane of neurons and their terminals along the nociceptive pathways and are closely associated with the modulation of pain transmission. GPCRs that can produce analgesia upon activation include opioid, cannabinoid, α2-adrenergic, muscarinic acetylcholine, γ-aminobutyric acidB (GABAB), group II and III metabotropic glutamate, and somatostatin receptors. Recent studies have led to a better understanding of the role of these GPCRs in the regulation of pain transmission. Here, we review the current knowledge about the cellular and molecular mechanisms that underlie the analgesic actions of GPCR agonists, with a focus on their effects on ion channels expressed on nociceptive sensory neurons and on synaptic transmission at the spinal cord level. PMID:17959251

G protein-coupled receptor kinase-2-deficient mice are protected from dextran sodium sulfate-induced acute colitis.

PubMed

Steury, Michael D; Kang, Ho Jun; Lee, Taehyung; Lucas, Peter C; McCabe, Laura R; Parameswaran, Narayanan

2018-06-01

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase and plays a key role in different disease processes. Previously, we showed that GRK2 knockdown enhances wound healing in colonic epithelial cells. Therefore, we hypothesized that ablation of GRK2 would protect mice from dextran sodium sulfate (DSS)-induced acute colitis. To test this, we administered DSS to wild-type (GRK2 +/+ ) and GRK2 heterozygous (GRK +/- ) mice in their drinking water for 7 days. As predicted, GRK2 +/- mice were protected from colitis as demonstrated by decreased weight loss (20% loss in GRK2 +/+ vs. 11% loss in GRK2 +/- ). lower disease activity index (GRK2 +/+ 9.1 vs GRK2 +/- 4.1), and increased colon lengths (GRK2 +/+ 4.7 cm vs GRK2 +/- 5.3 cm). To examine the mechanisms by which GRK2 +/- mice are protected from colitis, we investigated expression of inflammatory genes in the colon as well as immune cell profiles in colonic lamina propria, mesenteric lymph node, and in bone marrow. Our results did not reveal differences in immune cell profiles between the two genotypes. However, expression of inflammatory genes was significantly decreased in DSS-treated GRK2 +/- mice compared with GRK2 +/+ . To understand the mechanisms, we generated myeloid-specific GRK2 knockout mice and subjected them to DSS-induced colitis. Similar to whole body GRK2 heterozygous knockout mice, myeloid-specific knockout of GRK2 was sufficient for the protection from DSS-induced colitis. Together our results indicate that deficiency of GRK2 protects mice from DSS-induced colitis and further suggests that the mechanism of this effect is likely via GRK2 regulation of inflammatory genes in the myeloid cells.

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Suppressed G-protein-coupled receptor kinase 2 activity protects female diabetic-mouse aorta against endothelial dysfunction.

PubMed

Taguchi, K; Matsumoto, T; Kamata, K; Kobayashi, T

2013-01-01

Pre-menopausal women have less cardiovascular disease and lower cardiovascular morbidity and mortality than men the same age. Previously, we noted in mice that G-protein-coupled receptor kinase 2 (GRK2) negatively regulates the Akt/eNOS pathway in male diabetic aortas and that endothelial function via the Akt/eNOS pathway is less affected in female diabetic aortas. The cellular mechanisms underlying these sex differences remain unclear. We aimed to investigate the ways in which GRK2 might modulate vascular functions in male and female diabetic mice (DM). Vascular functions were examined in aortic rings. GRK2, β-arrestin 2 and Akt/eNOS-signalling-pathway protein levels and activities were assayed by Western blotting. Phenylephrine-induced contraction was greater, while both clonidine-induced and insulin-induced relaxations were weaker (vs. male controls), in aortas from male type 2 DM, suggesting impairments of the Akt/eNOS pathway and α-adrenoceptor function. GRK2-inhibitor reversed only the impairment in Akt/eNOS-pathway-mediated relaxation in male DM. Increases in GRK2 activity, GRK2 expression in the membrane, plasma Ang II and systolic blood pressure were seen in male DM (vs. male controls) but not in female DM; these increases were attenuated by GRK2-inhibitor treatment. Repeatedly obtaining clonidine concentration-response curves led to reduced relaxation in male and in female DM aortas, indicating similar desensitization between female DM and male DM. This effect was reversed by GRK2-inhibitor in both sexes. GRK2 plays a key role in modulating the aortic vasodilator effect of clonidine by selectively affecting the Akt/eNOS pathway. This action of GRK2 is more powerful in male than in female DM. © 2012 The Authors Acta Physiologica © 2012 Scandinavian Physiological Society.

Serial femtosecond crystallography datasets from G protein-coupled receptors

PubMed Central

White, Thomas A.; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A.; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R.; Yoon, Chun Hong; Yefanov, Oleksandr M.; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E.; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

2016-01-01

We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data. PMID:27479354

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

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

Palmitoylation and membrane cholesterol stabilize μ-opioid receptor homodimerization and G protein coupling

PubMed Central

2012-01-01

Background A cholesterol-palmitoyl interaction has been reported to occur in the dimeric interface of the β2-adrenergic receptor crystal structure. We sought to investigate whether a similar phenomenon could be observed with μ-opioid receptor (OPRM1), and if so, to assess the role of cholesterol in this class of G protein-coupled receptor (GPCR) signaling. Results C3.55(170) was determined to be the palmitoylation site of OPRM1. Mutation of this Cys to Ala did not affect the binding of agonists, but attenuated receptor signaling and decreased cholesterol associated with the receptor signaling complex. In addition, both attenuation of receptor palmitoylation (by mutation of C3.55[170] to Ala) and inhibition of cholesterol synthesis (by treating the cells with simvastatin, a HMG-CoA reductase inhibitor) impaired receptor signaling, possibly by decreasing receptor homodimerization and Gαi2 coupling; this was demonstrated by co-immunoprecipitation, immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) analyses. A computational model of the OPRM1 homodimer structure indicated that a specific cholesterol-palmitoyl interaction can facilitate OPRM1 homodimerization at the TMH4-TMH4 interface. Conclusions We demonstrate that C3.55(170) is the palmitoylation site of OPRM1 and identify a cholesterol-palmitoyl interaction in the OPRM1 complex. Our findings suggest that this interaction contributes to OPRM1 signaling by facilitating receptor homodimerization and G protein coupling. This conclusion is supported by computational modeling of the OPRM1 homodimer. PMID:22429589

Regulation of prostate cancer by hormone-responsive G protein-coupled receptors.

PubMed

Wang, Wei; Chen, Zhao-Xia; Guo, Dong-Yu; Tao, Ya-Xiong

2018-06-15

Regulation of prostate cancer by androgen and androgen receptor (AR), and blockade of AR signaling by AR antagonists and steroidogenic enzyme inhibitors have been extensively studied. G protein-coupled receptors (GPCRs) are a family of membrane receptors that regulate almost all physiological processes. Nearly 40% of FDA-approved drugs in the market target GPCRs. A variety of GPCRs that mediate reproductive function have been demonstrated to be involved in the regulation of prostate cancer. These GPCRs include gonadotropin-releasing hormone receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, relaxin receptor, ghrelin receptor, and kisspeptin receptor. We highlight here GPCR regulation of prostate cancer by these GPCRs. Further therapeutic approaches targeting these GPCRs for the treatment of prostate cancer are summarized. Copyright © 2018. Published by Elsevier Inc.

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

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

Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs)

PubMed Central

Yang, Kai; Jackson, Michael F.; MacDonald, John F.

2014-01-01

G Protein Coupled Receptors (GPCRs) are the largest family of receptors whose ligands constitute nearly a third of prescription drugs in the market. They are widely involved in diverse physiological functions including learning and memory. NMDA receptors (NMDARs), which belong to the ionotropic glutamate receptor family, are likewise ubiquitously expressed in the central nervous system (CNS) and play a pivotal role in learning and memory. Despite its critical contribution to physiological and pathophysiological processes, few pharmacological interventions aimed directly at regulating NMDAR function have been developed to date. However, it is well established that NMDAR function is precisely regulated by cellular signalling cascades recruited downstream of G protein coupled receptor (GPCR) stimulation. Accordingly, the downstream regulation of NMDARs likely represents an important determinant of outcome following treatment with neuropsychiatric agents that target selected GPCRs. Importantly, the functional consequence of such regulation on NMDAR function varies, based not only on the identity of the GPCR, but also on the cell type in which relevant receptors are expressed. Indeed, the mechanisms responsible for regulating NMDARs by GPCRs involve numerous intracellular signalling molecules and regulatory proteins that vary from one cell type to another. In the present article, we highlight recent findings from studies that have uncovered novel mechanisms by which selected GPCRs regulate NMDAR function and consequently NMDAR-dependent plasticity. PMID:24562329

Constitutive phospholipid scramblase activity of a G protein-coupled receptor

NASA Astrophysics Data System (ADS)

Goren, Michael A.; Morizumi, Takefumi; Menon, Indu; Joseph, Jeremiah S.; Dittman, Jeremy S.; Cherezov, Vadim; Stevens, Raymond C.; Ernst, Oliver P.; Menon, Anant K.

2014-10-01

Opsin, the rhodopsin apoprotein, was recently shown to be an ATP-independent flippase (or scramblase) that equilibrates phospholipids across photoreceptor disc membranes in mammalian retina, a process required for disc homoeostasis. Here we show that scrambling is a constitutive activity of rhodopsin, distinct from its light-sensing function. Upon reconstitution into vesicles, discrete conformational states of the protein (rhodopsin, a metarhodopsin II-mimic, and two forms of opsin) facilitated rapid (>10,000 phospholipids per protein per second) scrambling of phospholipid probes. Our results indicate that the large conformational changes involved in converting rhodopsin to metarhodopsin II are not required for scrambling, and that the lipid translocation pathway either lies near the protein surface or involves membrane packing defects in the vicinity of the protein. In addition, we demonstrate that β2-adrenergic and adenosine A2A receptors scramble lipids, suggesting that rhodopsin-like G protein-coupled receptors may play an unexpected moonlighting role in re-modelling cell membranes.

Modelling and simulation of biased agonism dynamics at a G protein-coupled receptor.

PubMed

Bridge, L J; Mead, J; Frattini, E; Winfield, I; Ladds, G

2018-04-07

Theoretical models of G protein-coupled receptor (GPCR) concentration-response relationships often assume an agonist producing a single functional response via a single active state of the receptor. These models have largely been analysed assuming steady-state conditions. There is now much experimental evidence to suggest that many GPCRs can exist in multiple receptor conformations and elicit numerous functional responses, with ligands having the potential to activate different signalling pathways to varying extents-a concept referred to as biased agonism, functional selectivity or pluri-dimensional efficacy. Moreover, recent experimental results indicate a clear possibility for time-dependent bias, whereby an agonist's bias with respect to different pathways may vary dynamically. Efforts towards understanding the implications of temporal bias by characterising and quantifying ligand effects on multiple pathways will clearly be aided by extending current equilibrium binding and biased activation models to include G protein activation dynamics. Here, we present a new model of time-dependent biased agonism, based on ordinary differential equations for multiple cubic ternary complex activation models with G protein cycle dynamics. This model allows simulation and analysis of multi-pathway activation bias dynamics at a single receptor for the first time, at the level of active G protein (α GTP ), towards the analysis of dynamic functional responses. The model is generally applicable to systems with N G G proteins and N* active receptor states. Numerical simulations for N G =N * =2 reveal new insights into the effects of system parameters (including cooperativities, and ligand and receptor concentrations) on bias dynamics, highlighting new phenomena including the dynamic inter-conversion of bias direction. Further, we fit this model to 'wet' experimental data for two competing G proteins (G i and G s ) that become activated upon stimulation of the adenosine A 1

Molecular evolution of a chordate specific family of G protein-coupled receptors

PubMed Central

2011-01-01

Background Chordate evolution is a history of innovations that is marked by physical and behavioral specializations, which led to the development of a variety of forms from a single ancestral group. Among other important characteristics, vertebrates obtained a well developed brain, anterior sensory structures, a closed circulatory system and gills or lungs as blood oxygenation systems. The duplication of pre-existing genes had profound evolutionary implications for the developmental complexity in vertebrates, since mutations modifying the function of a duplicated protein can lead to novel functions, improving the evolutionary success. Results We analyzed here the evolution of the GPRC5 family of G protein-coupled receptors by comprehensive similarity searches and found that the receptors are only present in chordates and that the size of the receptor family expanded, likely due to genome duplication events in the early history of vertebrate evolution. We propose that a single GPRC5 receptor coding gene originated in a stem chordate ancestor and gave rise by duplication events to a gene family comprising three receptor types (GPRC5A-C) in vertebrates, and a fourth homologue present only in mammals (GPRC5D). Additional duplications of GPRC5B and GPRC5C sequences occurred in teleost fishes. The finding that the expression patterns of the receptors are evolutionarily conserved indicates an important biological function of these receptors. Moreover, we found that expression of GPRC5B is regulated by vitamin A in vivo, confirming previous findings that linked receptor expression to retinoic acid levels in tumor cell lines and strengthening the link between the receptor expression and the development of a complex nervous system in chordates, known to be dependent on retinoic acid signaling. Conclusions GPRC5 receptors, a class of G protein-coupled receptors with unique sequence characteristics, may represent a molecular novelty that helped non-chordates to become

Signaling, physiological functions and clinical relevance of the G protein-coupled estrogen receptor GPER.

PubMed

Prossnitz, Eric R; Barton, Matthias

2009-09-01

GPR30, now named GPER1 (G protein-coupled estrogen receptor1) or GPER here, was first identified as an orphan 7-transmembrane G protein-coupled receptor by multiple laboratories using either homology cloning or differential expression and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. The actions of estrogen are extensive in the body and are thought to be mediated predominantly by classical nuclear estrogen receptors that act as transcription factors/regulators. Nevertheless, certain aspects of estrogen function remain incompatible with the generally accepted mechanisms of classical estrogen receptor action. Many recent studies have revealed that GPER contributes to some of the actions of estrogen, including rapid signaling events and rapid transcriptional activation. With the introduction of GPER-selective ligands and GPER knockout mice, the functions of GPER are becoming more clearly defined. In many cases, there appears to be a complex interplay between the two receptor systems, suggesting that estrogen-mediated physiological responses may be mediated by either receptor or a combination of both receptor types, with important medical implications.

Homology Modeling of Class A G Protein-Coupled Receptors

PubMed Central

Costanzi, Stefano

2012-01-01

G protein-coupled receptors (GPCRs) are a large superfamily of membrane bound signaling proteins that hold great pharmaceutical interest. Since experimentally elucidated structures are available only for a very limited number of receptors, homology modeling has become a widespread technique for the construction of GPCR models intended to study the structure-function relationships of the receptors and aid the discovery and development of ligands capable of modulating their activity. Through this chapter, various aspects involved in the constructions of homology models of the serpentine domain of the largest class of GPCRs, known as class A or rhodopsin family, are illustrated. In particular, the chapter provides suggestions, guidelines and critical thoughts on some of the most crucial aspect of GPCR modeling, including: collection of candidate templates and a structure-based alignment of their sequences; identification and alignment of the transmembrane helices of the query receptor to the corresponding domains of the candidate templates; selection of one or more templates receptor; election of homology or de novo modeling for the construction of specific extracellular and intracellular domains; construction of the three-dimensional models, with special consideration to extracellular regions, disulfide bridges, and interhelical cavity; validation of the models through controlled virtual screening experiments. PMID:22323225

Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

PubMed Central

Walther, Cornelia

2015-01-01

The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

PubMed

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

2014-08-29

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-[(3)H]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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Stereochemistry of an agonist determines coupling preference of beta2-adrenoceptor to different G proteins in cardiomyocytes.

PubMed

Woo, Anthony Yiu-Ho; Wang, Tian-Bing; Zeng, Xiaokun; Zhu, Weizhong; Abernethy, Darrell R; Wainer, Irving W; Xiao, Rui-Ping

2009-01-01

A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that although most beta(2)-adrenoceptor agonists activate both G(s) and G(i) proteins, fenoterol, a full agonist of beta(2)-adrenoceptor, selectively activates G(s) protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,R isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R isomers. It is noteworthy that although (R,R)-fenoterol and (R,R)-methoxyfenoterol preferentially activate G(s) signaling, their S,R isomers were able to activate both G(s) and G(i) proteins as evidenced by the robust pertussis toxin sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on G(s),G(i2), and G(i3) proteins. The inefficient G(i) signaling with the R,R isomers is not caused by the inability of the R,R isomers to trigger the protein kinase A (PKA)-mediated phosphorylation of the beta(2)-adrenoceptor, because the R,R isomers also markedly increased phosphorylation of the receptor at serine 262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

PubMed Central

Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L.; Hille, Bertil

2016-01-01

Activated Gq protein–coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to

Functional autoantibodies targeting G protein-coupled receptors in rheumatic diseases.

PubMed

Cabral-Marques, Otavio; Riemekasten, Gabriela

2017-11-01

G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of integral membrane proteins that participate in different physiological processes such as the regulation of the nervous and immune systems. Besides the endogenous ligands of GPCRs, functional autoantibodies are also able to bind GPCRs to trigger or block intracellular signalling pathways, resulting in agonistic or antagonistic effects, respectively. In this Review, the effects of functional GPCR-targeting autoantibodies on the pathogenesis of autoimmune diseases, including rheumatic diseases, are discussed. Autoantibodies targeting β1 and β2 adrenergic receptors, which are expressed by cardiac and airway smooth muscle cells, respectively, have an important role in the development of asthma and cardiovascular diseases. In addition, high levels of autoantibodies against the muscarinic acetylcholine receptor M3 as well as those targeting endothelin receptor type A and type 1 angiotensin II receptor have several implications in the pathogenesis of rheumatic diseases such as Sjögren syndrome and systemic sclerosis. Expanding the knowledge of the pathophysiological roles of autoantibodies against GPCRs will shed light on the biology of these receptors and open avenues for new therapeutic approaches.

Regulation of neuronal communication by G protein-coupled receptors.

PubMed

Huang, Yunhong; Thathiah, Amantha

2015-06-22

Neuronal communication plays an essential role in the propagation of information in the brain and requires a precisely orchestrated connectivity between neurons. Synaptic transmission is the mechanism through which neurons communicate with each other. It is a strictly regulated process which involves membrane depolarization, the cellular exocytosis machinery, neurotransmitter release from synaptic vesicles into the synaptic cleft, and the interaction between ion channels, G protein-coupled receptors (GPCRs), and downstream effector molecules. The focus of this review is to explore the role of GPCRs and G protein-signaling in neurotransmission, to highlight the function of GPCRs, which are localized in both presynaptic and postsynaptic membrane terminals, in regulation of intrasynaptic and intersynaptic communication, and to discuss the involvement of astrocytic GPCRs in the regulation of neuronal communication. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

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

PubMed

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

2008-12-01

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

Overlapping and Opposing Functions of G Protein-coupled Receptor Kinase 2 (GRK2) and GRK5 during Heart Development*

PubMed Central

Philipp, Melanie; Berger, Ina M.; Just, Steffen; Caron, Marc G.

2014-01-01

G protein-coupled receptor kinases 2 (GRK2) and 5 (GRK5) are fundamental regulators of cardiac performance in adults but are less well characterized for their function in the hearts of embryos. GRK2 and -5 belong to different subfamilies and function as competitors in the control of certain receptors and signaling pathways. In this study, we used zebrafish to investigate whether the fish homologs of GRK2 and -5, Grk2/3 and Grk5, also have unique, complementary, or competitive roles during heart development. We found that they differentially regulate the heart rate of early embryos and equally facilitate heart function in older embryos and that both are required to develop proper cardiac morphology. A loss of Grk2/3 results in dilated atria and hypoplastic ventricles, and the hearts of embryos depleted in Grk5 present with a generalized atrophy. This Grk5 morphant phenotype was associated with an overall decrease of early cardiac progenitors as well as a reduction in the area occupied by myocardial progenitor cells. In the case of Grk2/3, the progenitor decrease was confined to a subset of precursor cells with a committed ventricular fate. We attempted to rescue the GRK loss-of-function heart phenotypes by downstream activation of Hedgehog signaling. The Grk2/3 loss-of-function embryos were rescued by this approach, but Grk5 embryos failed to respond. In summary, we found that GRK2 and GRK5 control cardiac function as well as morphogenesis during development although with different morphological outcomes. PMID:25104355

Conformational Fluctuations in G-Protein-Coupled Receptors

NASA Astrophysics Data System (ADS)

Brown, Michael F.

2014-03-01

G-protein-coupled receptors (GPCRs) comprise almost 50% of pharmaceutical drug targets, where rhodopsin is an important prototype and occurs naturally in a lipid membrane. Rhodopsin photoactivation entails 11-cis to all-trans isomerization of the retinal cofactor, yielding an equilibrium between inactive Meta-I and active Meta-II states. Two important questions are: (1) Is rhodopsin is a simple two-state switch? Or (2) does isomerization of retinal unlock an activated conformational ensemble? For an ensemble-based activation mechanism (EAM) a role for conformational fluctuations is clearly indicated. Solid-state NMR data together with theoretical molecular dynamics (MD) simulations detect increased local mobility of retinal after light activation. Resultant changes in local dynamics of the cofactor initiate large-scale fluctuations of transmembrane helices that expose recognition sites for the signal-transducing G-protein. Time-resolved FTIR studies and electronic spectroscopy further show the conformational ensemble is strongly biased by the membrane lipid composition, as well as pH and osmotic pressure. A new flexible surface model (FSM) describes how the curvature stress field of the membrane governs the energetics of active rhodopsin, due to the spontaneous monolayer curvature of the lipids. Furthermore, influences of osmotic pressure dictate that a large number of bulk water molecules are implicated in rhodopsin activation. Around 60 bulk water molecules activate rhodopsin, which is much larger than the number of structural waters seen in X-ray crystallography, or inferred from studies of bulk hydrostatic pressure. Conformational selection and promoting vibrational motions of rhodopsin lead to activation of the G-protein (transducin). Our biophysical data give a paradigm shift in understanding GPCR activation. The new view is: dynamics and conformational fluctuations involve an ensemble of substates that activate the cognate G-protein in the amplified visual

The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

PubMed

Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

2017-08-11

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

G-protein-coupled receptors participate in cytokinesis

PubMed Central

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

2014-01-01

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 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, 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. PMID:22888021

Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors.

PubMed

Zhou, X Edward; He, Yuanzheng; de Waal, Parker W; Gao, Xiang; Kang, Yanyong; Van Eps, Ned; Yin, Yanting; Pal, Kuntal; Goswami, Devrishi; White, Thomas A; Barty, Anton; Latorraca, Naomi R; Chapman, Henry N; Hubbell, Wayne L; Dror, Ron O; Stevens, Raymond C; Cherezov, Vadim; Gurevich, Vsevolod V; Griffin, Patrick R; Ernst, Oliver P; Melcher, Karsten; Xu, H Eric

2017-07-27

G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs. Copyright © 2017 Elsevier Inc. All rights reserved.

Modeling G Protein-Coupled Receptors: a Concrete Possibility

PubMed Central

Costanzi, Stefano

2010-01-01

G protein-coupled receptors (GPCRs) are a large superfamily of membrane bound signaling proteins that are involved in the regulation of a wide range of physiological functions and constitute the most common target for therapeutic intervention. Due to the paucity of crystal structures, homology modeling has become a widespread technique for the construction of GPCR models, which have been applied to the study of their structure-function relationships and to the identification of lead ligands through virtual screening. Rhodopsin has been for years the only available template. However, recent breakthroughs in GPCR crystallography have led to the solution of the structures of a few additional receptors. In light of these newly elucidated crystal structures, we have been able to produce a substantial amount of data to demonstrate that accurate models of GPCRs in complex with their ligands can be constructed through homology modeling followed by fully flexible molecular docking. These results have been confirmed by our success in the first blind assessment of GPCR modeling and docking, organized in coordination with the solution of the X-ray structure of the adenosine A2A receptor. Taken together, these data indicate that: a) the transmembrane helical bundle can be modeled with considerable accuracy; b) predicting the binding mode of a ligand, although doable, is challenging; c) modeling of the extracellular and intracellular loops is still problematic. PMID:21253444

Spatially restricted G protein-coupled receptor activity via divergent endocytic compartments.

PubMed

Jean-Alphonse, Frederic; Bowersox, Shanna; Chen, Stanford; Beard, Gemma; Puthenveedu, Manojkumar A; Hanyaloglu, Aylin C

2014-02-14

Postendocytic sorting of G protein-coupled receptors (GPCRs) is driven by their interactions between highly diverse receptor sequence motifs with their interacting proteins, such as postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) (PDZ) domain proteins. However, whether these diverse interactions provide an underlying functional specificity, in addition to driving sorting, is unknown. Here we identify GPCRs that recycle via distinct PDZ ligand/PDZ protein pairs that exploit their recycling machinery primarily for targeted endosomal localization and signaling specificity. The luteinizing hormone receptor (LHR) and β2-adrenergic receptor (B2AR), two GPCRs sorted to the regulated recycling pathway, underwent divergent trafficking to distinct endosomal compartments. Unlike B2AR, which traffics to early endosomes (EE), LHR internalizes to distinct pre-early endosomes (pre-EEs) for its recycling. Pre-EE localization required interactions of the LHR C-terminal tail with the PDZ protein GAIP-interacting protein C terminus, inhibiting its traffic to EEs. Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling. Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment. We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.

Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

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

Booe, Jason M.; Walker, Christopher S.; Barwell, James

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

Structural basis for receptor activity-modifying protein-dependent selective peptide recognition by a G protein-coupled receptor

DOE PAGES

Booe, Jason M.; Walker, Christopher S.; Barwell, James; ...

2015-05-14

Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less

When Heterotrimeric G Proteins Are Not Activated by G Protein-Coupled Receptors: Structural Insights and Evolutionary Conservation.

PubMed

DiGiacomo, Vincent; Marivin, Arthur; Garcia-Marcos, Mikel

2018-01-23

Heterotrimeric G proteins are signal-transducing switches conserved across eukaryotes. In humans, they work as critical mediators of intercellular communication in the context of virtually any physiological process. While G protein regulation by G protein-coupled receptors (GPCRs) is well-established and has received much attention, it has become recently evident that heterotrimeric G proteins can also be activated by cytoplasmic proteins. However, this alternative mechanism of G protein regulation remains far less studied than GPCR-mediated signaling. This Viewpoint focuses on recent advances in the characterization of a group of nonreceptor proteins that contain a sequence dubbed the "Gα-binding and -activating (GBA) motif". So far, four proteins present in mammals [GIV (also known as Girdin), DAPLE, CALNUC, and NUCB2] and one protein in Caenorhabditis elegans (GBAS-1) have been described as possessing a functional GBA motif. The GBA motif confers guanine nucleotide exchange factor activity on Gαi subunits in vitro and activates G protein signaling in cells. The importance of this mechanism of signal transduction is highlighted by the fact that its dysregulation underlies human diseases, such as cancer, which has made the proteins attractive new candidates for therapeutic intervention. Here we discuss recent discoveries on the structural basis of GBA-mediated activation of G proteins and its evolutionary conservation and compare them with the better-studied mechanism mediated by GPCRs.

G Protein–Coupled Receptor-Type G Proteins Are Required for Light-Dependent Seedling Growth and Fertility in Arabidopsis[W

PubMed Central

Jaffé, Felix W.; Freschet, Gian-Enrico C.; Valdes, Billy M.; Runions, John; Terry, Matthew J.; Williams, Lorraine E.

2012-01-01

G protein–coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein–coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1pro:GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development. PMID:23001037

Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

PubMed

Mnpotra, Jagjeet S; Qiao, Zhuanhong; Cai, Jian; Lynch, Diane L; Grossfield, Alan; Leioatts, Nicholas; Hurst, Dow P; Pitman, Michael C; Song, Zhao-Hui; Reggio, Patricia H

2014-07-18

In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

GPCRdb: an information system for G protein-coupled receptors

PubMed Central

Isberg, Vignir; Mordalski, Stefan; Munk, Christian; Rataj, Krzysztof; Harpsøe, Kasper; Hauser, Alexander S.; Vroling, Bas; Bojarski, Andrzej J.; Vriend, Gert; Gloriam, David E.

2016-01-01

Recent developments in G protein-coupled receptor (GPCR) structural biology and pharmacology have greatly enhanced our knowledge of receptor structure-function relations, and have helped improve the scientific foundation for drug design studies. The GPCR database, GPCRdb, serves a dual role in disseminating and enabling new scientific developments by providing reference data, analysis tools and interactive diagrams. This paper highlights new features in the fifth major GPCRdb release: (i) GPCR crystal structure browsing, superposition and display of ligand interactions; (ii) direct deposition by users of point mutations and their effects on ligand binding; (iii) refined snake and helix box residue diagram looks; and (iii) phylogenetic trees with receptor classification colour schemes. Under the hood, the entire GPCRdb front- and back-ends have been re-coded within one infrastructure, ensuring a smooth browsing experience and development. GPCRdb is available at http://www.gpcrdb.org/ and it's open source code at https://bitbucket.org/gpcr/protwis. PMID:26582914

Chaperoning G Protein-Coupled Receptors: From Cell Biology to Therapeutics

PubMed Central

Conn, P. Michael

2014-01-01

G protein-coupled receptors (GPCRs) are membrane proteins that traverse the plasma membrane seven times (hence, are also called 7TM receptors). The polytopic structure of GPCRs makes the folding of GPCRs difficult and complex. Indeed, many wild-type GPCRs are not folded optimally, and defects in folding are the most common cause of genetic diseases due to GPCR mutations. Both general and receptor-specific molecular chaperones aid the folding of GPCRs. Chemical chaperones have been shown to be able to correct the misfolding in mutant GPCRs, proving to be important tools for studying the structure-function relationship of GPCRs. However, their potential therapeutic value is very limited. Pharmacological chaperones (pharmacoperones) are potentially important novel therapeutics for treating genetic diseases caused by mutations in GPCR genes that resulted in misfolded mutant proteins. Pharmacoperones also increase cell surface expression of wild-type GPCRs; therefore, they could be used to treat diseases that do not harbor mutations in GPCRs. Recent studies have shown that indeed pharmacoperones work in both experimental animals and patients. High-throughput assays have been developed to identify new pharmacoperones that could be used as therapeutics for a number of endocrine and other genetic diseases. PMID:24661201

The structural basis of arrestin-mediated regulation of G-protein-coupled receptors

PubMed Central

Gurevich, Vsevolod V.; Gurevich, Eugenia V.

2008-01-01

The 4 mammalian arrestins serve as almost universal regulators of the largest known family of signaling proteins, G-protein-coupled receptors (GPCRs). Arrestins terminate receptor interactions with G proteins, redirect the signaling to a variety of alternative pathways, and orchestrate receptor internalization and subsequent intracellular trafficking. The elucidation of the structural basis and fine molecular mechanisms of the arrestin–receptor interaction paved the way to the targeted manipulation of this interaction from both sides to produce very stable or extremely transient complexes that helped to understand the regulation of many biologically important processes initiated by active GPCRs. The elucidation of the structural basis of arrestin interactions with numerous non-receptor-binding partners is long overdue. It will allow the construction of fully functional arrestins in which the ability to interact with individual partners is specifically disrupted or enhanced by targeted mutagenesis. These “custom-designed” arrestin mutants will be valuable tools in defining the role of various interactions in the intricate interplay of multiple signaling pathways in the living cell. The identification of arrestin-binding sites for various signaling molecules will also set the stage for designing molecular tools for therapeutic intervention that may prove useful in numerous disorders associated with congenital or acquired disregulation of GPCR signaling. PMID:16460808

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

PubMed

Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P; Burgoyne, Robert D; Mayans, Olga; Derrick, Jeremy P; Lian, Lu-Yun

2015-07-24

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions*

PubMed Central

Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P.; Burgoyne, Robert D.; Mayans, Olga; Derrick, Jeremy P.; Lian, Lu-Yun

2015-01-01

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca2+-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca2+/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca2+/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178–Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca2+/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178–Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. PMID:25979333

The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors*

PubMed Central

Smith, Jeffrey S.; Rajagopal, Sudarshan

2016-01-01

The β-arrestins (βarrs) are versatile, multifunctional adapter proteins that are best known for their ability to desensitize G protein-coupled receptors (GPCRs), but also regulate a diverse array of cellular functions. To signal in such a complex fashion, βarrs adopt multiple conformations and are regulated at multiple levels to differentially activate downstream pathways. Recent structural studies have demonstrated that βarrs have a conserved structure and activation mechanism, with plasticity of their structural fold, allowing them to adopt a wide array of conformations. Novel roles for βarrs continue to be identified, demonstrating the importance of these dynamic regulators of cellular signaling. PMID:26984408

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

PubMed

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

2016-05-01

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

Overlapping and opposing functions of G protein-coupled receptor kinase 2 (GRK2) and GRK5 during heart development.

PubMed

Philipp, Melanie; Berger, Ina M; Just, Steffen; Caron, Marc G

2014-09-19

G protein-coupled receptor kinases 2 (GRK2) and 5 (GRK5) are fundamental regulators of cardiac performance in adults but are less well characterized for their function in the hearts of embryos. GRK2 and -5 belong to different subfamilies and function as competitors in the control of certain receptors and signaling pathways. In this study, we used zebrafish to investigate whether the fish homologs of GRK2 and -5, Grk2/3 and Grk5, also have unique, complementary, or competitive roles during heart development. We found that they differentially regulate the heart rate of early embryos and equally facilitate heart function in older embryos and that both are required to develop proper cardiac morphology. A loss of Grk2/3 results in dilated atria and hypoplastic ventricles, and the hearts of embryos depleted in Grk5 present with a generalized atrophy. This Grk5 morphant phenotype was associated with an overall decrease of early cardiac progenitors as well as a reduction in the area occupied by myocardial progenitor cells. In the case of Grk2/3, the progenitor decrease was confined to a subset of precursor cells with a committed ventricular fate. We attempted to rescue the GRK loss-of-function heart phenotypes by downstream activation of Hedgehog signaling. The Grk2/3 loss-of-function embryos were rescued by this approach, but Grk5 embryos failed to respond. In summary, we found that GRK2 and GRK5 control cardiac function as well as morphogenesis during development although with different morphological outcomes. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

An activation switch in the rhodopsin family of G protein-coupled receptors: the thyrotropin receptor.

PubMed

Urizar, Eneko; Claeysen, Sylvie; Deupí, Xavier; Govaerts, Cedric; Costagliola, Sabine; Vassart, Gilbert; Pardo, Leonardo

2005-04-29

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of approximately 14 and approximately 10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of approximately 13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways*

PubMed Central

Lecat, Sandra; Matthes, Hans W.D.; Pepperkok, Rainer; Simpson, Jeremy C.; Galzi, Jean-Luc

2015-01-01

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. PMID:25759509

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways.

PubMed

Lecat, Sandra; Matthes, Hans W D; Pepperkok, Rainer; Simpson, Jeremy C; Galzi, Jean-Luc

2015-05-01

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Conformational dynamics of activation for the pentameric complex of dimeric G protein – coupled receptor and heterotrimeric G protein

PubMed Central

Orban, Tivadar; Jastrzebska, Beata; Gupta, Sayan; Wang, Benlian; Miyagi, Masaru; Chance, Mark R.; Palczewski, Krzysztof

2012-01-01

Summary Photoactivation of rhodopsin (Rho), a G protein-coupled receptor (GPCR), causes conformational changes that provide a specific binding site for the rod G protein, Gt. In this work we employed structural mass spectrometry (MS) techniques to elucidate the structural changes accompanying transition of ground state Rho to photoactivated Rho (Rho*) and in the pentameric complex between dimeric Rho* and heterotrimeric Gt. Observed differences in hydroxyl radical labeling and deuterium uptake between Rho* and the (Rho*)2-Gt complex suggest that photoactivation causes structural relaxation of Rho following its initial tightening upon Gt coupling. In contrast, nucleotide-free Gt in the complex is significantly more accessible to deuterium uptake allowing it to accept GTP and mediating complex dissociation. Thus, we provide direct evidence that in the critical step of signal amplification, Rho* and Gt exhibit dissimilar conformational changes when they are coupled in the (Rho*)2-Gt complex. PMID:22579250

Stimulation of the p38 Mitogen-activated Protein Kinase Pathway in Neonatal Rat Ventricular Myocytes by the G Protein–coupled Receptor Agonists, Endothelin-1 and Phenylephrine: A Role in Cardiac Myocyte Hypertrophy?

PubMed Central

Clerk, Angela; Michael, Ashour; Sugden, Peter H.

1998-01-01

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein–coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by ∼12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 μM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response. PMID:9679149

Exploring G protein-coupled receptor signaling networks using SILAC-based phosphoproteomics

PubMed Central

Williams, Grace R.; Bethard, Jennifer R.; Berkaw, Mary N.; Nagel, Alexis K.; Luttrell, Louis M.; Ball, Lauren E.

2015-01-01

The type 1 parathyroid hormone receptor (PTH1R) is a key regulator of calcium homeostasis and bone turnover. Here, we employed SILAC-based quantitative mass spectrometry combined with bioinformatic pathways analysis to examine global changes in protein phosphorylation following short-term stimulation of endogenously expressed PTH1R in osteoblastic cells in vitro. Following 5 min exposure to the conventional agonist, PTH(1-34), we detected significant changes in the phosphorylation of 224 distinct proteins. Kinase substrate motif enrichment demonstrated that consensus motifs for PKA and CAMK2 were the most heavily upregulated within the phosphoproteome, while consensus motifs for mitogen-activated protein kinases were strongly downregulated. Signaling pathways analysis identified ERK1/2 and AKT as important nodal kinases in the downstream network and revealed strong regulation of small GTPases involved in cytoskeletal rearrangement, cell motility, and focal adhesion complex signaling. Our data illustrate the utility of quantitative mass spectrometry in measuring dynamic changes in protein phosphorylation following GPCR activation. PMID:26160508

An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

PubMed

Wittenberger, T; Schaller, H C; Hellebrand, S

2001-03-30

We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families. Copyright 2001 Academic Press.

G-protein-coupled receptor structures were not built in a day.

PubMed

Blois, Tracy M; Bowie, James U

2009-07-01

Among the most exciting recent developments in structural biology is the structure determination of G-protein-coupled receptors (GPCRs), which comprise the largest class of membrane proteins in mammalian cells and have enormous importance for disease and drug development. The GPCR structures are perhaps the most visible examples of a nascent revolution in membrane protein structure determination. Like other major milestones in science, however, such as the sequencing of the human genome, these achievements were built on a hidden foundation of technological developments. Here, we describe some of the methods that are fueling the membrane protein structure revolution and have enabled the determination of the current GPCR structures, along with new techniques that may lead to future structures.

Involvement of epidermal growth factor receptor signaling in estrogen inhibition of oocyte maturation mediated through the G protein-coupled estrogen receptor (Gper) in zebrafish (Danio rerio).

PubMed

Peyton, Candace; Thomas, Peter

2011-07-01

Oocyte maturation (OM) in teleosts is under precise hormonal control by progestins and estrogens. We show here that estrogens activate an epidermal growth factor receptor (Egfr) signaling pathway in fully grown, denuded zebrafish (Danio rerio) oocytes through the G protein-coupled estrogen receptor (Gper; also known as GPR30) to maintain oocyte meiotic arrest in a germinal vesicle breakdown (GVBD) bioassay. A GPER-specific antagonist, G-15, increased spontaneous OM, indicating that the inhibitory estrogen actions on OM are mediated through Gper. Estradiol-17beta-bovine serum albumin, which cannot enter oocytes, decreased GVBD, whereas treatment with actinomycin D did not block estrogen's inhibitory effects, suggesting that estrogens act at the cell surface via a nongenomic mechanism to prevent OM. The intracellular tyrosine kinase (Src) inhibitor, PP2, blocked estrogen inhibition of OM. Expression of egfr mRNA and Egfr protein were detected in denuded zebrafish oocytes. The matrix metalloproteinase (MMP) inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor, increased spontaneous OM, whereas the MMP activator, interleukin-1alpha, decreased spontaneous OM. Moreover, inhibitors of EGFR (ErbB1) and extracellular-related kinase 1 and 2 (Erk1/2; official symbol Mapk3/1) increased spontaneous OM. In addition, estradiol-17beta and the GPER agonist, G-1, increased phosphorylation of Erk, and this was abrogated by simultaneous treatment with the EGFR inhibitor. Taken together, these results suggest that estrogens act through Gper to maintain meiotic arrest via an Src kinase-dependent G-protein betagamma subunit signaling pathway involving transactivation of egfr and phosphorylation of Mapk3/1. To our knowledge, this is the first evidence that EGFR signaling in vertebrate oocytes can prevent meiotic progression.

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

Purine inhibitors of protein kinases, G proteins and polymerases

DOEpatents

Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

2001-07-03

The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

Pharmacology, signaling and physiological relevance of the G protein-coupled receptor 55.

PubMed

Balenga, Nariman A B; Henstridge, Christopher M; Kargl, Julia; Waldhoer, Maria

2011-01-01

According to The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), ∼70 million European adults have consumed cannabis on at least one occasion. Cannabis consumption leads to a variety of psychoactive effects due to the presence of the constituent Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Δ(9)-THC interacts with the endocannabinoid system (ECS), which consists of the seven transmembrane spanning (7TM)/G protein-coupled receptors (GPCRs) CB(1) and CB(2), their respective ligands (endocannabinoids), and enzymes involved in their biosynthesis and degradation. This system plays a critical role in many physiological processes such as learning and memory, appetite control, pain sensation, motor coordination, lipogenesis, modulation of immune response, and the regulation of bone mass. Therefore, a huge effort has been spent trying to fully elucidate the composition and function of the ECS. The G protein-coupled receptor 55 (GPR55) was recently proposed as a novel component of this system; however, its classification as a cannabinoid receptor has been significantly hampered by its complex pharmacology, signaling, and cellular function. GPR55 is phylogenetically distinct from the traditional cannabinoid receptors, but in some experimental paradigms, it is activated by endocannabinoids, phytocannabinoids, and synthetic cannabinoid ligands. However, the most potent compound appears to be a lysophospholipid known as lysophosphatidylinositol (LPI). Here, we provide a comprehensive evaluation of the current pharmacology and signaling of GPR55 and review the proposed role of this receptor in a number of physiological and pathophysiological processes. Copyright © 2011 Elsevier Inc. All rights reserved.

The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors.

PubMed

Smith, Jeffrey S; Rajagopal, Sudarshan

2016-04-22

The β-arrestins (βarrs) are versatile, multifunctional adapter proteins that are best known for their ability to desensitize G protein-coupled receptors (GPCRs), but also regulate a diverse array of cellular functions. To signal in such a complex fashion, βarrs adopt multiple conformations and are regulated at multiple levels to differentially activate downstream pathways. Recent structural studies have demonstrated that βarrs have a conserved structure and activation mechanism, with plasticity of their structural fold, allowing them to adopt a wide array of conformations. Novel roles for βarrs continue to be identified, demonstrating the importance of these dynamic regulators of cellular signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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 M 2 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 M 2 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 M 2 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.

Phosphorylation and regulation of a Gq/11-coupled receptor by casein kinase 1alpha.

PubMed

Budd, D C; McDonald, J E; Tobin, A B

2000-06-30

Agonist-mediated receptor phosphorylation by one or more of the members of the G-protein receptor kinase (GRK) family is an established model for G-protein-coupled receptor (GPCR) phosphorylation resulting in receptor desensitization. Our recent studies have, however, suggested that an alternative route to GPCR phosphorylation may be an operation involving casein kinase 1alpha (CK1alpha). In the current study we investigate the involvement of CK1alpha in the phosphorylation of the human m3-muscarinic receptor in intact cells. We show that expression of a catalytically inactive mutant of CK1alpha, designed to act in a dominant negative manner, inhibits agonist-mediated receptor phosphorylation by approximately 40% in COS-7 and HEK-293 cells. Furthermore, we present evidence that a peptide corresponding to the third intracellular loop of the m3-muscarinic receptor (Ser(345)-Leu(463)) is an inhibitor of CK1alpha due to its ability to both act as a pseudo-substrate for CK1alpha and form a high affinity complex with CK1alpha. Expression of this peptide was able to reduce both basal and agonist-mediated m3-muscarinic receptor phosphorylation in intact cells. These results support the notion that CK1alpha is able to mediate GPCR phosphorylation in an agonist-dependent manner and that this may provide a novel mechanism for GPCR phosphorylation. The functional role of phosphorylation was investigated using a mutant of the m3-muscarinic receptor that showed an approximately 80% reduction in agonist-mediated phosphorylation. Surprisingly, this mutant underwent agonist-mediated desensitization suggesting that, unlike many GPCRs, desensitization of the m3-muscarinic receptor is not mediated by receptor phosphorylation. The inositol (1,4, 5)-trisphosphate response did, however, appear to be dramatically potentiated in the phosphorylation-deficient mutant indicating that phosphorylation may instead control the magnitude of the initial inositol phosphate response.

The calcium-sensing receptor and its interacting proteins

PubMed Central

Huang, Chunfa; Miller, R Tyler

2007-01-01

Abstract Seven membrane-spanning, or G protein-coupled receptors were originally thought to act through het-erotrimeric G proteins that in turn activate intracellular enzymes or ion channels, creating relatively simple, linear signalling pathways. Although this basic model remains true in that this family does act via a relatively small number of G proteins, these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors. These additional proteins give receptors their unique signalling ‘personalities’. The extracellular Ca-sensing receptor (CaR) signals via Gαi, Gαq and Gα12/13, but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects. Additional structural or signalling proteins that interact with the CaR may explain its behaviour more fully. Although the CaR is less well studied in this respect than other receptors, several CaR-interacting proteins such as filamin, a potential scaffolding protein, receptor activity modifying proteins (RAMPs) and potassium channels may contribute to the unique characteristics of the CaR. The CaR also appears to interact with additional proteins common to other G protein-coupled receptors such as arrestins, G protein receptor kinases, protein kinase C, caveolin and proteins in the ubiquitination pathway. These proteins probably represent a few initial members of CaR-based signalling complex. These and other proteins may not all be associated with the CaR in all tissues, but they form the basis for understanding the complete nature of CaR signalling. PMID:17979874

Sexually Dimorphic Role of G Protein-Coupled Estrogen Receptor (GPER) in Modulating Energy Homeostasis

PubMed Central

Davis, Kathryn E.; Carstens, Elizabeth J.; Irani, Boman G.; Gent, Lana M.; Hahner, Lisa M.; Clegg, Deborah J.

2014-01-01

The classical estrogen receptors, estrogen receptor-α and estrogen receptor-β are well established in the regulation of body weight and energy homeostasis in both male and female mice, whereas, the role for G protein-coupled estrogen receptor 1 (GPER) as a modulator of energy homeostasis remains controversial. This study sought to determine whether gene deletion of GPER (GPER KO) alters body weight, body adiposity, food intake, and energy homeostasis in both males and females. Male mice lacking GPER developed moderate obesity and larger adipocyte size beginning at 8 weeks of age, with significant reductions in energy expenditure, but not food intake or adipocyte number. Female GPER KO mice developed increased body weight relative to WT females a full 6 weeks later than the male GPER KO mice. Female GPER KO mice also had reductions in energy expenditure, but not significant increases in body fat content. Consistent with their decrease in energy expenditure, GPER KO males and females showed significant reductions in two brown fat thermogenic proteins. GPER KO females, prior to their divergence in body weight, were less sensitive than WT females to the feeding-inhibitory effects of leptin and CCK. Additionally, body weight was not as modulated by ovariectomy or estradiol replacement in GPER KO mice. Estradiol treatment activated phosphorylated extracellular signal-regulated kinase (pERK) in WT but not GPER KO females. For the first time, GPER expression was found in the adipocyte but not the stromal fraction of adipose tissue. Together, these results provide new information elucidating a sexual dimorphism in GPER function in the development of postpubertal energy balance. PMID:24560890

Imaging of persistent cAMP signaling by internalized G protein-coupled receptors.

PubMed

Calebiro, Davide; Nikolaev, Viacheslav O; Lohse, Martin J

2010-07-01

G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. They mediate the effects of several endogenous cues and serve as important pharmacological targets. Although many biochemical events involved in GPCR signaling have been characterized in great detail, little is known about their spatiotemporal dynamics in living cells. The recent advent of optical methods based on fluorescent resonance energy transfer allows, for the first time, to directly monitor GPCR signaling in living cells. Utilizing these methods, it has been recently possible to show that the receptors for two protein/peptide hormones, the TSH and the parathyroid hormone, continue signaling to cAMP after their internalization into endosomes. This type of intracellular signaling is persistent and apparently triggers specific cellular outcomes. Here, we review these recent data and explain the optical methods used for such studies. Based on these findings, we propose a revision of the current model of the GPCR-cAMP signaling pathway to accommodate receptor signaling at endosomes.

Role of detergents in conformational exchange of a G protein-coupled receptor.

PubMed

Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K; Prosser, R Scott

2012-10-19

The G protein-coupled β(2)-adrenoreceptor (β(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and β-arrestin. As such, the energy landscape of β(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β(2)AR with a trifluoromethyl probe, (19)F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

Constitutive Gαi coupling activity of very large G protein-coupled receptor 1 (VLGR1) and its regulation by PDZD7 protein.

PubMed

Hu, Qiao-Xia; Dong, Jun-Hong; Du, Hai-Bo; Zhang, Dao-Lai; Ren, Hong-Ze; Ma, Ming-Liang; Cai, Yuan; Zhao, Tong-Chao; Yin, Xiao-Lei; Yu, Xiao; Xue, Tian; Xu, Zhi-Gang; Sun, Jin-Peng

2014-08-29

The very large G protein-coupled receptor 1 (VLGR1) is a core component in inner ear hair cell development. Mutations in the vlgr1 gene cause Usher syndrome, the symptoms of which include congenital hearing loss and progressive retinitis pigmentosa. However, the mechanism of VLGR1-regulated intracellular signaling and its role in Usher syndrome remain elusive. Here, we show that VLGR1 is processed into two fragments after autocleavage at the G protein-coupled receptor proteolytic site. The cleaved VLGR1 β-subunit constitutively inhibited adenylate cyclase (AC) activity through Gαi coupling. Co-expression of the Gαiq chimera with the VLGR1 β-subunit changed its activity to the phospholipase C/nuclear factor of activated T cells signaling pathway, which demonstrates the Gαi protein coupling specificity of this subunit. An R6002A mutation in intracellular loop 2 of VLGR1 abolished Gαi coupling, but the pathogenic VLGR1 Y6236fsx1 mutant showed increased AC inhibition. Furthermore, overexpression of another Usher syndrome protein, PDZD7, decreased the AC inhibition of the VLGR1 β-subunit but showed no effect on the VLGR1 Y6236fsx1 mutant. Taken together, we identified an independent Gαi signaling pathway of the VLGR1 β-subunit and its regulatory mechanisms that may have a role in the development of Usher syndrome. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Involvement of Epidermal Growth Factor Receptor Signaling in Estrogen Inhibition of Oocyte Maturation Mediated Through the G Protein-Coupled Estrogen Receptor (Gper) in Zebrafish (Danio rerio)1

PubMed Central

Peyton, Candace; Thomas, Peter

2011-01-01

Oocyte maturation (OM) in teleosts is under precise hormonal control by progestins and estrogens. We show here that estrogens activate an epidermal growth factor receptor (Egfr) signaling pathway in fully grown, denuded zebrafish (Danio rerio) oocytes through the G protein-coupled estrogen receptor (Gper; also known as GPR30) to maintain oocyte meiotic arrest in a germinal vesicle breakdown (GVBD) bioassay. A GPER-specific antagonist, G-15, increased spontaneous OM, indicating that the inhibitory estrogen actions on OM are mediated through Gper. Estradiol-17beta-bovine serum albumin, which cannot enter oocytes, decreased GVBD, whereas treatment with actinomycin D did not block estrogen's inhibitory effects, suggesting that estrogens act at the cell surface via a nongenomic mechanism to prevent OM. The intracellular tyrosine kinase (Src) inhibitor, PP2, blocked estrogen inhibition of OM. Expression of egfr mRNA and Egfr protein were detected in denuded zebrafish oocytes. The matrix metalloproteinase (MMP) inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor, increased spontaneous OM, whereas the MMP activator, interleukin-1alpha, decreased spontaneous OM. Moreover, inhibitors of EGFR (ErbB1) and extracellular-related kinase 1 and 2 (Erk1/2; official symbol Mapk3/1) increased spontaneous OM. In addition, estradiol-17beta and the GPER agonist, G-1, increased phosphorylation of Erk, and this was abrogated by simultaneous treatment with the EGFR inhibitor. Taken together, these results suggest that estrogens act through Gper to maintain meiotic arrest via an Src kinase-dependent G-protein betagamma subunit signaling pathway involving transactivation of egfr and phosphorylation of Mapk3/1. To our knowledge, this is the first evidence that EGFR signaling in vertebrate oocytes can prevent meiotic progression. PMID:21349822

Identification of a G protein coupled receptor induced in activated T cells.

PubMed

Kaplan, M H; Smith, D I; Sundick, R S

1993-07-15

Many genes are induced after T cell activation to make a cell competent for proliferation and ultimately, function. Many of these genes encode surface receptors for growth factors that signal a cell to proliferate. We have cloned a novel gene (clone 6H1) that codes for a member of the G protein-coupled receptor superfamily. This gene was isolated from a chicken activated T cell cDNA library by low level hybridization to mammalian IL-2 cDNA probes. The 308 amino acid open reading frame has seven hydrophobic, presumably transmembrane domains and a consensus site for interaction with G proteins. Tissue distribution studies suggest that gene expression is restricted to activated T cells. The message appears by 1 h after activation and is maintained for at least 45 h. Transcription of 6H1 is induced by a number of T cell stimuli and is inhibited by cyclosporin A, but not by cycloheximide. This is the first description of a member of this superfamily expressed specifically in activated T cells. The gene product may provide a link between T cell growth factors and G protein activation.

A G Protein-biased Designer G Protein-coupled Receptor Useful for Studying the Physiological Relevance of Gq/11-dependent Signaling Pathways.

PubMed

Hu, Jianxin; Stern, Matthew; Gimenez, Luis E; Wanka, Lizzy; Zhu, Lu; Rossi, Mario; Meister, Jaroslawna; Inoue, Asuka; Beck-Sickinger, Annette G; Gurevich, Vsevolod V; Wess, Jürgen

2016-04-08

Designerreceptorsexclusivelyactivated by adesignerdrug (DREADDs) are clozapine-N-oxide-sensitive designer G protein-coupled receptors (GPCRs) that have emerged as powerful novel chemogenetic tools to study the physiological relevance of GPCR signaling pathways in specific cell types or tissues. Like endogenous GPCRs, clozapine-N-oxide-activated DREADDs do not only activate heterotrimeric G proteins but can also trigger β-arrestin-dependent (G protein-independent) signaling. To dissect the relative physiological relevance of G protein-mediatedversusβ-arrestin-mediated signaling in different cell types or physiological processes, the availability of G protein- and β-arrestin-biased DREADDs would be highly desirable. In this study, we report the development of a mutationally modified version of a non-biased DREADD derived from the M3muscarinic receptor that can activate Gq/11with high efficacy but lacks the ability to interact with β-arrestins. We also demonstrate that this novel DREADD is activein vivoand that cell type-selective expression of this new designer receptor can provide novel insights into the physiological roles of G protein (Gq/11)-dependentversusβ-arrestin-dependent signaling in hepatocytes. Thus, this novel Gq/11-biased DREADD represents a powerful new tool to study the physiological relevance of Gq/11-dependent signaling in distinct tissues and cell types, in the absence of β-arrestin-mediated cellular effects. Such studies should guide the development of novel classes of functionally biased ligands that show high efficacy in various pathophysiological conditions but display a reduced incidence of side effects. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Purine inhibitors of protein kinases, G proteins and polymerases

DOEpatents

Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

2004-10-12

The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.

PubMed

Harding, Peter J; Attrill, Helen; Boehringer, Jonas; Ross, Simon; Wadhams, George H; Smith, Eleanor; Armitage, Judith P; Watts, Anthony

2009-02-01

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.

Tethered agonists: a new mechanism underlying adhesion G protein-coupled receptor activation.

PubMed

Schöneberg, Torsten; Liebscher, Ines; Luo, Rong; Monk, Kelly R; Piao, Xianhua

2015-06-01

The family of adhesion G protein-coupled receptors (aGPCRs) comprises 33 members in the human genome, which are subdivided into nine subclasses. Many aGPCRs undergo an autoproteolytic process via their GPCR Autoproteolysis-INducing (GAIN) domain during protein maturation to generate an N- and a C-terminal fragments, NTF and CTF, respectively. The NTF and CTF are non-covalently reassociated on the plasma membrane to form a single receptor unit. How aGPCRs are activated upon ligand binding remains one of the leading questions in the field of aGPCR research. Recent work from our labs and others shows that ligand binding can remove the NTF from the plasma membrane-bound CTF, exposing a tethered agonist which potently activates downstream signaling.

Gene transfer of heterologous G protein-coupled receptors to cardiomyocytes: differential effects on contractility.

PubMed

Laugwitz, K L; Weig, H J; Moretti, A; Hoffmann, E; Ueblacker, P; Pragst, I; Rosport, K; Schömig, A; Ungerer, M

2001-04-13

In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to beta-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective beta-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V(2) vasopressin receptors (rV(2)-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant beta(2)-adrenergic receptors (rbeta(2)-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V(2)-Rs are uniquely coupled to Gs, PTH1-Rs and beta(2)-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rbeta(2)-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV(2)-R-expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of beta(2)-ARS: In the absence of receptor agonists, rPTH1-Rs and rbeta(2)-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV(2)-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V(2)-Rs might be more suited to test the effects of c

G-Protein/β-Arrestin-Linked Fluctuating Network of G-Protein-Coupled Receptors for Predicting Drug Efficacy and Bias Using Short-Term Molecular Dynamics Simulation

PubMed Central

Ichikawa, Osamu; Fujimoto, Kazushi; Yamada, Atsushi; Okazaki, Susumu; Yamazaki, Kazuto

2016-01-01

The efficacy and bias of signal transduction induced by a drug at a target protein are closely associated with the benefits and side effects of the drug. In particular, partial agonist activity and G-protein/β-arrestin-biased agonist activity for the G-protein-coupled receptor (GPCR) family, the family with the most target proteins of launched drugs, are key issues in drug discovery. However, designing GPCR drugs with appropriate efficacy and bias is challenging because the dynamic mechanism of signal transduction induced by ligand—receptor interactions is complicated. Here, we identified the G-protein/β-arrestin-linked fluctuating network, which initiates large-scale conformational changes, using sub-microsecond molecular dynamics (MD) simulations of the β2-adrenergic receptor (β2AR) with a diverse collection of ligands and correlation analysis of their G protein/β-arrestin efficacy. The G-protein-linked fluctuating network extends from the ligand-binding site to the G-protein-binding site through the connector region, and the β-arrestin-linked fluctuating network consists of the NPxxY motif and adjacent regions. We confirmed that the averaged values of fluctuation in the fluctuating network detected are good quantitative indexes for explaining G protein/β-arrestin efficacy. These results indicate that short-term MD simulation is a practical method to predict the efficacy and bias of any compound for GPCRs. PMID:27187591

Design, Synthesis, and Evaluation of N- and C-Terminal Protein Bioconjugates as G Protein-Coupled Receptor Agonists.

PubMed

Healey, Robert D; Wojciechowski, Jonathan P; Monserrat-Martinez, Ana; Tan, Susan L; Marquis, Christopher P; Sierecki, Emma; Gambin, Yann; Finch, Angela M; Thordarson, Pall

2018-02-21

A G protein-coupled receptor (GPCR) agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; N-terminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for N-terminal specific bioconjugations.

Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support

PubMed Central

Di Bartolo, Natalie; Compton, Emma L. R.; Warne, Tony; Edwards, Patricia C.; Tate, Christopher G.; Schertler, Gebhard F. X.; Booth, Paula J.

2016-01-01

The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40–70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes. PMID:26982879

Modulation of CaV1.2 calcium channel by neuropeptide W regulates vascular myogenic tone via G protein-coupled receptor 7.

PubMed

Ji, Li; Zhu, Huayuan; Chen, Hong; Fan, Wenyong; Chen, Junjie; Chen, Jing; Zhu, Guoqing; Wang, Juejin

2015-12-01

Neuropeptide W (NPW), an endogenous ligand for the G protein-coupled receptor 7 (GPR7), was first found to make important roles in central nerve system. In periphery, NPW was also present and regulated intracellular calcium homeostasis by L-type calcium channels. This study was designed to discover the effects of NPW-GPR7 on the function of CaV1.2 calcium channels in the vascular smooth muscle cells (VSMCs) and vasotone of arterial vessels. By whole-cell patch clamp, we studied the effects of NPW-23, the active form of NPW, on the CaV1.2 channels in the heterologously transfected human embryonic kidney 293 cells and VSMCs isolated from rat. Living system was used to explore the physiological function of NPW-23 in arterial myogenic tone. To investigate the pathological relevance, NPW mRNA level of mesenteric arteries was measured in the hypertensive and normotensive rats. NPW's receptor GPR7 was coexpressed with CaV1.2 channels in arterial smooth muscle. NPW-23 increased the ICa,L in transfected human embryonic kidney 293 cells and VSMCs via GPR7, which could be abrogated by phospholipase C (PLC)/protein kinase C (PKC) inhibitors, not protein kinase A or protein kinase G inhibitor. After NPW-23 application, the expression of pan phospho-PKC was increased; moreover, intracellular diacylglycerol level, the second messenger catalyzed by PLC, was increased 1.5-2-fold. Application with NPW-23 increased pressure-induced vasotone of the rat mesenteric arteries. Importantly, the expression of NPW was decreased in the hypertensive rats. NPW-23 regulates ICa,L via GPR7, which is mediated by PLC/PKC signaling, and such a mechanism plays a role in modulating vascular myogenic tone, which may involve in the development of vascular hypertension.

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

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

Mochimaru, Yuta; Azuma, Morio; Oshima, Natsuki

2015-02-20

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

Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy.

PubMed

Rahmeh, Rita; Damian, Marjorie; Cottet, Martin; Orcel, Hélène; Mendre, Christiane; Durroux, Thierry; Sharma, K Shivaji; Durand, Grégory; Pucci, Bernard; Trinquet, Eric; Zwier, Jurriaan M; Deupi, Xavier; Bron, Patrick; Banères, Jean-Louis; Mouillac, Bernard; Granier, Sébastien

2012-04-24

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

The mapping of yeast's G-protein coupled receptor with an atomic force microscope

NASA Astrophysics Data System (ADS)

Takenaka, Musashi; Miyachi, Yusuke; Ishii, Jun; Ogino, Chiaki; Kondo, Akihiko

2015-03-01

An atomic force microscope (AFM) can measure the adhesion force between a sample and a cantilever while simultaneously applying a rupture force during the imaging of a sample. An AFM should be useful in targeting specific proteins on a cell surface. The present study proposes the use of an AFM to measure the adhesion force between targeting receptors and their ligands, and to map the targeting receptors. In this study, Ste2p, one of the G protein-coupled receptors (GPCRs), was chosen as the target receptor. The specific force between Ste2p on a yeast cell surface and a cantilever modified with its ligand, α-factor, was measured and found to be approximately 250 pN. In addition, through continuous measuring of the cell surface, a mapping of the receptors on the cell surface could be performed, which indicated the differences in the Ste2p expression levels. Therefore, the proposed AFM system is accurate for cell diagnosis.

Sexually dimorphic role of G protein-coupled estrogen receptor (GPER) in modulating energy homeostasis.

PubMed

Davis, Kathryn E; Carstens, Elizabeth J; Irani, Boman G; Gent, Lana M; Hahner, Lisa M; Clegg, Deborah J

2014-06-01

This article is part of a Special Issue "Energy Balance". The classical estrogen receptors, estrogen receptor-α and estrogen receptor-β are well established in the regulation of body weight and energy homeostasis in both male and female mice, whereas, the role for G protein-coupled estrogen receptor 1 (GPER) as a modulator of energy homeostasis remains controversial. This study sought to determine whether gene deletion of GPER (GPER KO) alters body weight, body adiposity, food intake, and energy homeostasis in both males and females. Male mice lacking GPER developed moderate obesity and larger adipocyte size beginning at 8 weeks of age, with significant reductions in energy expenditure, but not food intake or adipocyte number. Female GPER KO mice developed increased body weight relative to WT females a full 6 weeks later than the male GPER KO mice. Female GPER KO mice also had reductions in energy expenditure, but no significant increases in body fat content. Consistent with their decrease in energy expenditure, GPER KO males and females showed significant reductions in two brown fat thermogenic proteins. GPER KO females, prior to their divergence in body weight, were less sensitive than WT females to the feeding-inhibitory effects of leptin and CCK. Additionally, body weight was not as modulated by ovariectomy or estradiol replacement in GPER KO mice. Estradiol treatment activated phosphorylated extracellular signal-regulated kinase (pERK) in WT but not GPER KO females. For the first time, GPER expression was found in the adipocyte but not the stromal fraction of adipose tissue. Together, these results provide new information elucidating a sexual dimorphism in GPER function in the development of postpubertal energy balance. Copyright © 2014 Elsevier Inc. All rights reserved.

Agonist-induced conformational changes in the G-protein-coupling domain of the β2 adrenergic receptor

PubMed Central

Ghanouni, Pejman; Steenhuis, Jacqueline J.; Farrens, David L.; Kobilka, Brian K.

2001-01-01

The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the β2 adrenergic receptor (β2AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-β2AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the β2AR, which may reflect the different energetics of activation by diffusible ligands. PMID:11353823

Characterization of B61, the ligand for the Eck receptor protein-tyrosine kinase.

PubMed

Shao, H; Pandey, A; O'Shea, K S; Seldin, M; Dixit, V M

1995-03-10

B61 was originally described as a novel secreted tumor necrosis factor-alpha-inducible gene product in endothelial cells (Holzman, L. B., Marks, R. M., and Dixit, V. M. (1990) Mol. Cell. Biol. 10, 5830-5838). It was recently discovered that soluble recombinant B61 could serve as a ligand for the Eck receptor protein-tyrosine kinase, a member of the Eph/Eck subfamily of receptor protein-tyrosine kinases (Bartley, T.D., Hunt, R. W., Welcher, A. A., Boyle, W. J., Parker, V. P., Lindberg, R. A., Lu, H. S., Colombero, A. M., Elliott, R. L., Guthrie, R. A., Holst, P. L., Skrine, J. D., Toso, R. J., Zhang, M., Fernandez, E., Trail, G., Yarnum, B., Yarden, Y., Hunter, T., and Fox, G. M. (1994) Nature 368, 558-560). We now show that B61 can also exist as a cell surface glycosylphosphatidyl-inositol-linked protein that is capable of activating the Eck receptor protein-tyrosine kinase, the first such report of a receptor protein-tyrosine kinase ligand that is glycosylphosphatidylinositol-linked. In addition, the expression patterns of B61 and Eck during mouse ontogeny were determined by in situ hybridization. Both were found to be highly expressed in the developing lung and gut, while Eck was preferentially expressed in the thymus. Finally, the gene for B61 was localized to a specific position on mouse chromosome 3 by interspecific back-cross analysis.

Quasi-elastic neutron scattering reveals ligand-induced protein dynamics of a G-protein-coupled receptor

DOE PAGES

Shrestha, Utsab R.; Perera, Suchithranga M. D. C.; Bhowmik, Debsindhu; ...

2016-09-15

Light activation of the visual G-protein-coupled receptor (GPCR) rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane yielding the activation of cognate G-protein (transducin), which initiates biological signaling. Here, we report a quasi-elastic neutron scattering study of the activation of rhodopsin as a GPCR prototype. Our results reveal a broadly distributed relaxation of hydrogen atom dynamics of rhodopsin on a picosecond–nanosecond time scale, crucial for protein function, as only observed for globular proteins previously. Interestingly, the results suggest significant differences in the intrinsic protein dynamics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. These differencesmore » can be attributed to the influence of the covalently bound retinal ligand. Moreover, an idea of the generic free-energy landscape is used to explain the GPCR dynamics of ligand-binding and ligand-free protein conformations, which can be further applied to other GPCR systems.« less

Quasi-elastic neutron scattering reveals ligand-induced protein dynamics of a G-protein-coupled receptor

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

Shrestha, Utsab R.; Perera, Suchithranga M. D. C.; Bhowmik, Debsindhu

Light activation of the visual G-protein-coupled receptor (GPCR) rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane yielding the activation of cognate G-protein (transducin), which initiates biological signaling. Here, we report a quasi-elastic neutron scattering study of the activation of rhodopsin as a GPCR prototype. Our results reveal a broadly distributed relaxation of hydrogen atom dynamics of rhodopsin on a picosecond–nanosecond time scale, crucial for protein function, as only observed for globular proteins previously. Interestingly, the results suggest significant differences in the intrinsic protein dynamics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. These differencesmore » can be attributed to the influence of the covalently bound retinal ligand. Moreover, an idea of the generic free-energy landscape is used to explain the GPCR dynamics of ligand-binding and ligand-free protein conformations, which can be further applied to other GPCR systems.« less

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. Copyright 2010 Elsevier Ltd. All rights reserved.

Variable Dependence of Signaling Output on Agonist Occupancy of Ste2p, a G Protein-coupled Receptor in Yeast.

PubMed

Sridharan, Rajashri; Connelly, Sara M; Naider, Fred; Dumont, Mark E

2016-11-11

We report here on the relationship between ligand binding and signaling responses in the yeast pheromone response pathway, a well characterized G protein-coupled receptor system. Responses to agonist (α-factor) by cells expressing widely varying numbers of receptors depend primarily on fractional occupancy, not the absolute number of agonist-bound receptors. Furthermore, the concentration of competitive antagonist required to inhibit α-factor-dependent signaling is more than 10-fold higher than predicted based on the known ligand affinities. Thus, responses to a particular number of agonist-bound receptors can vary greatly, depending on whether there are unoccupied or antagonist-bound receptors present on the same cell surface. This behavior does not appear to be due to pre-coupling of receptors to G protein or to the Sst2p regulator of G protein signaling. The results are consistent with a signaling response that is determined by the integration of positive signals from agonist-occupied receptors and inhibitory signals from unoccupied receptors, where the inhibitory signals can be diminished by antagonist binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Myocardial Ablation of G Protein-Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway.

PubMed

Fan, Qian; Chen, Mai; Zuo, Lin; Shang, Xiying; Huang, Maggie Z; Ciccarelli, Michele; Raake, Philip; Brinks, Henriette; Chuprun, Kurt J; Dorn, Gerald W; Koch, Walter J; Gao, Erhe

2013-01-01

Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

Tracking G-protein-coupled receptor activation using genetically encoded infrared probes.

PubMed

Ye, Shixin; Zaitseva, Ekaterina; Caltabiano, Gianluigi; Schertler, Gebhard F X; Sakmar, Thomas P; Deupi, Xavier; Vogel, Reiner

2010-04-29

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

Customizing G Protein-coupled receptor models for structure-based virtual screening.

PubMed

de Graaf, Chris; Rognan, Didier

2009-01-01

This review will focus on the construction, refinement, and validation of G Protein-coupled receptor models for the purpose of structure-based virtual screening. Practical tips and tricks derived from concrete modeling and virtual screening exercises to overcome the problems and pitfalls associated with the different steps of the receptor modeling workflow will be presented. These examples will not only include rhodopsin-like (class A), but also secretine-like (class B), and glutamate-like (class C) receptors. In addition, the review will present a careful comparative analysis of current crystal structures and their implication on homology modeling. The following themes will be discussed: i) the use of experimental anchors in guiding the modeling procedure; ii) amino acid sequence alignments; iii) ligand binding mode accommodation and binding cavity expansion; iv) proline-induced kinks in transmembrane helices; v) binding mode prediction and virtual screening by receptor-ligand interaction fingerprint scoring; vi) extracellular loop modeling; vii) virtual filtering schemes. Finally, an overview of several successful structure-based screening shows that receptor models, despite structural inaccuracies, can be efficiently used to find novel ligands.

Coupling to protein kinases A and C of adenosine A2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens.

PubMed

Queiroz, Glória; Quintas, Clara; Talaia, Carlos; Gonçalves, Jorge

2004-08-01

In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.

Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.

PubMed

Devost, Dominic; Sleno, Rory; Pétrin, Darlaine; Zhang, Alice; Shinjo, Yuji; Okde, Rakan; Aoki, Junken; Inoue, Asuka; Hébert, Terence E

2017-03-31

Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gα q , Gα 11 , Gα 12 , and Gα 13 or β-arrestins, we showed that Gα q and Gα 11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor*

PubMed Central

Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K.; Prosser, R. Scott

2012-01-01

The G protein-coupled β2-adrenoreceptor (β2AR) signals through the heterotrimeric G proteins Gs and Gi and β-arrestin. As such, the energy landscape of β2AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β2AR with a trifluoromethyl probe, 19F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β2AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β2AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor. PMID:22893704

G protein γ (Gγ) subtype dependent targeting of GRK2 to M3 receptor by Gβγ.

PubMed

Samaradivakara, Saroopa; Kankanamge, Dinesh; Senarath, Kanishka; Ratnayake, Kasun; Karunarathne, Ajith

2018-06-11

Interactions of cytosolic G protein coupled receptor kinase 2 (GRK2) with activated G protein coupled receptors (GPCRs) induce receptor phosphorylation and desensitization. GRK2 is recruited to active M3-muscarinic receptors (M3R) with the participation of the receptor, Gαq and Gβγ. Since we have shown that signaling efficacy of Gβγ is governed by its Gγ subtype identity, the present study examined whether recruitment of GRK2 to M3R is also Gγ subtype dependent. To capture the dynamics of GRK2-recruitment concurrently with GPCR-G protein activation, we employed live cell confocal imaging and a novel assay based on Gβγ translocation. Data show that M3R activation-induced GRK2 recruitment is Gγ subtype dependent in which Gβγ dimers with low PM-affinity Gγ9 exhibited a two-fold higher GRK2-recruitment compared to high PM affinity Gγ3 expressing cells. Since 12-mammalian Gγ types exhibit a cell and tissue specific expressions and the PM-affinity of a Gγ is linked to its subtype identity, our results indicate a mechanism by which Gγ profile of a cell controls GRK2 signaling and GPCR desensitization. Copyright © 2018 Elsevier Inc. All rights reserved.

Molecular recognition of parathyroid hormone by its G protein-coupled receptor

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

Pioszak, Augen A.; Xu, H. Eric

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 engineeredmore » 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.« less

Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes

PubMed Central

Dalet, Farfán-García Eunice; Guadalupe, Trujillo-Ferrara José; María del Carmen, Castillo-Hernández; Humberto, Guerra-Araiza Christian; Antonio, Soriano-Ursúa Marvin

2013-01-01

In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selectivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disorders of the central nervous system. Specifically, new possibilities are explored in relation to allosteric and orthosteric binding sites on dopamine receptors for the treatment of Parkinson's disease, and on muscarinic receptors for Alzheimer's disease. Future research can seek to identify ligands that can bind to more than one site on the same receptor, or simultaneously bind to two receptors and form a dimer. For example, the design of bivalent drugs that can reach homo/hetero-dimers of D2 dopamine receptor holds promise as a relevant therapeutic strategy for Parkinson's disease. Regarding the treatment of Alzheimer's disease, the design of dualsteric ligands for mono-oligomeric rinic receptors could increase therapeutic effectiveness by generating potent compounds that could activate more than one signaling pathway. PMID:25206539

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.

Differential expression of G-protein-coupled estrogen receptor-30 in human myometrial and uterine leiomyoma smooth muscle.

PubMed

Tian, Ruijuan; Wang, Zengyong; Shi, Zhan; Li, Dong; Wang, Yuebing; Zhu, Yingjun; Lin, Wanjun; Gui, Yu; Zheng, Xi-Long

2013-01-01

To determine differential expression of G-protein-coupled receptor 30 (GPR30) in uterine leiomyoma and its matched myometrium. GPR30 expression examined in both tissues and cultured cells. Research laboratories. Women 35 to 50 years old with uterine leiomyomas. Hysterectomy. GPR30 expression profile. Using Western blot and real-time quantitative polymerase chain reaction analyses, we found that GPR30 was highly expressed in uterine leiomyomas compared with their matched myometrium. In only three out of nine patients examined was GPR30 protein detectable by Western blot analysis in myometrial tissues, but at statistically significantly lower levels than in their leiomyomas. Confocal microscopy revealed the nuclear localization of GPR30 in leiomyoma tissues and cultured leiomyoma smooth muscle cells (SMCs). Treatment with 0.1 μM 17β-estradiol increased mRNA expression of GPR30 in leiomyoma SMCs but decreased expression in myometrial SMCs. Treatment with G-1, a GPR30 agonist, stimulated phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) in both SMC types. PD98059, the MEK inhibitor, completely inhibited G-1-induced phosphorylation of p44/42 in myometrium SMCs, but not in SMCs from leiomyoma. GPR30 is abundantly expressed in uterine leiomyomas, likely resulting from estrogen stimulation. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.

PubMed

Kishore, Ayush; Hall, Randy A

2017-06-09

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα 12/13 -mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gα q/11 -mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Roles of G protein-coupled estrogen receptor GPER in metabolic regulation.

PubMed

Sharma, Geetanjali; Mauvais-Jarvis, Franck; Prossnitz, Eric R

2018-02-01

Metabolic homeostasis is differentially regulated in males and females. The lower incidence of obesity and associated diseases in pre-menopausal females points towards the beneficial role of the predominant estrogen, 17β-estradiol (E2). The actions of E2 are elicited by nuclear and extra-nuclear estrogen receptor (ER) α and ERβ, as well as the G protein-coupled estrogen receptor (GPER, previously termed GPR30). The roles of GPER in the regulation of metabolism are only beginning to emerge and much remains unclear. The present review highlights recent advances implicating the importance of GPER in metabolic regulation. Assessment of the specific metabolic roles of GPER employing GPER-deficient mice and highly selective GPER-targeted pharmacological agents, agonist G-1 and antagonists G-15 and G36, is also presented. Evidence from in vitro and in vivo studies involving either GPER deficiency or selective activation suggests that GPER is involved in body weight regulation, glucose and lipid homeostasis as well as inflammation. The therapeutic potential of activating GPER signaling through selective ligands for the treatment of obesity and diabetes is also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ligand-induced dynamical change of G-protein-coupled receptor revealed by neutron scattering

NASA Astrophysics Data System (ADS)

Shrestha, Utsab R.; Bhowmik, Debsindhu; Mamontov, Eugene; Chu, Xiang-Qiang

Light activation of the visual G-protein-coupled receptor rhodopsin leads to the significant change in protein conformation and structural fluctuations, which further activates the cognate G-protein (transducin) and initiates the biological signaling. In this work, we studied the rhodopsin activation dynamics using state-of-the-art neutron scattering technique. Our quasi-elastic neutron scattering (QENS) results revealed a broadly distributed relaxation rate of the hydrogen atom in rhodopsin on the picosecond to nanosecond timescale (beta-relaxation region), which is crucial for the protein function. Furthermore, the application of mode-coupling theory to the QENS analysis uncovers the subtle changes in rhodopsin dynamics due to the retinal cofactor. Comparing the dynamics of the ligand-free apoprotein, opsin versus the dark-state rhodopsin, removal of the retinal cofactor increases the relaxation time in the beta-relaxation region, which is due to the possible open conformation. Moreover, we utilized the concept of free-energy landscape to explain our results for the dark-state rhodopsin and opsin dynamics, which can be further applied to other GPCR systems to interpret various dynamic behaviors in ligand-bound and ligand-free protein.

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

PubMed Central

Naganathan, Saranga; Grunbeck, Amy; Tian, He; Huber, Thomas; Sakmar, Thomas P.

2013-01-01

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes. PMID:24056801

Internalization of G-protein-coupled receptors: Implication in receptor function, physiology and diseases.

PubMed

Calebiro, Davide; Godbole, Amod

2018-04-01

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and mediate the effects of numerous hormones and neurotransmitters. The nearly 1000 GPCRs encoded by the human genome regulate virtually all physiological functions and are implicated in the pathogenesis of prevalent human diseases such as thyroid disorders, hypertension or Parkinson's disease. As a result, 30-50% of all currently prescribed drugs are targeting these receptors. Once activated, GPCRs induce signals at the cell surface. This is often followed by internalization, a process that results in the transfer of receptors from the plasma membrane to membranes of the endosomal compartment. Internalization was initially thought to be mainly implicated in signal desensitization, a mechanism of adaptation to prolonged receptor stimulation. However, several unexpected functions have subsequently emerged. Most notably, accumulating evidence indicates that internalization can induce prolonged receptor signaling on intracellular membranes, which is apparently required for at least some biological effects of hormones like TSH, LH and adrenaline. These findings reveal an even stronger connection between receptor internalization and signaling than previously thought. Whereas new studies are just beginning to reveal an important physiological role for GPCR signaling after internalization and ways to exploit it for therapeutic purposes, future investigations will be required to explore its involvement in human disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Structural–Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work

PubMed Central

Kleinau, Gunnar; Worth, Catherine L.; Kreuchwig, Annika; Biebermann, Heike; Marcinkowski, Patrick; Scheerer, Patrick; Krause, Gerd

2017-01-01

The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to

G protein-coupled estrogen receptor and estrogen receptor ligands regulate colonic motility and visceral pain.

PubMed

Zielińska, M; Fichna, J; Bashashati, M; Habibi, S; Sibaev, A; Timmermans, J-P; Storr, M

2017-07-01

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a functional gastrointestinal (GI) disorder, which occurs more frequently in women than men. The aim of our study was to determine the role of activation of classical estrogen receptors (ER) and novel membrane receptor, G protein-coupled estrogen receptor (GPER) in human and mouse tissue and to assess the possible cross talk between these receptors in the GI tract. Immunohistochemistry was used to determine the expression of GPER in human and mouse intestines. The effect of G-1, a GPER selective agonist, and estradiol, a non-selective ER agonist, on muscle contractility was characterized in isolated preparations of the human and mouse colon. To characterize the effect of G-1 and estradiol in vivo, colonic bead expulsion test was performed. G-1 and estradiol activity on the visceral pain signaling was assessed in the mustard oil-induced abdominal pain model. GPER is expressed in the human colon and in the mouse colon and ileum. G-1 and estradiol inhibited muscle contractility in vitro in human and mouse colon. G-1 or estradiol administered intravenously at the dose of 20 mg/kg significantly prolonged the time to bead expulsion in females. Moreover, G-1 prolonged the time to bead expulsion and inhibited GI hypermotility in both genders. The injection of G-1 or estradiol resulted in a significant reduction in the number of pain-induced behaviors in mice. GPER and ER receptors are involved in the regulation of GI motility and visceral pain. Both may thus constitute an important pharmacological target in the IBS-D therapy. © 2017 John Wiley & Sons Ltd.

Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane.

PubMed

Filardo, E; Quinn, J; Pang, Y; Graeber, C; Shaw, S; Dong, J; Thomas, P

2007-07-01

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17beta-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17beta-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

Microgravity and Signaling Molecules in Rat Osteoblasts: Downstream of Receptor Tyrosine Kinase, G-Protein-Coupled Receptor, and Small GTP-Binding Proteins

NASA Technical Reports Server (NTRS)

Kumel, Yasuhiro; Shimokawa, Hitoyata; Morita, Sadao; Katano, Hisako; Akiyama, Hideo; Hirano, Masahiko; Ohya, Keiichi; Sams, Clarence F.; Whitson, Peggy A.

2005-01-01

Rat osteoblasts were cultured for 4 and 5 days aboard Space Shuttle and solubilized on board. The mRNA levels of the post-receptor signaling molecules were analyzed by quantitative RT-PCR. The G-protein alpha subunit G(alpha)q mRNA levels were elevated 3-fold by microgravity. G(alpha)q stimulates PLC(beta), and then PKC. PKC(delta) and PKC(theta) mRNA levels were increased 2- to 5-fold by microgravity The mRNA levels of SOS and Ras GRF were increased 4 to 5-fold by microgravity, while Ras GAP was not altered. Spaceflight-induced bone loss might be attributed to microgravity modulation of the signaling pathway in osteoblasts.

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

PubMed Central

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

2012-01-01

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

Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

PubMed

Chaturvedi, Madhu; Schilling, Justin; Beautrait, Alexandre; Bouvier, Michel; Benovic, Jeffrey L; Shukla, Arun K

2018-05-04

G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting β-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology. Copyright © 2018 Elsevier Ltd. All rights reserved.

Selective targeting of G-protein-coupled receptor subtypes with venom peptides.

PubMed

Näreoja, K; Näsman, J

2012-02-01

The G-protein-coupled receptor (GPCR) family is one of the largest gene superfamilies with approx. 370 members responding to endogenous ligands in humans and a roughly equal amount of receptors sensitive to external stimuli from the surrounding. A number of receptors from this superfamily are well recognized targets for medical treatment of various disease conditions, whereas for many others the potential medical benefit of interference is still obscure. A general problem associated with GPCR research and therapeutics is the insufficient specificity of available ligands to differentiate between closely homologous receptor subtypes. In this context, venom peptides could make a significant contribution to the development of more specific drugs. Venoms from certain animals specialized in biochemical hunting contain a mixture of molecules that are directed towards a variety of membrane proteins. Peptide toxins isolated from these mixtures usually exhibit high specificity for their targets. Muscarinic toxins found from mamba snakes attracted much attention during the 1990s. These are 65-66 amino acid long peptides with a structural three-finger folding similar to the α-neurotoxins and they target the muscarinic acetylcholine receptors in a subtype-selective manner. Recently, several members of the three-finger toxins from mamba snakes as well as conotoxins from marine cone snails have been shown to selectively interact with subtypes of adrenergic receptors. In this review, we will discuss the GPCR-directed peptide toxins found from different venoms and how some of these can be useful in exploring specific roles of receptor subtypes. © 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.

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

Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli.

PubMed

Link, A James; Skretas, Georgios; Strauch, Eva-Maria; Chari, Nandini S; Georgiou, George

2008-10-01

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.

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.

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

PubMed

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

2014-11-04

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

A physiologically required G protein-coupled receptor (GPCR)-regulator of G protein signaling (RGS) interaction that compartmentalizes RGS activity.

PubMed

Croft, Wayne; Hill, Claire; McCann, Eilish; Bond, Michael; Esparza-Franco, Manuel; Bennett, Jeannette; Rand, David; Davey, John; Ladds, Graham

2013-09-20

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.

The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

PubMed

Pack, Thomas F; Orlen, Margo I; Ray, Caroline; Peterson, Sean M; Caron, Marc G

2018-04-20

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and β-arrestins (βarrs). Selectively engaging either the G protein or βarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to βarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates βarr recruitment, bridge these pathways. Therefore, βarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on βarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or βarrs as well as a βarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the βarr-preferring D2R achieves βarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the βarr-preferring D2R and also contributes to βarr recruitment at the WT D2R. Additionally, we found an additive interaction between the βarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving βarr-biased signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

PubMed

Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.

PubMed

Roubalova, Lenka; Vosahlikova, Miroslava; Brejchova, Jana; Sykora, Jan; Rudajev, Vladimir; Svoboda, Petr

2015-01-01

HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between �

Bombyx neuropeptide G protein-coupled receptor A7 is the third cognate receptor for short neuropeptide F from silkworm.

PubMed

Ma, Qiang; Cao, Zheng; Yu, Yena; Yan, Lili; Zhang, Wenjuan; Shi, Ying; Zhou, Naiming; Huang, Haishan

2017-12-15

The short neuropeptide F (sNPF) neuropeptides, closely related to vertebrate neuropeptide Y (NPY), have been suggested to exert pleiotropic effects on many physiological processes in insects. In the silkworm ( Bombyx mori ) two orphan G protein-coupled receptors, Bombyx neuropeptide G protein-coupled receptor (BNGR) A10 and A11, have been identified as cognate receptors for sNPFs, but other sNPF receptors and their signaling mechanisms in B. mori remain unknown. Here, we cloned the full-length cDNA of the orphan receptor BNGR-A7 from the brain of B. mori larvae and identified it as a receptor for Bombyx sNPFs. Further characterization of signaling and internalization indicated that BNGR-A7, -A10, and -A11 are activated by direct interaction with synthetic Bombyx sNPF-1 and -3 peptides. This activation inhibited forskolin or adipokinetic hormone-induced adenylyl cyclase activity and intracellular Ca 2+ mobilization via a G i/o -dependent pathway. Upon activation by sNPFs, BNGR-A7, -A10, and -A11 evoked ERK1/2 phosphorylation and underwent internalization. On the basis of these findings, we designated the receptors BNGR-A7, -A10, and -A11 as Bommo -sNPFR-1, -2, and -3, respectively. Moreover, the results obtained with quantitative RT-PCR analysis revealed that the three Bombyx sNPF receptor subtypes exhibit differential spatial and temporal expression patterns, suggesting possible roles of sNPF signaling in the regulation of a wide range of biological processes. Our findings provide the first in-depth information on sNPF signaling for further elucidation of the roles of the Bombyx sNPF/sNPFR system in the regulation of physiological activities. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

G protein-coupled estrogen receptor is required for the neuritogenic mechanism of 17β-estradiol in developing hippocampal neurons.

PubMed

Ruiz-Palmero, Isabel; Hernando, Maria; Garcia-Segura, Luis M; Arevalo, Maria-Angeles

2013-06-15

Estradiol promotes neuritogenesis in developing hippocampal neurons by a mechanism involving the upregulation of neurogenin 3, a Notch-regulated transcription factor. In this study we have explored whether G-protein coupled estrogen receptor 1 (GPER) participates in this hormonal action. GPER agonists (17β-estradiol, G1, ICI 182,780) increased neurogenin 3 expression and neuritogenesis in mouse primary hippocampal neurons and this effect was blocked by the GPER antagonist G15 and by a siRNA for GPER. In addition, GPER agonists increased Akt phosphorylation in ser473, which is indicative of the activation of phosphoinositide-3-kinase (PI3K). G15 or GPER silencing prevented the estrogenic induction of Akt phosphorylation. Furthermore, the PI3K inhibitor wortmannin prevented the effect of G1 and estradiol on neurogenin 3 expression and the effect of estradiol on neuritogenesis. These findings suggest that GPER participates in the control of hippocampal neuritogenesis by a mechanism involving the activation of PI3K signaling. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Mechanisms of Disease: the first kiss-a crucial role for kisspeptin-1 and its receptor, G-protein-coupled receptor 54, in puberty and reproduction.

PubMed

Seminara, Stephanie B

2006-06-01

Although the hypothalamic secretion of gonadotropin-releasing hormone (GnRH) is the defining hormonal event of puberty, the physiologic mechanisms that drive secretion of GnRH at the time of sexual maturation have been difficult to identify. After puberty is initiated, the factors that modulate the frequency and amplitude of GnRH secretion in rapidly changing sex-steroid environments (i.e. the female menstrual cycle) also remain unknown. The discovery that, in both humans and mouse models, loss-of-function mutations in the gene that encodes G-protein-coupled receptor 54 result in phenotypes of hypogonadotropic hypogonadism with an absence of pubertal development has unearthed a novel pathway regulating GnRH secretion. Ligands for G-protein-coupled receptor 54 (KiSS-1R), including metastin (derived from the parent compound, kisspeptin-1) and metastin's C-terminal peptide fragments, have been shown to be powerful stimulants for GnRH release in vivo via their stimulation of G-protein-coupled receptor 54. This article reviews the discovery of the GPR54 gene, places it into the appropriate biological context, and explores the data from in vitro and in vivo studies that point to this ligand-receptor system as a major driver of GnRH secretion.

G Protein-Coupled Estrogen Receptor-Selective Ligands Modulate Endometrial Tumor Growth

PubMed Central

Petrie, Whitney K.; Dennis, Megan K.; Dai, Donghai; Arterburn, Jeffrey B.; Smith, Harriet O.; Hathaway, Helen J.; Prossnitz, Eric R.

2013-01-01

Endometrial carcinoma is the most common cancer of the female reproductive tract. GPER/GPR30 is a 7-transmembrane spanning G protein-coupled receptor that has been identified as the third estrogen receptor, in addition to ERα and ERβ. High GPER expression is predictive of poor survival in endometrial and ovarian cancer, but despite this, the estrogen-mediated signaling pathways and specific estrogen receptors involved in endometrial cancer remain unclear. Here, employing ERα-negative Hec50 endometrial cancer cells, we demonstrate that GPER mediates estrogen-stimulated activation of ERK and PI3K via matrix metalloproteinase activation and subsequent transactivation of the EGFR and that ER-targeted therapeutic agents (4-hydroxytamoxifen, ICI182,780/fulvestrant, and Raloxifene), the phytoestrogen genistein, and the “ERα-selective” agonist propylpyrazole triol also function as GPER agonists. Furthermore, xenograft tumors of Hec50 cells yield enhanced growth with G-1 and estrogen, the latter being inhibited by GPER-selective pharmacologic antagonism with G36. These results have important implications with respect to the use of putatively ER-selective ligands and particularly for the widespread long-term use of “ER-targeted” therapeutics. Moreover, our findings shed light on the potential mechanisms of SERM/SERD side effects reported in many clinical studies. Finally, our results provide the first demonstration that pharmacological inhibition of GPER activity in vivo prevents estrogen-mediated tumor growth. PMID:24379833

G protein-coupled estrogen receptor-selective ligands modulate endometrial tumor growth.

PubMed

Petrie, Whitney K; Dennis, Megan K; Hu, Chelin; Dai, Donghai; Arterburn, Jeffrey B; Smith, Harriet O; Hathaway, Helen J; Prossnitz, Eric R

2013-01-01

Endometrial carcinoma is the most common cancer of the female reproductive tract. GPER/GPR30 is a 7-transmembrane spanning G protein-coupled receptor that has been identified as the third estrogen receptor, in addition to ERα and ERβ. High GPER expression is predictive of poor survival in endometrial and ovarian cancer, but despite this, the estrogen-mediated signaling pathways and specific estrogen receptors involved in endometrial cancer remain unclear. Here, employing ERα-negative Hec50 endometrial cancer cells, we demonstrate that GPER mediates estrogen-stimulated activation of ERK and PI3K via matrix metalloproteinase activation and subsequent transactivation of the EGFR and that ER-targeted therapeutic agents (4-hydroxytamoxifen, ICI182,780/fulvestrant, and Raloxifene), the phytoestrogen genistein, and the "ERα-selective" agonist propylpyrazole triol also function as GPER agonists. Furthermore, xenograft tumors of Hec50 cells yield enhanced growth with G-1 and estrogen, the latter being inhibited by GPER-selective pharmacologic antagonism with G36. These results have important implications with respect to the use of putatively ER-selective ligands and particularly for the widespread long-term use of "ER-targeted" therapeutics. Moreover, our findings shed light on the potential mechanisms of SERM/SERD side effects reported in many clinical studies. Finally, our results provide the first demonstration that pharmacological inhibition of GPER activity in vivo prevents estrogen-mediated tumor growth.

Extent of Vascular Remodeling Is Dependent on the Balance Between Estrogen Receptor α and G-Protein-Coupled Estrogen Receptor.

PubMed

Gros, Robert; Hussain, Yasin; Chorazyczewski, Jozef; Pickering, J Geoffrey; Ding, Qingming; Feldman, Ross D

2016-11-01

Estrogens are important regulators of cardiovascular function. Some of estrogen's cardiovascular effects are mediated by a G-protein-coupled receptor mechanism, namely, G-protein-coupled estrogen receptor (GPER). Estradiol-mediated regulation of vascular cell programmed cell death reflects the balance of the opposing actions of GPER versus estrogen receptor α (ERα). However, the significance of these opposing actions on the regulation of vascular smooth muscle cell proliferation or migration in vitro is unclear, and the significance in vivo is unknown. To determine the effects of GPER activation in vitro, we studied rat aortic vascular smooth muscle cells maintained in primary culture. GPER was reintroduced using adenoviral gene transfer. Both estradiol and G1, a GPER agonist, inhibited both proliferation and cell migration effects that were blocked by the GPER antagonist, G15. To determine the importance of the GPER-ERα balance in regulating vascular remodeling in a rat model of carotid ligation, we studied the effects of upregulation of GPER expression versus downregulation of ERα. Reintroduction of GPER significantly attenuated the extent of medial hypertrophy and attenuated the extent of CD45 labeling. Downregulation of ERα expression comparably attenuated the extent of medial hypertrophy and inflammation after carotid ligation. These studies demonstrate that the balance between GPER and ERα regulates vascular remodeling. Receptor-specific modulation of estrogen's effects may be an important new approach in modifying vascular remodeling in both acute settings like vascular injury and perhaps in longer term regulation like in hypertension. © 2016 American Heart Association, Inc.

Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

PubMed

Fischer, Jana; Kleinau, Gunnar; Rutz, Claudia; Zwanziger, Denise; Khajavi, Noushafarin; Müller, Anne; Rehders, Maren; Brix, Klaudia; Worth, Catherine L; Führer, Dagmar; Krude, Heiko; Wiesner, Burkhard; Schülein, Ralf; Biebermann, Heike

2018-06-01

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

Do Plants Contain G Protein-Coupled Receptors?1[C][W][OPEN

PubMed Central

Taddese, Bruck; Upton, Graham J.G.; Bailey, Gregory R.; Jordan, Siân R.D.; Abdulla, Nuradin Y.; Reeves, Philip J.; Reynolds, Christopher A.

2014-01-01

Whether G protein-coupled receptors (GPCRs) exist in plants is a fundamental biological question. Interest in deorphanizing new GPCRs arises because of their importance in signaling. Within plants, this is controversial, as genome analysis has identified 56 putative GPCRs, including G protein-coupled receptor1 (GCR1), which is reportedly a remote homolog to class A, B, and E GPCRs. Of these, GCR2 is not a GPCR; more recently, it has been proposed that none are, not even GCR1. We have addressed this disparity between genome analysis and biological evidence through a structural bioinformatics study, involving fold recognition methods, from which only GCR1 emerges as a strong candidate. To further probe GCR1, we have developed a novel helix-alignment method, which has been benchmarked against the class A-class B-class F GPCR alignments. In addition, we have presented a mutually consistent set of alignments of GCR1 homologs to class A, class B, and class F GPCRs and shown that GCR1 is closer to class A and/or class B GPCRs than class A, class B, or class F GPCRs are to each other. To further probe GCR1, we have aligned transmembrane helix 3 of GCR1 to each of the six GPCR classes. Variability comparisons provide additional evidence that GCR1 homologs have the GPCR fold. From the alignments and a GCR1 comparative model, we have identified motifs that are common to GCR1, class A, B, and E GPCRs. We discuss the possibilities that emerge from this controversial evidence that GCR1 has a GPCR fold. PMID:24246381

Free energy landscape of G-protein coupled receptors, explored by accelerated molecular dynamics.

PubMed

Miao, Yinglong; Nichols, Sara E; McCammon, J Andrew

2014-04-14

G-protein coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. They are known to adopt multiple conformational states (e.g., inactive, intermediate and active) during their modulation of various cell signaling pathways. Here, the free energy landscape of GPCRs is explored using accelerated molecular dynamics (aMD) simulations as demonstrated on the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes. Free energy profiles of important structural motifs that undergo conformational transitions upon GPCR activation and allosteric signaling are analyzed in detail, including the Arg(3.50)-Glu(6.30) ionic lock, the Trp(6.48) toggle switch and the hydrogen interactions between Tyr(5.58)-Tyr(7.53).

Fusion Partner Toolchest for the Stabilization and Crystallization of G Protein-Coupled Receptors

PubMed Central

Chun, Eugene; Thompson, Aaron A.; Liu, Wei; Roth, Christopher B.; Griffith, Mark T.; Katritch, Vsevolod; Kunken, Joshua; Xu, Fei; Cherezov, Vadim; Hanson, Michael A.; Stevens, Raymond C.

2012-01-01

SUMMARY Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of the T4 lysozyme fusion partner that was inserted into the third intracellular loop. Using chimeras of the human β2-adrenergic and human A2A adenosine receptors, we present the methodology and data for the selection of five new fusion partners for crystallizing GPCRs. In particular, the use of the thermostabilized apocytochrome b562RIL as a fusion partner displays certain advantages over the previously utilized T4 lysozyme, resulting in a significant improvement in stability and structure in GPCR-fusion constructs. PMID:22681902

The G-protein coupled estrogen receptor, GPER: The inside and inside-out story.

PubMed

Gaudet, H M; Cheng, S B; Christensen, E M; Filardo, E J

2015-12-15

GPER possesses structural and functional characteristics shared by members of the G-protein-coupled receptor (GPCR) superfamily, the largest class of plasma membrane receptors. This newly appreciated estrogen receptor is localized predominately within intracellular membranes in most, but not all, cell types and its surface expression is modulated by steroid hormones and during tissue injury. An intracellular staining pattern is not unique among GPCRs, which employ a diverse array of molecular mechanisms that restrict cell surface expression and effectively regulating receptor binding and activation. The finding that GPER displays an intracellular predisposition has created some confusion as the estrogen-inducible transcription factors, ERα and ERβ, also reside intracellularly, and has led to complex suggestions of receptor interaction. GPER undergoes constitutive retrograde trafficking from the plasma membrane to the endoplasmic reticulum and recent studies indicate its interaction with PDZ binding proteins that sort transmembrane receptors to synaptosomes and endosomes. Genetic targeting and selective ligand approaches as well as cell models that express GPER in the absence of ERs clearly supports GPER as a bonafide "stand alone" receptor. Here, the molecular details that regulate GPER action, its cell biological activities and its implicated roles in physiological and pathological processes are reviewed. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

PubMed

Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor

PubMed Central

Cai, Yingying; Liu, Yuting; Culhane, Kelly J.; DeVree, Brian T.; Yang, Yang; Sunahara, Roger K.; Yan, Elsa C. Y.

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms. PMID:28609478

The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells.

PubMed

Ariazi, Eric A; Brailoiu, Eugen; Yerrum, Smitha; Shupp, Heather A; Slifker, Michael J; Cunliffe, Heather E; Black, Michael A; Donato, Anne L; Arterburn, Jeffrey B; Oprea, Tudor I; Prossnitz, Eric R; Dun, Nae J; Jordan, V Craig

2010-02-01

The G protein-coupled receptor GPR30 binds 17beta-estradiol (E(2)) yet differs from classic estrogen receptors (ERalpha and ERbeta). GPR30 can mediate E(2)-induced nongenomic signaling, but its role in ERalpha-positive breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERalpha-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol (DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expression studies, E(2) and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. In Ca(2+) mobilization studies, GPR30, but not ERalpha, mediated E(2)-induced Ca(2+) responses because E(2), 4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca(2+) increases not only in MCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E(2)-induced and G-1-induced Ca(2+) mobilization, but ERalpha depletion did not. Interestingly, GPR30-coupled Ca(2+) responses were sustained and inositol triphosphate receptor mediated in ER-positive MCF-7 cells but transitory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that the role of GPR30 was to promote SKBr3 cell growth but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G(1) phase. Thus, GPR30 antagonizes growth of ERalpha-positive breast cancer and may represent a new target to combat this disease.

Dehydroepiandrosterone Activation of G-protein-coupled Estrogen Receptor Rapidly Stimulates MicroRNA-21 Transcription in Human Hepatocellular Carcinoma Cells.

PubMed

Teng, Yun; Radde, Brandie N; Litchfield, Lacey M; Ivanova, Margarita M; Prough, Russell A; Clark, Barbara J; Doll, Mark A; Hein, David W; Klinge, Carolyn M

2015-06-19

Little is known about the regulation of the oncomiR miR-21 in liver. Dehydroepiandrosterone (DHEA) regulates gene expression as a ligand for a G-protein-coupled receptor and as a precursor for steroids that activate nuclear receptor signaling. We report that 10 nm DHEA increases primary miR-21 (pri-miR-21) transcription and mature miR-21 expression in HepG2 cells in a biphasic manner with an initial peak at 1 h followed by a second, sustained response from 3-12 h. DHEA also increased miR-21 in primary human hepatocytes and Hep3B cells. siRNA, antibody, and inhibitor studies suggest that the rapid DHEA-mediated increase in miR-21 involves a G-protein-coupled estrogen receptor (GPER/GPR30), estrogen receptor α-36 (ERα36), epidermal growth factor receptor-dependent, pertussis toxin-sensitive pathway requiring activation of c-Src, ERK1/2, and PI3K. GPER antagonist G-15 attenuated DHEA- and BSA-conjugated DHEA-stimulated pri-miR-21 transcription. Like DHEA, GPER agonists G-1 and fulvestrant increased pri-miR-21 in a GPER- and ERα36-dependent manner. DHEA, like G-1, increased GPER and ERα36 mRNA and protein levels. DHEA increased ERK1/2 and c-Src phosphorylation in a GPER-responsive manner. DHEA increased c-Jun, but not c-Fos, protein expression after 2 h. DHEA increased androgen receptor, c-Fos, and c-Jun recruitment to the miR-21 promoter. These results suggest that physiological concentrations of DHEA activate a GPER intracellular signaling cascade that increases pri-miR-21 transcription mediated at least in part by AP-1 and androgen receptor miR-21 promoter interaction. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Dehydroepiandrosterone Activation of G-protein-coupled Estrogen Receptor Rapidly Stimulates MicroRNA-21 Transcription in Human Hepatocellular Carcinoma Cells*

PubMed Central

Teng, Yun; Radde, Brandie N.; Litchfield, Lacey M.; Ivanova, Margarita M.; Prough, Russell A.; Clark, Barbara J.; Doll, Mark A.; Hein, David W.; Klinge, Carolyn M.

2015-01-01

Little is known about the regulation of the oncomiR miR-21 in liver. Dehydroepiandrosterone (DHEA) regulates gene expression as a ligand for a G-protein-coupled receptor and as a precursor for steroids that activate nuclear receptor signaling. We report that 10 nm DHEA increases primary miR-21 (pri-miR-21) transcription and mature miR-21 expression in HepG2 cells in a biphasic manner with an initial peak at 1 h followed by a second, sustained response from 3–12 h. DHEA also increased miR-21 in primary human hepatocytes and Hep3B cells. siRNA, antibody, and inhibitor studies suggest that the rapid DHEA-mediated increase in miR-21 involves a G-protein-coupled estrogen receptor (GPER/GPR30), estrogen receptor α-36 (ERα36), epidermal growth factor receptor-dependent, pertussis toxin-sensitive pathway requiring activation of c-Src, ERK1/2, and PI3K. GPER antagonist G-15 attenuated DHEA- and BSA-conjugated DHEA-stimulated pri-miR-21 transcription. Like DHEA, GPER agonists G-1 and fulvestrant increased pri-miR-21 in a GPER- and ERα36-dependent manner. DHEA, like G-1, increased GPER and ERα36 mRNA and protein levels. DHEA increased ERK1/2 and c-Src phosphorylation in a GPER-responsive manner. DHEA increased c-Jun, but not c-Fos, protein expression after 2 h. DHEA increased androgen receptor, c-Fos, and c-Jun recruitment to the miR-21 promoter. These results suggest that physiological concentrations of DHEA activate a GPER intracellular signaling cascade that increases pri-miR-21 transcription mediated at least in part by AP-1 and androgen receptor miR-21 promoter interaction. PMID:25969534

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

PubMed

Prossnitz, Eric R; Arterburn, Jeffrey B

2015-07-01

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. Copyright �

Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

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

Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp; Sekiguchi, Toshio; Nagata, Sayaka

2016-02-19

Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding andmore » AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.« less

Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway.

PubMed

Rajagopal, Senthilkumar; Kumar, Divya P; Mahavadi, Sunila; Bhattacharya, Sayak; Zhou, Ruizhe; Corvera, Carlos U; Bunnett, Nigel W; Grider, John R; Murthy, Karnam S

2013-03-01

The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5(-/-) mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2'-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser(188). TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

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

PubMed

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

2008-08-01

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

Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists.

PubMed

Cheng, Han; Lear-Rooney, Calli M; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W; Olinger, Gene G; Rong, Lijun

2015-10-01

Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious

Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists

PubMed Central

Cheng, Han; Lear-Rooney, Calli M.; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W.; Olinger, Gene G.

2015-01-01

ABSTRACT Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. IMPORTANCE Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of

Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor.

PubMed

Weiss, Dahlia R; Ahn, SeungKirl; Sassano, Maria F; Kleist, Andrew; Zhu, Xiao; Strachan, Ryan; Roth, Bryan L; Lefkowitz, Robert J; Shoichet, Brian K

2013-05-17

A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency.

Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

PubMed

Martin, Adam L; Steurer, Michael A; Aronstam, Robert S

2015-01-01

The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.

Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

PubMed Central

Martin, Adam L.; Steurer, Michael A.; Aronstam, Robert S.

2015-01-01

The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention. PMID:26384023

Twenty years of the G protein-coupled estrogen receptor GPER: Historical and personal perspectives.

PubMed

Barton, Matthias; Filardo, Edward J; Lolait, Stephen J; Thomas, Peter; Maggiolini, Marcello; Prossnitz, Eric R

2018-02-01

Estrogens play a critical role in many aspects of physiology, particularly female reproductive function, but also in pathophysiology, and are associated with protection from numerous diseases in premenopausal women. Steroids and the effects of estrogen have been known for ∼90 years, with the first evidence for a receptor for estrogen presented ∼50 years ago. The original ancestral steroid receptor, extending back into evolution more than 500 million years, was likely an estrogen receptor, whereas G protein-coupled receptors (GPCRs) trace their origins back into history more than one billion years. The classical estrogen receptors (ERα and ERβ) are ligand-activated transcription factors that confer estrogen sensitivity upon many genes. It was soon apparent that these, or novel receptors may also be responsible for the "rapid"/"non-genomic" membrane-associated effects of estrogen. The identification of an orphan GPCR (GPR30, published in 1996) opened a new field of research with the description in 2000 that GPR30 expression is required for rapid estrogen signaling. In 2005-2006, the field was greatly stimulated by two studies that described the binding of estrogen to GPR30-expressing cell membranes, followed by the identification of a GPR30-selective agonist (that lacked binding and activity towards ERα and ERβ). Renamed GPER (G protein-coupled estrogen receptor) by IUPHAR in 2007, the total number of articles in PubMed related to this receptor recently surpassed 1000. In this article, the authors present personal perspectives on how they became involved in the discovery and/or advancement of GPER research. These areas include non-genomic effects on vascular tone, receptor cloning, molecular and cellular biology, signal transduction mechanisms and pharmacology of GPER, highlighting the roles of GPER and GPER-selective compounds in diseases such as obesity, diabetes, and cancer and the obligatory role of GPER in propagating cardiovascular aging, arterial

Arsenite and Cadmium Activate MAPK/ERK via Membrane Estrogen Receptors and G-Protein Coupled Estrogen Receptor Signaling in Human Lung Adenocarcinoma Cells.

PubMed

Huff, Mary O; Todd, Sarah L; Smith, Aaron L; Elpers, Julie T; Smith, Alexander P; Murphy, Robert D; Bleser-Shartzer, Allison S; Hoerter, Jacob E; Radde, Brandie N; Klinge, Carolyn M

2016-07-01

Epidemiological evidence indicates that cadmium and arsenic exposure increase lung cancer risk. Cadmium and arsenic are environmental contaminants that act as endocrine disruptors (EDs) by activating estrogen receptors (ERs) in breast and other cancer cell lines but their activity as EDs in lung cancer is untested. Here, we examined the effect of cadmium chloride (CdCl2) and sodium arsenite (NaAsO2) on the proliferation of human lung adenocarcinoma cell lines. Results demonstrated that both CdCl2 and NaAsO2 stimulated cell proliferation at environmentally relevant nM concentrations in a similar manner to 17β-estradiol (E2) in H1793, H2073, and H1944 cells but not in H1792 or H1299 cells. Further studies in H1793 cells showed that 100 nM CdCl2 and NaAsO2 rapidly stimulated mitogen-activated protein kinase (MAPK, extracellular-signal-regulated kinases) phosphorylation with a peak detected at 15 min. Inhibitor studies suggest that rapid MAPK phosphorylation by NaAsO2, CdCl2, and E2 involves ER, Src, epidermal growth factor receptor, and G-protein coupled ER (GPER) in a pertussis toxin-sensitive pathway. CdCl2 and E2 activation of MAPK may also involve ERβ. This study supports the involvement of membrane ER and GPER signaling in mediating cellular responses to environmentally relevant nM concentrations of CdCl2 and NaAsO2 in lung adenocarcinoma cells. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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