The Experts below are selected from a list of 18378 Experts worldwide ranked by ideXlab platform
Vladlen Z. Slepak - One of the best experts on this subject based on the ideXlab platform.
-
differential effects of the gβ5 rgs7 complex on muscarinic m3 receptor induced ca2 influx and release
Molecular Pharmacology, 2014Co-Authors: Darla Karpinskysemper, Claude-henry Volmar, Vladlen Z. SlepakAbstract:The G protein β subunit Gβ5 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Gγ. Although the Gβ5-RGS7 complex attenuates Ca2+ signaling mediated by the muscarinic M3 receptor (M3R), the route of Ca2+ entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here, we show that, in addition to suppressing carbachol-stimulated Ca2+ release, Gβ5-RGS7 enhanced Ca2+ influx. This novel effect of Gβ5-RGS7 was blocked by nifedipine and 2-aminoethoxydiphenyl borate. Experiments with pertussis toxin, an RGS domain–deficient mutant of RGS7, and UBO-QIC {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4- methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)}, a novel inhibitor of Gq, showed that Gβ5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gβ5-RGS7, independent of RGS7 GTPase-accelerating protein activity, couples M3R to a nifedipine-sensitive Ca2+ channel. We also compared the action of Gβ5-RGS7 on M3R-induced Ca2+ influx and release elicited by different muscarinic agonists. Responses to Oxo-M [oxotremorine methiodide N,N,N,-trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn-1-ammonium iodide] were insensitive to Gβ5-RGS7. Pilocarpine responses consisted of a large release and modest influx components, of which the former was strongly inhibited whereas the latter was insensitive to Gβ5-RGS7. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride] was the only compound whose total Ca2+ response was enhanced by Gβ5-RGS7, attributed to, in part, by the relatively small Ca2+ release this partial agonist stimulated. Together, these results show that distinct agonists not only have differential M3R functional selectivity, but also confer specific sensitivity to the Gβ5-RGS7 complex.
-
Effects of the Gβ5-RGS7 complex on muscarinic M3 receptor-induced Ca2+ signaling and insulin secretion (843.12)
The FASEB Journal, 2014Co-Authors: Vladlen Z. Slepak, Darla Karpinsky-semper, Qiang WangAbstract:The G protein subunit Gβ5 forms obligatory heterodimers with regulators of G protein signaling (RGS) proteins of R7 family (RGS6, 7, 9 and 11). These complexes are expressed in neurons and glands. Previously, we showed that Gβ5-RGS7 attenuated signaling downstream of muscarinic M3 receptor, M3R. A predicted consequence of Gβ5 ablation on pancreatic beta cells would be an increase in insulin secretion. However, we found that Gβ5-R7 knockout strongly attenuated, while its overexperession in Min6 insulinoma cells enhanced M3R-induced insulin secretion. We studied isolated mouse islets, transfected Min6 and CHO-K1 cells, measuring insulin secretion and changes in intracellular Ca2+ in the presence of various pharmacological agents, muscarinic agonists and functional mutants of Gβ5-RGS7 and M3R. The results showed that Gβ5-RGS7 has a dual effect on M3R-mediated Ca2+ transients. While it attenuated Ca2+ release from internal stores, it stimulated Ca2+ influx across the plasma membranes. This pathway involves Gq...
-
Intramolecular interaction between the DEP domain of RGS7 and the Gbeta5 subunit.
Biochemistry, 2007Co-Authors: Vijaya Narayanan, Qiang Wang, Konstantin Levay, Tal Keren-raifman, Simone L. Sandiford, Vladlen Z. SlepakAbstract:The R7 family of RGS proteins (RGS6, -7, -9, -11) is characterized by the presence of three domains: DEP, GGL, and RGS. The RGS domain interacts with Galpha subunits and exhibits GAP activity. The GGL domain permanently associates with Gbeta5. The DEP domain interacts with the membrane anchoring protein, R7BP. Here we provide evidence for a novel interaction within this complex: between the DEP domain and Gbeta5. GST fusion of the RGS7 DEP domain (GST-R7DEP) binds to both native and recombinant Gbeta5-RGS7, recombinant Gbetagamma complexes, and monomeric Gbeta5 and Gbeta1 subunits. Co-immunoprecipitation and FRET assays supported the GST pull-down experiments. GST-R7DEP reduced FRET between CFP-Gbeta5 and YFP-RGS7, indicating that the DEP-Gbeta5 interaction is dynamic. In transfected cells, R7BP had no effect on the Gbeta5/RGS7 pull down by GST-R7DEP. The DEP domain of RGS9 did not bind to Gbeta5. Substitution of RGS7 Glu-73 and Asp-74 for the corresponding Ser and Gly residues (ED/SG mutation) of RGS9 diminished the DEP-Gbeta5 interaction. In the absence of R7BP both the wild-type RGS7 and the ED/SG mutant attenuated muscarinic M3 receptor-mediated Ca2+ mobilization. In the presence of R7BP, wild-type RGS7 lost this inhibitory activity, whereas the ED/SG mutant remained active. Taken together, our results are consistent with the following model. The Gbeta5-RGS7 molecule can exist in two conformations: "closed" and "open", when the DEP domain and Gbeta5 subunit either do or do not interact. The closed conformation appears to be less active with respect to its effect on Gq-mediated signaling than the open conformation.
-
Phosphorylation of the regulator of G protein signaling RGS9-1 by protein kinase a is a potential mechanism of light- and Ca2+-mediated regulation of G protein function in photoreceptors
Biochemistry, 2001Co-Authors: Nagaraj Balasubramanian, Konstantin Levay, Tal Keren-raifman, Eva Faurobert, Vladlen Z. SlepakAbstract:In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90%, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein β subunit Gβ5. Phosphorylated RGS9-1 rem...
-
Expression levels of RGS7 and RGS4 proteins determine the mode of regulation of the G protein-activated K+ channel and control regulation of RGS7 by Gβ5
FEBS Letters, 2001Co-Authors: Tal Keren-raifman, D. S. Witherow, Vladlen Z. Slepak, A K Bera, D Zveig, S Peleg, N DascalAbstract:Regulators of G protein signaling RGS4 and RGS7 accelerate the kinetics of K+ channels (GIRKs) in the Xenopus oocyte system. Here, via quantitative analysis of RGS expression, we reveal biphasic effects of RGSs on GIRK regulation. At low concentrations, RGS4 inhibited basal GIRK activity, but stimulated it at high concentrations. RGS7, which is associated with the G protein subunit Gβ5, is regulated by Gβ5 by two distinct mechanisms. First, Gβ5 augments RGS7 activity, and second, it increases its expression. These dual effects resolve previous controversies regarding RGS4 and RGS7 function and indicate that they modulate signaling by mechanisms supplementary to their GTPase-activating protein activity.
Richard R Neubig - One of the best experts on this subject based on the ideXlab platform.
-
Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins
The Journal of biological chemistry, 2013Co-Authors: Buxin Chen, Richard R Neubig, David P Siderovski, Mark A. Lawson, Joann TrejoAbstract:The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.
-
a nanomolar potency small molecule inhibitor of regulator of g protein signaling proteins
Biochemistry, 2011Co-Authors: Levi L Blazer, Haoming Zhang, Emma M Casey, Stephen M Husbands, Richard R NeubigAbstract:Regulators of G-protein signaling (RGS) proteins are potent negative modulators of signal transduction through G-protein-coupled receptors. They function by binding to activated (GTP-bound) Gα subunits and accelerating the rate of GTP hydrolysis. Modulation of RGS activity by small molecules is an attractive mechanism for fine-tuning GPCR signaling for therapeutic and research purposes. Here we describe the pharmacologic properties and mechanism of action of CCG-50014, the most potent small molecule RGS inhibitor to date. It has an IC(50) for RGS4 of 30 nM and is >20-fold selective for RGS4 over other RGS proteins. CCG-50014 binds covalently to the RGS, forming an adduct on two cysteine residues located in an allosteric regulatory site. It is not a general cysteine alkylator as it does not inhibit activity of the cysteine protease papain at concentrations >3000-fold higher than those required to inhibit RGS4 function. It is also >1000-fold more potent as an RGS4 inhibitor than are the cysteine alkylators N-ethylmaleimide and iodoacetamide. Analysis of the cysteine reactivity of the compound shows that compound binding to Cys(107) in RGS8 inhibits Gα binding in a manner that can be reversed by cleavage of the compound-RGS disulfide bond. If the compound reacts with Cys(160) in RGS8, the adduct induces RGS denaturation, and activity cannot be restored by removal of the compound. The high potency and good selectivity of CCG-50014 make it a useful tool for studying the functional roles of RGS4.
-
novel peptide ligands of rgs4 from a focused one bead one compound library
Chemical Biology & Drug Design, 2008Co-Authors: Rebecca A Roof, David L. Roman, Katarzyna Sobczykkojiro, Levi L Blazer, Anjanette J Turbiak, Richard R Neubig, Irina D. Pogozheva, Henry I MosbergAbstract:: Regulators of G protein signaling accelerate GTP hydrolysis by G alpha subunits and profoundly inhibit signaling by G protein-coupled receptors. The distinct expression patterns and pathophysiologic regulation of regulators of G protein signaling proteins suggest that inhibitors may have therapeutic potential. We previously reported the design, mechanistic evaluation, and structure-activity relationships of a disulfide-containing cyclic peptide inhibitor of RGS4, YJ34 (Ac-Val-Lys-c[Cys-Thr-Gly-Ile-Cys]-Glu-NH(2), S-S) (Roof et al., Chem Biol Drug Des, 67, 2006, 266). Using a focused one-bead, one-compound peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2 (Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH(2), S-S with a free or acetylated N-terminus) inhibited RGS4-stimulated G alpha(o) GTPase activity at 25-50 microM. They selectively inhibit RGS4 but not RGS7, RGS16, and RGS19. Their inhibition of RGS4 does not depend on cysteine-modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation.
-
n terminal residues control proteasomal degradation of rgs2 rgs4 and RGS5 in human embryonic kidney 293 cells
Molecular Pharmacology, 2007Co-Authors: Johannes Bodenstein, Roger K. Sunahara, Richard R NeubigAbstract:Regulator of G protein signaling (RGS) proteins modulate G protein-coupled receptor (GPCR) signaling. The N termini of some RGS4-family proteins provide receptor specificity and also contain an N-end rule determinant that results in ubiquitylation and decreased protein expression. The relevance of these mechanisms to other RGS proteins is not fully understood. Thus we examined function, receptor specificity, and expression of R4 subfamily RGS proteins (RGS2, -3, -4, -5, and -8). Although the N terminus plays a key role in protein stability in human embryonic kidney (HEK) 293 cells, we were unable to demonstrate specificity of RGS2, -3, -4, -5, or -8 for muscarinic receptors (M1, M3, and M5). However, cellular RGS activity (8 = 3 > 2) was strongly correlated with expression; RGS4 and -5 had minimal expression and activity. Stabilizing mutations of RGS4 and -5 (C2S) enhanced expression and function with a greater influence on RGS4 than on RGS5. We were surprised to find that a predicted destabilizing mutation in RGS8 (A2C) did not markedly affect expression and had no effect on function. In contrast, a destabilizing mutation in RGS2 (RGS2-Q2L) recently identified as a rare N-terminal genetic variant in a Japanese hypertensive cohort ( J Hypertens 23: 1497–1505, 2005) showed significantly reduced expression and inhibition of angiotensin II (AT1) receptor-stimulated accumulation of inositol phosphates. We were surprised to find that RGS2-Q2R, also predicted to be destabilizing, showed nearly normal expression and function. Thus, proteasomal regulation of RGS expression in HEK293 cells strongly controls RGS function and a novel RGS2 mutation with decreased protein expression could be relevant to the pathophysiology of hypertension in humans.
-
identification of small molecule inhibitors of rgs4 using a high throughput flow cytometry protein interaction assay
Molecular Pharmacology, 2007Co-Authors: David L. Roman, Jeffery N Talbot, Rebecca A Roof, John R Traynor, Roger K. Sunahara, Richard R NeubigAbstract:Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein α-subunits (Gα) and thus shorten the time course and reduce the magnitude of G-protein α- and βγ-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess Gα-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1, methyl N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/Gαo binding with 3 to 5 μM potency. It binds to RGS4, inhibits RGS4 stimulation of Gαo GTPase activity in vitro, and prevents RGS4 regulation of μ-opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/Gα protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.
John H Kehrl - One of the best experts on this subject based on the ideXlab platform.
-
RGS5 targeting leads to chronic low blood pressure and a lean body habitus
Molecular and Cellular Biology, 2008Co-Authors: Hyeseon Cho, Chung Park, Ilyoung Hwang, Sangbae Han, Dan Schimel, Daryl Despres, John H KehrlAbstract:RGS5 is a potent GTPase-activating protein for G(ialpha) and G(qalpha) that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated RGS5-deficient mice. The RGS5(-/-) mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, RGS5(-/-) mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of RGS5(-/-) mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the RGS5(-/-) mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or sphingosine 1-phosphate. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.
-
abnormal b cell responses to chemokines disturbed plasma cell localization and distorted immune tissue architecture in rgs1 mice
Molecular and Cellular Biology, 2004Co-Authors: Chantal Moratz, Russell J Hayman, John H KehrlAbstract:The migration of developing lymphocytes into lymphoid tissues, the recirculation of lymphocytes, as well as organization of secondary immune structures such as lymphoid follicles results from a regulated configuration of cell surface adhesion molecules and chemoattractant receptors as well as a spatial overlay of multiple chemoattractant gradients (1, 23, 29, 32). Mechanisms that allow a cell, such as a B-lymphocyte, to interrupt, modify, and prioritize signals from such a milieu of stimuli are essential for efficient and normal immune function. Inappropriate interpretations by cells within such a complex environment is deleterious, leading to inappropriate trafficking, survival, and activation of cells (10, 12, 28, 37). Previous work has detailed B-cell migratory patterns following antigen challenge, including the establishment of germinal centers; cycling within germinal centers; the exiting of memory and antibody-secreting B cells (ASCs) from germinal centers; and the trafficking of ASCs to bone marrow, lamina propria, and other mucosal sites (5, 16, 22, 27, 30, 42, 45). Much of this work has focused on chemokines and their receptors and upon adhesion molecules, while less is known about the signaling mechanisms that allow B cells to efficiently negotiate the complex chemokine gradients likely present in tissues. Most chemoattracant receptors and all chemokine receptors couple to heterotrimeric G-proteins (1). Activated receptors trigger Gα subunits to exchange GTP for GDP, which dissociates the Gα subunit from βγ heterodimers, leading to the activation of downstream effectors. However, Gα subunits possess an intrinsic GTPase activity that limits the duration of their remaining GTP bound. GTP hydrolysis allows the heterotrimer to reform, and signaling ceases (18, 35). Also limiting the duration of Gα subunits' remaining GTP bound, members of the regulator of G protein signaling (RGS) protein family dramatically increase the intrinsic Gα GTPase activity, a property that defines them as GTPase activating proteins (GAPs). Genetic studies in Saccharomyces cerevisiae, Caenorhabditis elegans, and Aspergillus nidulans first identified such proteins (7, 21, 25). Independently, a mammalian protein termed GAIP was discovered to interact with a Gα subunit (6) and four mammalian proteins designated RGS1, RGS2, RGS3, and RGS4 substituted to various degrees for Sst2p, a yeast protein involved in the desensitization of pheromone signaling (9). Approximately 25 human RGS proteins have now been identified. When tested in standard in vitro GAP assays, most RGS proteins possess GAP activity for the α subunits of the Gi and Gq subfamilies (3, 19, 44). Since chemokine receptors use Gi and perhaps Gq to transduce intracellular signals, the presence of an RGS protein in target cells could substantially alter the response to chemokine stimulation (20). B-lymphocytes, especially following B-cell activation through their antigen receptors, express RGS1. Consistent with a role for RGS1 in regulating the B-cell responses to chemokines, the expression of RGS1 in B-cell lines dramatically impairs their migratory response to CXCL12 and CXCL13 (4, 33, 38). Because the normal trafficking of B cells depends upon the ligand receptor pairs CXCL12-CXCR4 (34, 46) and CXCL13-CXCR5 (11, 15, 26), a deficiency of Rgs1 in vivo could alter B-cell development and/or the organization of B cells in lymphoid tissues. To test that possibility we generated Rgs1−/− mice.
-
the aorta and heart differentially express rgs regulators of g protein signalling proteins that selectively regulate sphingosine 1 phosphate angiotensin ii and endothelin 1 signalling
Biochemical Journal, 2003Co-Authors: Hyeseon Cho, Kathleen Harrison, Owen Schwartz, John H KehrlAbstract:Normal cardiovascular development and physiology depend in part upon signalling through G-protein-coupled receptors (GPCRs), such as the angiotensin II type 1 (AT(1)) receptor, sphingosine 1-phosphate (S1P) receptors and endothelin-1 (ET-1) receptor. Since regulator of G-protein signalling (RGS) proteins function as GTPase-activating proteins for the G alpha subunit of heterotrimeric G-proteins, these proteins undoubtedly have functional roles in the cardiovascular system. In the present paper, we show that human aorta and heart differentially express RGS1, RGS2, RGS3S (short-form), RGS3L (long-form), PDZ-RGS3 (PDZ domain-containing) and RGS4. The aorta prominently expresses mRNAs for all these RGS proteins except PDZ-RGS3. Various stimuli that are critical for both cardiovascular development and function regulate dynamically the mRNA levels of several of these RGS proteins in primary human aortic smooth muscle cells. Both RGS1 and RGS3 inhibit signalling through the S1P(1) (formerly known as EDG-1), S1P(2) (formerly known as EDG-5) and S1P(3) (formerly known as EDG-3) receptors, whereas RGS2 and RGS4 selectively attenuate S1P(2)-and S1P(3)-receptor signalling respectively. All of the tested RGS proteins inhibit AT(1)-receptor signalling, whereas only RGS3 and, to a lesser extent, RGS4 inhibit ET(A)-receptor signalling. The conspicuous expression of RGS proteins in the cardiovascular system and their selective effects on relevant GPCR-signalling pathways provide additional evidence that they have functional roles in cardiovascular development and physiology.
-
rgs2 a multifunctional regulator of g protein signaling
The International Journal of Biochemistry & Cell Biology, 2002Co-Authors: John H Kehrl, Srikumar SinnarajahAbstract:Abstract Regulators of G-protein signaling (RGS) proteins enhance the intrinsic rate at which certain heterotrimeric G-protein α-subunits hydrolyze GTP to GDP, thereby limiting the duration that α-subunits activate downstream effectors. This activity defines them as GTPase activating proteins (GAPs). As do other RGS proteins RGS2 possesses a 120 amino acid RGS domain, which mediates its GAP activity. In addition, RGS2 shares an N-terminal membrane targeting domain with RGS4 and RGS16. Found in many cell types, RGS2 expression is highly regulated. Functionally, RGS2 blocks Gqα-mediated signaling, a finding consistent with its potent Gqα GAP activity. Surprisingly, RGS2 inhibits Gs signaling to certain adenylyl cyclases. Like other RGS proteins, RGS2 lacks Gsα GAP activity, however it directly inhibits the activity of several adenylyl cyclase isoforms. Targeted mutation of RGS2 in mice impairs anti-viral immunity, increases anxiety levels, and alters synaptic development in hippocampal CA1 neurons. RGS2 has emerged as a multifunctional RGS protein that regulates multiple G-protein linked signaling pathways.
-
molecules in focus rgs2 a multifunctional regulator of g protein signaling
2002Co-Authors: John H Kehrl, Srikumar SinnarajahAbstract:Regulators of G-protein signaling (RGS) proteins enhance the intrinsic rate at which certain heterotrimeric G-protein -subunits hydrolyze GTP to GDP, thereby limiting the duration that -subunits activate downstream effectors. This activity defines them as GTPase activating proteins (GAPs). As do other RGS proteins RGS2 possesses a 120 amino acid RGS domain, which mediates its GAP activity. In addition, RGS2 shares an N-terminal membrane targeting domain with RGS4 and RGS16. Found in many cell types, RGS2 expression is highly regulated. Functionally, RGS2 blocks Gq-mediated signaling, a finding consistent with its potent Gq GAP activity. Surprisingly, RGS2 inhibits Gs signaling to certain adenylyl cyclases. Like other RGS proteins, RGS2 lacks Gs GAP activity, however it directly inhibits the activity of several adenylyl cyclase isoforms. Targeted mutation of RGS2 in mice impairs anti-viral immunity, increases anxiety levels, and alters synaptic development in hippocampal CA1 neurons. RGS2 has emerged as a multifunctional RGS protein that regulates multiple G-protein linked signaling pathways. Published by Elsevier Science Ltd.
Qiang Wang - One of the best experts on this subject based on the ideXlab platform.
-
Effects of the Gβ5-RGS7 complex on muscarinic M3 receptor-induced Ca2+ signaling and insulin secretion (843.12)
The FASEB Journal, 2014Co-Authors: Vladlen Z. Slepak, Darla Karpinsky-semper, Qiang WangAbstract:The G protein subunit Gβ5 forms obligatory heterodimers with regulators of G protein signaling (RGS) proteins of R7 family (RGS6, 7, 9 and 11). These complexes are expressed in neurons and glands. Previously, we showed that Gβ5-RGS7 attenuated signaling downstream of muscarinic M3 receptor, M3R. A predicted consequence of Gβ5 ablation on pancreatic beta cells would be an increase in insulin secretion. However, we found that Gβ5-R7 knockout strongly attenuated, while its overexperession in Min6 insulinoma cells enhanced M3R-induced insulin secretion. We studied isolated mouse islets, transfected Min6 and CHO-K1 cells, measuring insulin secretion and changes in intracellular Ca2+ in the presence of various pharmacological agents, muscarinic agonists and functional mutants of Gβ5-RGS7 and M3R. The results showed that Gβ5-RGS7 has a dual effect on M3R-mediated Ca2+ transients. While it attenuated Ca2+ release from internal stores, it stimulated Ca2+ influx across the plasma membranes. This pathway involves Gq...
-
Intramolecular interaction between the DEP domain of RGS7 and the Gbeta5 subunit.
Biochemistry, 2007Co-Authors: Vijaya Narayanan, Qiang Wang, Konstantin Levay, Tal Keren-raifman, Simone L. Sandiford, Vladlen Z. SlepakAbstract:The R7 family of RGS proteins (RGS6, -7, -9, -11) is characterized by the presence of three domains: DEP, GGL, and RGS. The RGS domain interacts with Galpha subunits and exhibits GAP activity. The GGL domain permanently associates with Gbeta5. The DEP domain interacts with the membrane anchoring protein, R7BP. Here we provide evidence for a novel interaction within this complex: between the DEP domain and Gbeta5. GST fusion of the RGS7 DEP domain (GST-R7DEP) binds to both native and recombinant Gbeta5-RGS7, recombinant Gbetagamma complexes, and monomeric Gbeta5 and Gbeta1 subunits. Co-immunoprecipitation and FRET assays supported the GST pull-down experiments. GST-R7DEP reduced FRET between CFP-Gbeta5 and YFP-RGS7, indicating that the DEP-Gbeta5 interaction is dynamic. In transfected cells, R7BP had no effect on the Gbeta5/RGS7 pull down by GST-R7DEP. The DEP domain of RGS9 did not bind to Gbeta5. Substitution of RGS7 Glu-73 and Asp-74 for the corresponding Ser and Gly residues (ED/SG mutation) of RGS9 diminished the DEP-Gbeta5 interaction. In the absence of R7BP both the wild-type RGS7 and the ED/SG mutant attenuated muscarinic M3 receptor-mediated Ca2+ mobilization. In the presence of R7BP, wild-type RGS7 lost this inhibitory activity, whereas the ED/SG mutant remained active. Taken together, our results are consistent with the following model. The Gbeta5-RGS7 molecule can exist in two conformations: "closed" and "open", when the DEP domain and Gbeta5 subunit either do or do not interact. The closed conformation appears to be less active with respect to its effect on Gq-mediated signaling than the open conformation.
-
Complexes of the G Protein Subunit Gβ5 with the Regulators of G Protein Signaling RGS7 and RGS9 CHARACTERIZATION IN NATIVE TISSUES AND IN TRANSFECTED CELLS
The Journal of biological chemistry, 2000Co-Authors: D. S. Witherow, Qiang Wang, Konstantin Levay, Jorge L. Cabrera, Jiandong Chen, G. B. Willars, Vladlen Z. SlepakAbstract:Abstract A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Gα subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Gγ -like domain, also has the unique ability to interact with the G protein β subunit Gβ5. Here, we examined the behavior of Gβ5, RGS7, RGS9, and Gα in tissue extracts using immunoprecipitation and conventional chromatography. Native Gβ5 and RGS7 from brain, as well as photoreceptor-specific Gβ5L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gβ5 nor Gβ5-free RGS could be detected. Co-expression in COS-7 cells of Gβ5 dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gβ5:RGS stoichiometry in a manner similar to Gβγ complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysis of native Gβ5-RGS and their coupled expression argue that in vivo, Gβ5and Gγ-like domain-containing RGSs only exist as heterodimers. Native Gβ5-RGS7 did not co-precipitate or co-purify with Gαo or Gαq; nor did Gβ5 L-RGS9 with Gαt. However, in transfected cells, RGS7 and Gβ5-RGS7 inhibited Gαq-mediated Ca2+ response to muscarinic M3 receptor activation. Thus, Gβ5-RGS dimers differ from other RGS proteins in that they do not bind to Gα with high affinity, but they can still inhibit G protein signaling.
Sadao Kimura - One of the best experts on this subject based on the ideXlab platform.
-
Potent and selective inhibition of angiotensin AT1 receptor signaling by RGS2: roles of its N-terminal domain.
Cellular Signalling, 2011Co-Authors: Naoko Matsuzaki, Kayoko Moroi, Dan Song, Mariko Nishiyama, Sadao KimuraAbstract:Abstract Emerging evidence indicates that R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in functional regulation in the cardiovascular system. In this study, we compared effects of three R4/B subfamily proteins, RGS2, RGS4 and RGS5 on angiotensin AT1 receptor signaling, and investigated roles of the N-terminus of RGS2. In HEK293T cells expressing AT1 receptor stably, intracellular Ca 2+ responses induced by angiotensin II were much more strongly attenuated by RGS2 than by RGS4 and RGS5. N-terminally deleted RGS2 proteins lost this potent inhibitory effect. Replacement of the N-terminal residues 1–71 of RGS2 with the corresponding residues (1–51) of RGS5 decreased significantly the inhibitory effect. On the other hand, replacement of the residues 1–51 of RGS5 with the residues 1–71 of RGS2 increased the inhibitory effect dramatically. Furthermore, we investigated functional contribution of N-terminal subdomains of RGS2, namely, an N-terminal region (residues 16–55) with an amphipathic α helix domain (the subdomain N1), a probable non-specific membrane-targeting subdomain, and another region (residues 56–71) between the α helix and the RGS box (the subdomain N2), a probable GPCR-recognizing subdomain. RGS2 chimera proteins with the residues 1–33 or 34–52 of RGS5 showed weak inhibitory activity, and either of RGS5 chimera proteins with residues 1–55 or 56–71 of RGS2 showed strong inhibitory effects on AT1 receptor signaling. The present study indicates the essential roles of both N-terminal subdomains for the potent inhibitory activity of RGS2 on AT1 receptor signaling.
-
phosphorylation of ser166 in RGS5 by protein kinase c causes loss of rgs function
Life Sciences, 2007Co-Authors: Kayoko Moroi, Mariko Nishiyama, Shinichirou Kawabata, Hideaki Ichiba, Takehiko Yajima, Sadao KimuraAbstract:Abstract RGS5 is a member of regulators of G protein signaling (RGS) proteins that attenuate heterotrimeric G protein signaling by functioning as GTPase-activating proteins (GAPs). We investigated phosphorylation of RGS5 and the resulting change of its function. In 293T cells, transiently expressed RGS5 was phosphorylated by endogenous protein kinases in the basal state. The phosphorylation was enhanced by phorbol 12-myristate 13-acetate (PMA) and endothelin-1 (ET-1), and suppressed by protein kinase C (PKC) inhibitors, H7, calphostin C and staurosporine. These results suggest involvement of PKC in phosphorylation of RGS5. In in vitro experiments, PKC phosphorylated recombinant RGS5 protein at serine residues. RGS5 protein phosphorylated by PKC showed much lower binding capacity for and GAP activity toward Gα subunits than did the unphosphorylated RGS5. In cells expressing RGS5, the inhibitory effect of RGS5 on ET-1-induced Ca 2+ responses was enhanced by staurosporine. Mass spectrometric analysis of the phosphorylated RGS5 revealed that Ser166 was one of the predominant phosphorylation sites. Substitution of Ser166 by aspartic acid abolished the binding capacity to Gα subunits and the GAP activity, and markedly reduced the inhibitory effect on ET-1-induced Ca 2+ responses. These results indicate that phosphorylation at Ser166 of RGS5 by PKC causes loss of the function of RGS5 in G protein signaling. Since this serine residue is conserved in RGS domains of many RGS proteins, the phosphorylation at Ser166 by PKC might act as a molecular switch and have functional significance.
-
rgs4 and RGS5 are in vivo substrates of the n end rule pathway
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Takafumi Tasaki, Kayoko Moroi, Ilia V Davydov, Jee Young An, Sadao Kimura, Yong Tae KwonAbstract:The ATE1-encoded Arg-transferase mediates conjugation of Arg to N-terminal Asp, Glu, and Cys of certain eukaryotic proteins, yielding N-terminal Arg that can act as a degradation signal for the ubiquitin-dependent N-end rule pathway. We have previously shown that mouse ATE1–/– embryos die with defects in heart development and angiogenesis. Here, we report that the ATE1 Arg-transferase mediates the in vivo degradation of RGS4 and RGS5, which are negative regulators of specific G proteins whose functions include cardiac growth and angiogenesis. The proteolysis of these regulators of G protein signaling (RGS) proteins was perturbed either by hypoxia or in cells lacking ubiquitin ligases UBR1 and/or UBR2. Mutant RGS proteins in which the conserved Cys-2 residue could not become N-terminal were long-lived in vivo. We propose a model in which the sequential modifications of RGS4, RGS5, and RGS16 (N-terminal exposure of their Cys-2, its oxidation, and subsequent arginylation) act as a licensing mechanism in response to extracellular and intracellular signals before the targeting for proteolysis by UBR1 and UBR2. We also show that ATE1–/– embryos are impaired in the activation of extracellular signal-regulated kinase mitogen-activated protein kinases and in the expression of G protein-induced downstream effectors such as Jun, cyclin D1, and β-myosin heavy chain. These results establish RGS4 and RGS5 as in vivo substrates of the mammalian N-end rule pathway and also suggest that the O2-ATE1-UBR1/UBR2 proteolytic circuit plays a role in RGS-regulated G protein signaling in the cardiovascular system.
-
Characterization of RGS5 in Regulation of G Protein-Coupled Receptor Signaling
Life sciences, 2001Co-Authors: Jing Zhou, Kayoko Moroi, Mariko Nishiyama, Naohiko Seki, Hirokazu Usui, Junji Ishida, Akiyoshi Fukamizu, Sadao KimuraAbstract:RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (deltaN-RGS5). RGS5 bound to G alpha(i1), G alpha(i2), G alpha(i3), G alpha(o) and G alpha(q) but not to G alpha(s) and G alpha13 in the presence of GDP/AIF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha(i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT1a receptors (AT1a-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGS5 was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGS5 induced about 20-25% reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT1a receptors showed that RGS5 inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT1a receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT1a-293T cells, deltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and deltaN-RGS5 revealed that the N terminal (1-33) of RGS5 plays a role in targeting this protein to membranes, and that the N terminal region of RGS5 is not essential for exerting activities.