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Vladlen Z. Slepak - One of the best experts on this subject based on the ideXlab platform.
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Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein
The Journal of biological chemistry, 2016Co-Authors: Tayou, Kirill A. Martemyanov, Qiang Wang, Geeng Fu Jang, Alexey Pronin, Cesare Orlandi, John W. Crabb, Vladlen Z. SlepakAbstract:RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gβ5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gβ5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners.
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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.
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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...
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Differential Effects of the Gβ5-RGS7 Complex on Muscarinic M3 Receptor–Induced Ca2+ Influx and Release
Molecular pharmacology, 2014Co-Authors: Darla Karpinsky-semper, 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.
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Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells
Journal of neurochemistry, 2012Co-Authors: Evangelos Liapis, Qiang Wang, Konstantin Levay, Simone L. Sandiford, Gabriel Gaidosh, Dario Motti, Vladlen Z. SlepakAbstract:The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.
Kirill A. Martemyanov - One of the best experts on this subject based on the ideXlab platform.
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Striatal RGS7 regulates depression-related behaviors and stress-induced reinstatement of cocaine conditioned place preference.
eNeuro, 2021Co-Authors: Laurie P Sutton, Natalia Khalatyan, Jeffrey N Savas, Kirill A. MartemyanovAbstract:The striatum plays a key role in both reward-related and affective behaviors and disruptions to this circuit contributes to depression and drug addiction. However, our understanding of the molecular factors that facilitate and modify these processes are incomplete. Striatal function is modulated by G protein coupled receptors (GPCRs) that process vast neuromodulatory inputs. GPCR signaling is negatively regulated by Regulator of G protein Signaling (Rgs) proteins. In this study, we examine the role of striatal Rgs proteins in depressive-like and reward-related behaviors in male mice. Using a genetic mouse model with specific elimination of RGS7 in striatal neurons we found that these mice exhibit an anxiolytic-like and antidepressant-like phenotype. In contrast, knockout of Rgs9, an abundant Rgs protein in the same neuronal population did not affect the behavioral outcome in the depressive-like tests. Mice lacking striatal RGS7 did not show significant differences in cocaine-induced psychomotor activation, sensitization or conditional place preference (CPP). Interestingly, loss of RGS7 in the striatum made mice resilient to stress-induced but not drug-induced reinstatement of cocaine CPP. Analysis of striatal proteome revealed that loss of RGS7 selectively affected expression of several networks, most prominently including proteins involved in translation and vesicular exocytosis. Together, these findings begin to demonstrate the specific contribution of RGS7 acting in the striatum towards depression as it relates to stress-induced reinstatement of drug use.SIGNIFICANCE STATEMENT G protein coupled receptors (GPCRs) play a key role in modulating responses of striatal neurons that ultimately shape complex behaviors such as mood and reward. The extent of GPCR signaling is tightly controlled by Regulators of G protein signaling (Rgs). In this study, we report a key role of RGS7 in the striatum towards depression and reward-related behaviors, while addressing the effects of stress on these behavioral outcomes. Together our findings provide new insights into the molecular mechanisms involved in stress induced drug seeking behaviors.
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Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein
The Journal of biological chemistry, 2016Co-Authors: Tayou, Kirill A. Martemyanov, Qiang Wang, Geeng Fu Jang, Alexey Pronin, Cesare Orlandi, John W. Crabb, Vladlen Z. SlepakAbstract:RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gβ5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gβ5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners.
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Association with the Plasma Membrane Is Sufficient for Potentiating Catalytic Activity of Regulators of G Protein Signaling (RGS) Proteins of the R7 Subfamily
The Journal of biological chemistry, 2016Co-Authors: Brian S. Muntean, Kirill A. MartemyanovAbstract:Regulators of G protein Signaling (RGS) promote deactivation of heterotrimeric G proteins thus controlling the magnitude and kinetics of responses mediated by G protein-coupled receptors (GPCR). In the nervous system, RGS7 and RGS9-2 play essential role in vision, reward processing, and movement control. Both RGS7 and RGS9-2 belong to the R7 subfamily of RGS proteins that form macromolecular complexes with R7-binding protein (R7BP). R7BP targets RGS proteins to the plasma membrane and augments their GTPase-accelerating protein (GAP) activity, ultimately accelerating deactivation of G protein signaling. However, it remains unclear if R7BP serves exclusively as a membrane anchoring subunit or further modulates RGS proteins to increase their GAP activity. To directly answer this question, we utilized a rapidly reversible chemically induced protein dimerization system that enabled us to control RGS localization independent from R7BP in living cells. To monitor kinetics of Gα deactivation, we coupled this strategy with measuring changes in the GAP activity by bioluminescence resonance energy transfer-based assay in a cellular system containing μ-opioid receptor. This approach was used to correlate changes in RGS localization and activity in the presence or absence of R7BP. Strikingly, we observed that RGS activity is augmented by membrane recruitment, in an orientation independent manner with no additional contributions provided by R7BP. These findings argue that the association of R7 RGS proteins with the membrane environment provides a major direct contribution to modulation of their GAP activity.
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macromolecular composition dictates receptor and g protein selectivity of regulator of g protein signaling rgs 7 and 9 2 protein complexes in living cells
Journal of Biological Chemistry, 2013Co-Authors: Ikuo Masuho, Keqiang Xie, Kirill A. MartemyanovAbstract:Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.
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R7BP Modulates Opiate Analgesia and Tolerance but not Withdrawal
Neuropsychopharmacology, 2012Co-Authors: Dimitra Terzi, Kirill A. Martemyanov, Yan Cao, Ioanna Agrimaki, Venetia ZachariouAbstract:The adaptor protein R7 family binding protein (R7BP) modulates G protein coupled receptor (GPCR) signaling and desensitization by controlling the function of regulator of G protein signaling (RGS) proteins. R7BP is expressed throughout the brain and appears to modulate the membrane localization and stability of three proteins that belong to R7 RGS family: RGS6, RGS7, and RGS9-2. RGS9-2 is a potent negative modulator of opiate and psychostimulant addiction and promotes the development of analgesic tolerance to morphine, whereas the role of RGS6 and RGS7 in addiction remains unknown. Recent studies revealed that functional deletion of R7BP reduces R7 protein activity by preventing their anchoring to the cell membrane and enhances GPCR responsiveness in the basal ganglia. Here, we take advantage of R7BP knockout mice in order to examine the way interventions in R7 proteins function throughout the brain affect opiate actions. Our results suggest that R7BP is a negative modulator of the analgesic and locomotor activating actions of morphine. We also report that R7BP contributes to the development of morphine tolerance. Finally, our data suggest that although prevention of R7BP actions enhances the analgesic responses to morphine, it does not affect the severity of somatic withdrawal signs. Our data suggest that interventions in R7BP actions enhance the analgesic effect of morphine and prevent tolerance, without affecting withdrawal, pointing to R7BP complexes as potential new targets for analgesic drugs.
Konstantin Levay - One of the best experts on this subject based on the ideXlab platform.
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Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells
Journal of neurochemistry, 2012Co-Authors: Evangelos Liapis, Qiang Wang, Konstantin Levay, Simone L. Sandiford, Gabriel Gaidosh, Dario Motti, Vladlen Z. SlepakAbstract:The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.
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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.
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Spinal cord injury induces expression of RGS7 in microglia/macrophages in rats.
The European journal of neuroscience, 2002Co-Authors: Oliver N. Hausmann, D. S. Witherow, Qiang Wang, Konstantin Levay, Vladlen Z. Slepak, Tal Keren-raifman, Beata Frydel, John R. BetheaAbstract:RGS proteins regulate G protein-mediated signalling pathways through direct interaction with the Galpha subunits and facilitation of GTP hydrolysis. An RGS subfamily consisting of RGS 6, 7, 9, and 11 also interacts with the G protein beta subunit Gbeta5 via a characteristic Ggamma-like domain. Thus far, these complexes were found only in neurons, with RGS7 being the most widely distributed in the brain. Here we confirm the expression of RGS7 in spinal neurons and show as a novel finding that following an experimental spinal cord injury in rats, expression of RGS7 is induced in a subpopulation of other cells. Immunofluorescent confocal microscopy using a series of cell specific antibodies identified these RGS7 positive cells as activated microglia and/or invading peripheral macrophages. To rule out interference from the adjacent neurons and confirm the presence of RGS7-Gbeta5 complex in inflammatory cells, we performed immunocytochemistry, RT-PCR, Western blot, and immunoprecipitation using microglial (BV2) and peripheral macrophage (RAW) cell lines. Expression of RGS7 mRNA and protein are nearly undetectable in non-stimulated BV2 and RAW cells, but remarkably increased after stimulation with LPS or TNF-alpha In addition, RGS7-positive cells were also found in the perinodular rim in the rat spleen. Our findings show that RGS7-Gbeta5 complex is expressed in immunocompetent cells such as resident microglia and peripheral macrophages following spinal cord injury. This expression might contribute to the post-traumatic inflammatory responses in the central nervous system.
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spinal cord injury induces expression of RGS7 in microglia macrophages in rats
European Journal of Neuroscience, 2002Co-Authors: Oliver N. Hausmann, Qiang Wang, Konstantin Levay, Vladlen Z. Slepak, Beata Frydel, John R. Bethea, Tal Kerenraifman, Scott D WitherowAbstract:RGS proteins regulate G protein-mediated signalling pathways through direct interaction with the Ga subunits and facilitation of GTP hydrolysis. An RGS subfamily consisting of RGS 6, 7, 9, and 11 also interacts with the G protein b subunit Gb5 via a characteristic Gg-like domain. Thus far, these complexes were found only in neurons, with RGS7 being the most widely distributed in the brain. Here we confirm the expression of RGS7 in spinal neurons and show as a novel finding that following an experimental spinal cord injury in rats, expression of RGS7 is induced in a subpopulation of other cells. Immunofluorescent confocal microscopy using a series of cell specific antibodies identified these RGS7 positive cells as activated microglia and/or invading peripheral macrophages. To rule out interference from the adjacent neurons and confirm the presence of RGS7-Gb5 complex in inflammatory cells, we performed immunocytochemistry, RT-PCR, Western blot, and immunoprecipitation using microglial (BV2) and peripheral macrophage (RAW) cell lines. Expression of RGS7 mRNA and protein are nearly undetectable in non-stimulated BV2 and RAW cells, but remarkably increased after stimulation with LPS or TNF-a In addition, RGS7-positive cells were also found in the perinodular rim in the rat spleen. Our findings show that RGS7-Gb5 complex is expressed in immunocompetent cells such as resident microglia and peripheral macrophages following spinal cord injury. This expression might contribute to the post-traumatic inflammatory responses in the central nervous system.
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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...
Qiang Wang - One of the best experts on this subject based on the ideXlab platform.
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Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein
The Journal of biological chemistry, 2016Co-Authors: Tayou, Kirill A. Martemyanov, Qiang Wang, Geeng Fu Jang, Alexey Pronin, Cesare Orlandi, John W. Crabb, Vladlen Z. SlepakAbstract:RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gβ5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gβ5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners.
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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...
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Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells
Journal of neurochemistry, 2012Co-Authors: Evangelos Liapis, Qiang Wang, Konstantin Levay, Simone L. Sandiford, Gabriel Gaidosh, Dario Motti, Vladlen Z. SlepakAbstract:The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.
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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.
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Gβ5·RGS7 Inhibits Gαq-mediated Signaling via a Direct Protein-Protein Interaction
The Journal of biological chemistry, 2003Co-Authors: Steven C. Tovey, Qiang Wang, Gary B. Willars, Vladlen Z. SlepakAbstract:A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Ggamma-like domain through which they form obligatory dimers with the G protein subunit Gbeta5 in vivo. In Caenorhabditis elegans, orthologs of Gbeta5.RGS dimers are implicated in regulating both Galphai and Galphaq signaling, and in cell-based assays these dimers regulate Galphai/o- and Galphaq/11-mediated pathways. However, initial studies with purified Gbeta5.RGS6 or Gbeta5.RGS7 showed that they only serve as GTPase activating proteins for Galphao. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Galphao or Galphaq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7.Gbeta5 complex binds to Galphaq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gbeta5, and Galpha subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gbeta5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7.Gbeta5 complex and cyan fluorescence protein-tagged Galphaq, indicating a direct interaction between these molecules.
John R. Bethea - One of the best experts on this subject based on the ideXlab platform.
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Spinal cord injury induces expression of RGS7 in microglia/macrophages in rats.
The European journal of neuroscience, 2002Co-Authors: Oliver N. Hausmann, D. S. Witherow, Qiang Wang, Konstantin Levay, Vladlen Z. Slepak, Tal Keren-raifman, Beata Frydel, John R. BetheaAbstract:RGS proteins regulate G protein-mediated signalling pathways through direct interaction with the Galpha subunits and facilitation of GTP hydrolysis. An RGS subfamily consisting of RGS 6, 7, 9, and 11 also interacts with the G protein beta subunit Gbeta5 via a characteristic Ggamma-like domain. Thus far, these complexes were found only in neurons, with RGS7 being the most widely distributed in the brain. Here we confirm the expression of RGS7 in spinal neurons and show as a novel finding that following an experimental spinal cord injury in rats, expression of RGS7 is induced in a subpopulation of other cells. Immunofluorescent confocal microscopy using a series of cell specific antibodies identified these RGS7 positive cells as activated microglia and/or invading peripheral macrophages. To rule out interference from the adjacent neurons and confirm the presence of RGS7-Gbeta5 complex in inflammatory cells, we performed immunocytochemistry, RT-PCR, Western blot, and immunoprecipitation using microglial (BV2) and peripheral macrophage (RAW) cell lines. Expression of RGS7 mRNA and protein are nearly undetectable in non-stimulated BV2 and RAW cells, but remarkably increased after stimulation with LPS or TNF-alpha In addition, RGS7-positive cells were also found in the perinodular rim in the rat spleen. Our findings show that RGS7-Gbeta5 complex is expressed in immunocompetent cells such as resident microglia and peripheral macrophages following spinal cord injury. This expression might contribute to the post-traumatic inflammatory responses in the central nervous system.
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spinal cord injury induces expression of RGS7 in microglia macrophages in rats
European Journal of Neuroscience, 2002Co-Authors: Oliver N. Hausmann, Qiang Wang, Konstantin Levay, Vladlen Z. Slepak, Beata Frydel, John R. Bethea, Tal Kerenraifman, Scott D WitherowAbstract:RGS proteins regulate G protein-mediated signalling pathways through direct interaction with the Ga subunits and facilitation of GTP hydrolysis. An RGS subfamily consisting of RGS 6, 7, 9, and 11 also interacts with the G protein b subunit Gb5 via a characteristic Gg-like domain. Thus far, these complexes were found only in neurons, with RGS7 being the most widely distributed in the brain. Here we confirm the expression of RGS7 in spinal neurons and show as a novel finding that following an experimental spinal cord injury in rats, expression of RGS7 is induced in a subpopulation of other cells. Immunofluorescent confocal microscopy using a series of cell specific antibodies identified these RGS7 positive cells as activated microglia and/or invading peripheral macrophages. To rule out interference from the adjacent neurons and confirm the presence of RGS7-Gb5 complex in inflammatory cells, we performed immunocytochemistry, RT-PCR, Western blot, and immunoprecipitation using microglial (BV2) and peripheral macrophage (RAW) cell lines. Expression of RGS7 mRNA and protein are nearly undetectable in non-stimulated BV2 and RAW cells, but remarkably increased after stimulation with LPS or TNF-a In addition, RGS7-positive cells were also found in the perinodular rim in the rat spleen. Our findings show that RGS7-Gb5 complex is expressed in immunocompetent cells such as resident microglia and peripheral macrophages following spinal cord injury. This expression might contribute to the post-traumatic inflammatory responses in the central nervous system.