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Kirill A. Martemyanov - 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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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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rgs6 but not rgs4 is the dominant regulator of g protein signaling rgs modulator of the parasympathetic regulation of mouse heart rate
Journal of Biological Chemistry, 2014Co-Authors: Nicole Wydeven, Ekaterina Posokhova, Kirill A. Martemyanov, Kevin WickmanAbstract:Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K+ channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4−/−, Rgs6−/−, and Rgs4−/−:Rgs6−/− mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6−/− mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6−/− and Gβ5−/− mice, suggest that the partial rescue of phenotypes in Rgs4−/−:Rgs6−/− mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice.
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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.
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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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.
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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.
Rhodri Ceredig - One of the best experts on this subject based on the ideXlab platform.
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Differential expression of regulator of G-protein signalling transcripts and in vivo migration of CD4+ naïve and regulatory T cells.
Immunology, 2007Co-Authors: Fabien Agenes, Nabil Bosco, Laurent Mascarell, Sabrina Fritah, Rhodri CeredigAbstract:The immune response of T lymphocytes to pathogens is initiated in draining secondary lymphoid organs, and activated cells then migrate to the site of infection. Thus, control of naive and regulatory CD4+ T-cell migration is crucial; however, it is poorly understood in physiological and pathological conditions. We found that CD4+ subpopulations displayed characteristic regulator of G-protein signalling (RGS) gene expression profiles. Regulatory T cells express higher levels of RGS1, RGS9 and RGS16 than naive cells. These genes are up-regulated upon cell activation and their level of expression correlates with in vivo cell migration. Using parabiosis, we showed that regulatory T lymphocytes migrate less than naive T cells and that migrant naive T cells express even lower RGS levels than their static counterparts. Our results show an inverse correlation between the capacity to migrate and the levels of RGS1, RGS9 and RGS16 for both naive and regulatory T cells. Taken together, these results suggest a role for RGS molecules in chemokine-induced lymphocyte migration and demonstrate the peculiarity of regulatory T cells in terms of phenotype and migration ability, providing new insights into their function.
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Differential expression of regulator of G‐protein signalling transcripts and in vivo migration of CD4+ naïve and regulatory T cells
Immunology, 2005Co-Authors: Fabien Agenes, Nabil Bosco, Laurent Mascarell, Sabrina Fritah, Rhodri CeredigAbstract:The immune response of T lymphocytes to pathogens is initiated in draining secondary lymphoid organs, and activated cells then migrate to the site of infection. Thus, control of naive and regulatory CD4+ T-cell migration is crucial; however, it is poorly understood in physiological and pathological conditions. We found that CD4+ subpopulations displayed characteristic regulator of G-protein signalling (RGS) gene expression profiles. Regulatory T cells express higher levels of RGS1, RGS9 and RGS16 than naive cells. These genes are up-regulated upon cell activation and their level of expression correlates with in vivo cell migration. Using parabiosis, we showed that regulatory T lymphocytes migrate less than naive T cells and that migrant naive T cells express even lower RGS levels than their static counterparts. Our results show an inverse correlation between the capacity to migrate and the levels of RGS1, RGS9 and RGS16 for both naive and regulatory T cells. Taken together, these results suggest a role for RGS molecules in chemokine-induced lymphocyte migration and demonstrate the peculiarity of regulatory T cells in terms of phenotype and migration ability, providing new insights into their function.
Stephen J Gold - One of the best experts on this subject based on the ideXlab platform.
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differentially regulated expression of endogenous rgs4 and rgs7
Journal of Biological Chemistry, 2004Co-Authors: Andrejs M Krumins, Sheryll A Barker, Kan Yu, Thomas M Wilkie, Stephen J Gold, Chunfa Huang, Roger K. Sunahara, Susanne M MumbyAbstract:Regulators of G protein signaling (RGS proteins) constitute a family of newly appreciated components of G protein-mediated signal transduction. With few exceptions, most information available on mammalian RGS proteins was gained by transfection/overexpression or in vitro experiments, with relatively little known about the endogenous counterparts. Transfection studies, typically of tagged RGS proteins, have been conducted to overcome the low natural abundance of endogenous RGS proteins. Because transfection studies can lead to imprecise or erroneous conclusions, we have developed antibodies of high specificity and sensitivity to focus study on endogenous proteins. Expression of both RGS4 and RGS7 was detected in rat brain tissue and cultured PC12 and AtT-20 cells. Endogenous RGS4 presented as a single 27–28-kDa protein. By contrast, cultured cells transfected with a plasmid encoding RGS4 expressed two observable forms of the protein, apparently due to utilization of distinct sites of initiation of protein synthesis. Subcellular localization of endogenous RGS4 revealed predominant association with membrane fractions, rather than in cytosolic fractions, where most heterologously expressed RGS4 has been found. Endogenous levels of RGS7 exceeded RGS4 by 30 – 40-fold, and studies of cultured cells revealed regulatory differences between the two proteins. We observed that RGS4 mRNA and protein were concomitantly augmented with increased cell density and decreased by exposure of PC12M cells to nerve growth factor, whereas RGS7 was unaffected. Endogenous RGS7 was relatively stable, whereas proteolysis of endogenous RGS4 was a strong determinant of its lower level expression and short halflife. Although we searched without finding evidence for regulation of RGS4 proteolysis, the possibility remains that alterations in the degradation of this protein could provide a means to promptly alter patterns of signal transduction. G proteins transduce signals across the plasma membrane by sequential interactions with cell surface receptors and appropriate second messenger-producing effectors (e.g. enzymes and ion channels). These interactions are modulated by nucleotide-driven conformational changes in the subunits of heterotrimeric G proteins (G). 1 A ligand-bound receptor catalyzes the exchange of GDP for GTP on its cognate G and the dissociation of G from the complex of G protein and subunits (G). These dissociated subunits are competent to modulate the activity of effectors. The duration of G protein-mediated responses are dependent on the intrinsic GTPase rate of G and on extrinsic factors, such as regulators of G protein signaling (RGS proteins).
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Differentially Regulated Expression of Endogenous RGS4 and RGS7
The Journal of biological chemistry, 2003Co-Authors: Andrejs M Krumins, Sheryll A Barker, Thomas M Wilkie, Stephen J Gold, Chunfa Huang, Roger K. Sunahara, Susanne M MumbyAbstract:Regulators of G protein signaling (RGS proteins) constitute a family of newly appreciated components of G protein-mediated signal transduction. With few exceptions, most information available on mammalian RGS proteins was gained by transfection/overexpression or in vitro experiments, with relatively little known about the endogenous counterparts. Transfection studies, typically of tagged RGS proteins, have been conducted to overcome the low natural abundance of endogenous RGS proteins. Because transfection studies can lead to imprecise or erroneous conclusions, we have developed antibodies of high specificity and sensitivity to focus study on endogenous proteins. Expression of both RGS4 and RGS7 was detected in rat brain tissue and cultured PC12 and AtT-20 cells. Endogenous RGS4 presented as a single 27-28-kDa protein. By contrast, cultured cells transfected with a plasmid encoding RGS4 expressed two observable forms of the protein, apparently due to utilization of distinct sites of initiation of protein synthesis. Subcellular localization of endogenous RGS4 revealed predominant association with membrane fractions, rather than in cytosolic fractions, where most heterologously expressed RGS4 has been found. Endogenous levels of RGS7 exceeded RGS4 by 30-40-fold, and studies of cultured cells revealed regulatory differences between the two proteins. We observed that RGS4 mRNA and protein were concomitantly augmented with increased cell density and decreased by exposure of PC12M cells to nerve growth factor, whereas RGS7 was unaffected. Endogenous RGS7 was relatively stable, whereas proteolysis of endogenous RGS4 was a strong determinant of its lower level expression and short half-life. Although we searched without finding evidence for regulation of RGS4 proteolysis, the possibility remains that alterations in the degradation of this protein could provide a means to promptly alter patterns of signal transduction.
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Regulation of RGS proteins by chronic morphine in rat locus coeruleus.
The European journal of neuroscience, 2003Co-Authors: Stephen J Gold, Susanne M Mumby, Cindy M. Pudiak, Bryan W. Potts, Ming-hu Han, Amy E. Herman, George K. Aghajanian, Rong-jian Liu, Eric J NestlerAbstract:The present study explored a possible role for RGS (regulators of G protein signalling) proteins in the long term actions of morphine in the locus coeruleus (LC), a brainstem region implicated in opiate physical dependence and withdrawal. Morphine influences LC neurons through activation of micro -opioid receptors, which, being Gi/o-linked, would be expected to be modulated by RGS proteins. We focused on several RGS subtypes that are known to be expressed in this brain region. Levels of mRNAs encoding RGS2, -3, -4, -5, -7, -8 and -11 are unchanged following chronic morphine, but RGS2 and -4 mRNA levels are increased 2-3-fold 6 h following precipitation of opiate withdrawal. The increases in RGS2 and -4 mRNA peak after 6 h of withdrawal and return to control levels by 24 h. Immunoblot analysis of RGS4 revealed a striking divergence between mRNA and protein responses in LC: protein levels are elevated twofold following chronic morphine and decrease to control values by 6 h of withdrawal. In contrast, levels of RGS7 and -11 proteins, the only other subtypes for which antibodies are available, were not altered by these treatments. Intracellular application of wild-type RGS4, but not a GTPase accelerating-deficient mutant of RGS4, into LC neurons diminished electrophysiological responses to morphine. The observed subtype- and time-specific regulation of RGS4 protein and mRNA, and the diminished morphine-induced currents in the presence of elevated RGS4 protein levels, indicate that morphine induction of RGS4 could contribute to aspects of opiate tolerance and dependence displayed by LC neurons.
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Regulation of regulators of G protein signaling mRNA expression in rat brain by acute and chronic electroconvulsive seizures.
Journal of neurochemistry, 2002Co-Authors: Stephen J Gold, Boris D. Heifets, Cindy M. Pudiak, Bryan W. Potts, Eric J NestlerAbstract:G protein-coupled receptor (GPCR) signaling cascades may be key substrates for the antidepressant effects of chronic electroconvulsive seizures (ECS). To better understand changes in these signaling pathways, alterations in levels of mRNA's encoding regulators of G protein signaling (RGS) protein subtypes-2, -4, -7, -8 and -10 were evaluated in rat brain using northern blotting and in situ hybridization. In prefrontal cortex, RGS2 mRNA levels were increased several-fold 2 h following an acute ECS. Increases in RGS8 mRNA were of lesser magnitude (30%), and no changes were evident for the other RGS subtypes. At 24 h following a chronic ECS regimen, RGS4, -7, and -10 mRNA levels were reduced by 20–30%; only RGS10 was significantly reduced 24 h after acute ECS. Levels of RGS2 mRNA were unchanged 24 h following either acute or chronic ECS. In hippocampus, RGS2 mRNA levels were markedly increased 2 h following acute ECS. More modest increases were seen for RGS4 mRNA expression, whereas levels of the other RGS subtypes were unaltered. At 24 h following chronic ECS, RGS7, -8 and -10 mRNA levels were decreased in the granule cell layer, and RGS7 and -8 mRNA levels were decreased in the pyramidal cell layers. Only RGS8 and -10 mRNA levels were significantly reduced in hippocampus 24 h following an acute ECS. Paralleling neocortex, RGS2 mRNA content was unchanged in hippocampus 24 h following either acute or chronic ECS. In ventromedial hypothalamus, RGS4 mRNA content was increased 24 h following chronic ECS, whereas RGS7 mRNA levels were only increased 24 h following an acute ECS. The increased RGS4 mRNA levels in hypothalamus were significant by 2 h following an acute ECS. These studies demonstrate subtype-, time-, and region-specific regulation of RGS proteins by ECS, adaptations that may contribute to the antidepressant effects of this treatment.
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Cloning and Characterization of RGS9-2: A Striatal-Enriched Alternatively Spliced Product of the RGS9 Gene
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1999Co-Authors: Zia Rahman, Stephen J Gold, Theodore G. Wensel, Marc N. Potenza, Christopher W. Cowan, Eric J NestlerAbstract:Regulators of G-protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for α subunits of heterotrimeric G-proteins. Previous in situ hybridization analysis of mRNAs encoding RGS3–RGS11 revealed region-specific expression patterns in rat brain. RGS9 showed a particularly striking pattern of almost exclusive enrichment in striatum. In a parallel study, RGS9 cDNA, here referred to as RGS9-1, was cloned from retinal cDNA libraries, and the encoded protein was identified as a GAP for transducin (Gα t ) in rod outer segments. In the present study we identify a novel splice variant of RGS9, RGS9-2, cloned from a mouse forebrain cDNA library, which encodes a striatal-specific isoform of the protein. RGS9-2 is 191 amino acids longer than the retinal isoform, has a unique 3′ untranslated region, and is highly enriched in striatum, with much lower levels seen in other brain regions and no expression detectable in retina. Immunohistochemistry showed that RGS9-2 protein is restricted to striatal neuropil and absent in striatal terminal fields. The functional activity of RGS9-2 is supported by the finding that it, but not RGS9-1, dampens the G i/o -coupled μ-opioid receptor response in vitro . Characterization of a bacterial artificial chromosome genomic clone of ∼200 kb indicates that these isoforms represent alternatively spliced mRNAs from a single gene and that the RGS domain, conserved among all known RGS members, is encoded over three distinct exons. The distinct C-terminal domains of RGS9-2 and RGS9-1 presumably contribute to unique regulatory properties in the neural and retinal cells in which these proteins are selectively expressed.
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ontogenetic quinpirole treatment produces long lasting decreases in the expression of RGS9 but increases rgs17 in the striatum nucleus accumbens and frontal cortex
European Journal of Neuroscience, 2007Co-Authors: Amanda M Maple, Joshua P. Parlaman, Marla K. Perna, Gregg D. Stanwood, Russell W. BrownAbstract:Ontogenetic treatment of rats with the dopamine D 2 -like receptor agonist quinpirole produces a significant increase in dopamine D 2 receptor sensitivity that persists throughout the animal's lifetime, a phenomenon known as D 2 priming. The present study was designed to investigate the effects of priming of the D 2 receptor on the expression of three different members of the regulator of G-protein signaling (RGS) family: Rgs4, RGS9 and Rgs17. Male offspring were ontogenetically treated with quinpirole or saline from postnatal days (P)1-21 and raised to adulthood. On ∼P65, animals were given an acute quinipirole injection (0.1 mg/kg) and the number of yawns was recorded for 1 h after the injection. Yawning has been shown to be a behavioural event mediated by the dopamine D 2 /D 3 receptor. Animals ontogenetically treated with quinpirole demonstrated a significant 2.5-fold increase in yawning as compared to controls. Rgs transcripts were analysed through in situ hybridization several weeks later. Rats ontogenetically treated with quinpirole demonstrated a significant decrease in RGS9 expression in the frontal cortex, but a more robust decrease in the striatum and nucleus accumbens as compared to controls. Regarding Rgs17, ontogenetic quinpirole produced a modest but significant increase in expression in the same brain areas. There were no significant differences in Rgs4 expression produced by drug treatment in any of the brain regions analysed. This study demonstrates that ontogenetic quinpirole treatment, which results in priming of the D 2 receptor, results in significant decreases in RGS9, which has been shown to regulate G-protein coupling to D 2 receptors.
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Ontogenetic quinpirole treatment produces long-lasting decreases in the expression of RGS9, but increases Rgs17 in the striatum, nucleus accumbens and frontal cortex.
The European journal of neuroscience, 2007Co-Authors: Amanda M Maple, Joshua P. Parlaman, Marla K. Perna, Gregg D. Stanwood, Russell W. BrownAbstract:Ontogenetic treatment of rats with the dopamine D(2)-like receptor agonist quinpirole produces a significant increase in dopamine D(2) receptor sensitivity that persists throughout the animal's lifetime, a phenomenon known as D(2) priming. The present study was designed to investigate the effects of priming of the D(2) receptor on the expression of three different members of the regulator of G-protein signaling (RGS) family: Rgs4, RGS9 and Rgs17. Male offspring were ontogenetically treated with quinpirole or saline from postnatal days (P)1-21 and raised to adulthood. On approximately P65, animals were given an acute quinipirole injection (0.1 mg/kg) and the number of yawns was recorded for 1 h after the injection. Yawning has been shown to be a behavioural event mediated by the dopamine D(2)/D(3) receptor. Animals ontogenetically treated with quinpirole demonstrated a significant 2.5-fold increase in yawning as compared to controls. Rgs transcripts were analysed through in situ hybridization several weeks later. Rats ontogenetically treated with quinpirole demonstrated a significant decrease in RGS9 expression in the frontal cortex, but a more robust decrease in the striatum and nucleus accumbens as compared to controls. Regarding Rgs17, ontogenetic quinpirole produced a modest but significant increase in expression in the same brain areas. There were no significant differences in Rgs4 expression produced by drug treatment in any of the brain regions analysed. This study demonstrates that ontogenetic quinpirole treatment, which results in priming of the D(2) receptor, results in significant decreases in RGS9, which has been shown to regulate G-protein coupling to D(2) receptors.