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Vladlen Z. Slepak - One of the best experts on this subject based on the ideXlab platform.
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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.
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A Novel Kind of G Protein Heterodimer: The Gβ5-RGS Complex
Receptors & channels, 2003Co-Authors: D. Scott Witherow, Vladlen Z. SlepakAbstract:The fifth member of the G protein g the subunit family, G g 5, has been shown to bind exclusively to a subfamily of regulators of G protein signaling (RGS) including RGS6, RGS7, RGS9, and RGS11. This interaction occurs through a G protein gamma-like (GGL) domain present in members of this RGS subfamily and is the only reported instance in which a G g subunit is not bound to a G n subunit. The G g 5-RGS interaction has been demonstrated both in vitro and in vivo and has been shown to stabilize the dimer against proteolytic degradation. GTPase activating protein (GAP) assays suggest that G g 5-RGS7 acts specifically on G f o, however in cell-based assays it also inhibited G f i- and G f q-mediated signaling. The role of the dimer in signaling and the function of G g 5 moiety within the complex are poorly understood. This review summarizes the information about the assembly and function of G g 5-RGS dimers, as well as their posttranslational modifications and localization.
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Gβ5·RGS7 Inhibits Gαq-mediated Signaling via a Direct Protein-Protein Interaction
The Journal of biological chemistry, 2003Co-Authors: D. Scott Witherow, Qiang Wang, Steven C. Tovey, 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.
Kirill A. Martemyanov - One of the best experts on this subject based on the ideXlab platform.
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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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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.
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Type 5 G Protein β Subunit (Gβ5) Controls the Interaction of Regulator of G Protein Signaling 9 (RGS9) with Membrane Anchors
The Journal of biological chemistry, 2011Co-Authors: Ikuo Masuho, Ekaterina Posokhova, Hideko Wakasugi-masuho, Joseph R. Patton, Kirill A. MartemyanovAbstract:Abstract The R7 family of regulators of G protein signaling (RGS) proteins, comprising RGS6, RGS7, RGS9, and RGS11, regulate neuronal G protein signaling pathways. All members of the R7 RGS form trimeric complexes with the atypical G protein β subunit, Gβ5, and membrane anchor R7BP or R9AP. Association with Gβ5 and membrane anchors has been shown to be critical for maintaining proteolytic stability of the R7 RGS proteins. However, despite its functional importance, the mechanism of how R7 RGS forms complexes with Gβ5 and membrane anchors remains poorly understood. Here, we used protein-protein interaction, co-localization, and protein stability assays to show that association of RGS9 with membrane anchors requires Gβ5. We further establish that the recruitment of R7BP to the complex requires an intact interface between the N-terminal lobe of RGS9 and protein interaction surface of Gβ5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between Gβ5 and N-terminal Dishevelled, EGL-10, Pleckstrin/DEP Helical Extension (DEP/DHEY) domains are differentially involved in R7BP binding and proteolytic stabilization. On the basis of these findings, we conclude that Gβ5 contributes to the formation of the binding site to the membrane anchors and thus is playing a central role in the assembly of the proteolytically stable trimeric complex and its correct localization in the cell.
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R7BP Complexes With RGS9-2 and RGS7 in the Striatum Differentially Control Motor Learning and Locomotor Responses to Cocaine
Neuropsychopharmacology, 2010Co-Authors: Garret R Anderson, Kevin Wickman, Yan Cao, Steve Davidson, Hai V Truong, Marco Pravetoni, Mark J Thomas, Glenn J Giesler, Kirill A. MartemyanovAbstract:In the striatum, signaling through G protein-coupled dopamine receptors mediates motor and reward behavior, and underlies the effects of addictive drugs. The extent of receptor responses is determined by RGS9-2/G β 5 complexes, a striatally enriched regulator that limits the lifetime of activated G proteins. Recent studies suggest that the function of RGS9-2/G β 5 is controlled by the association with an additional subunit, R7BP, making elucidation of its contribution to striatal signaling essential for understanding molecular mechanisms of behaviors mediated by the striatum. In this study, we report that elimination of R7BP in mice results in motor coordination deficits and greater locomotor response to morphine administration, consistent with the essential role of R7BP in maintaining RGS9-2 expression in the striatum. However, in contrast to previously reported observations with RGS9-2 knockouts, mice lacking R7BP do not show higher sensitivity to locomotor-stimulating effects of cocaine. Using a striatum-specific knockdown approach, we show that the sensitivity of motor stimulation to cocaine is instead dependent on RGS7, whose complex formation with R7BP is dictated by RGS9-2 expression. These results indicate that dopamine signaling in the striatum is controlled by concerted interplay between two RGS proteins, RGS7 and RGS9-2, which are balanced by a common subunit, R7BP.
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The R7 RGS Protein Family: Multi-Subunit Regulators of Neuronal G Protein Signaling
Cell Biochemistry and Biophysics, 2009Co-Authors: Garret R Anderson, Ekaterina Posokhova, Kirill A. MartemyanovAbstract:G protein-coupled receptor signaling pathways mediate the transmission of signals from the extracellular environment to the generation of cellular responses, a process that is critically important for neurons and neurotransmitter action. The ability to promptly respond to rapidly changing stimulation requires timely inactivation of G proteins, a process controlled by a family of specialized proteins known as regulators of G protein signaling (RGS). The R7 group of RGS proteins (R7 RGS) has received special attention due to their pivotal roles in the regulation of a range of crucial neuronal processes such as vision, motor control, reward behavior, and nociception in mammals. Four proteins in this group, RGS6, RGS7, RGS9, and RGS11, share a common molecular organization of three modules: (i) the catalytic RGS domain, (ii) a GGL domain that recruits Gβ_5, an outlying member of the G protein beta subunit family, and (iii) a DEP/DHEX domain that mediates interactions with the membrane anchor proteins R7BP and R9AP. As heterotrimeric complexes, R7 RGS proteins not only associate with and regulate a number of G protein signaling pathway components, but have also been found to form complexes with proteins that are not traditionally associated with G protein signaling. This review summarizes our current understanding of the biology of the R7 RGS complexes including their structure/functional organization, protein–protein interactions, and physiological roles.
Kirk M Druey - One of the best experts on this subject based on the ideXlab platform.
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rgs4 overexpression in lung attenuates airway hyperresponsiveness in mice
American Journal of Respiratory Cell and Molecular Biology, 2018Co-Authors: Laura A Madigan, Gordon S Wong, Elizabeth M Gordon, Nariman Balenga, Stewart J Levine, Weisheng Chen, Reynold A Panettieri, Cynthia J Koziolwhite, Kirk M DrueyAbstract:A cardinal feature of asthma is airway hyperresponsiveness (AHR) to spasmogens, many of which activate G protein–coupled receptors (GPCRs) on airway smooth muscle (ASM) cells. Asthma subtypes associated with allergy are characterized by eosinophilic inflammation in the lung due to the type 2 immune response to allergens and proinflammatory mediators that promote AHR. The degree to which intrinsic abnormalities of ASM contribute to this phenotype remains unknown. The regulators of G protein signaling (RGS) proteins are a large group of intracellular proteins that inhibit GPCR signaling pathways. RGS2- and RGS5-deficient mice develop AHR spontaneously. Although RGS4 is upregulated in ASM from patients with severe asthma, the effects of increased RGS4 expression on AHR in vivo are unknown. Here, we examined the impact of forced RGS4 overexpression in lung on AHR using transgenic (Tg) mice. Tg RGS4 was expressed in bronchial epithelium and ASM in vivo, and protein expression in lung was increased at least 4-f...
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R4 Regulator of G Protein Signaling (RGS) Proteins in Inflammation and Immunity
The AAPS Journal, 2016Co-Authors: Eunice C. Chan, Kirk M DrueyAbstract:G protein-coupled receptors (GPCRs) have important functions in both innate and adaptive immunity, with the capacity to bridge interactions between the two arms of the host responses to pathogens through direct recognition of secreted microbial products or the by-products of host cells damaged by pathogen exposure. In the mid-1990s, a large group of intracellular proteins was discovered, the regulator of G protein signaling (RGS) family, whose main, but not exclusive, function appears to be to constrain the intensity and duration of GPCR signaling. The R4/B subfamily—the focus of this review—includes RGS1–5, 8, 13, 16, 18, and 21, which are the smallest RGS proteins in size, with the exception of RGS3. Prominent roles in the trafficking of B and T lymphocytes and macrophages have been described for RGS1, RGS13, and RGS16, while RGS18 appears to control platelet and osteoclast functions. Additional G protein independent functions of RGS13 have been uncovered in gene expression in B lymphocytes and mast cell-mediated allergic reactions. In this review, we discuss potential physiological roles of this RGS protein subfamily, primarily in leukocytes having central roles in immune and inflammatory responses. We also discuss approaches to target RGS proteins therapeutically, which represents a virtually untapped strategy to combat exaggerated immune responses leading to inflammation.
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RGS16 inhibits signalling through the Gα13-Rho axis
Nature cell biology, 2003Co-Authors: Eric N. Johnson, Abdul Waheed, Teresa L. Z. Jones, Tammy M. Seasholtz, Barry Kreutz, Nobuchika Suzuki, Tohru Kozasa, Joan Heller Brown, Kirk M DrueyAbstract:Gα13 stimulates the guanine nucleotide exchange factors (GEFs) for Rho, such as p115Rho-GEF1. Activated Rho induces numerous cellular responses, including actin polymerization, serum response element (SRE)-dependent gene transcription and transformation2. p115Rho-GEF contains a Regulator of G protein Signalling domain (RGS box) that confers GTPase activating protein (GAP) activity towards Gα12 and Gα13 (ref. 3). In contrast, classical RGS proteins (such as RGS16 and RGS4) exhibit RGS domain-dependent GAP activity on Gαi and Gαq, but not Gα12 or Gα13 (ref 4). Here, we show that RGS16 inhibits Gα13-mediated, RhoA-dependent reversal of stellation and SRE activation. The RGS16 amino terminus binds Gα13 directly, resulting in translocation of Gα13 to detergent-resistant membranes (DRMs) and reduced p115Rho-GEF binding. RGS4 does not bind Gα13 or attenuate Gα13-dependent responses, and neither RGS16 nor RGS4 affects Gα12-mediated signalling. These results elucidate a new mechanism whereby a classical RGS protein regulates Gα13-mediated signal transduction independently of the RGS box.
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Palmitoylation Regulates Regulator of G-protein Signaling (RGS) 16 Function II. PALMITOYLATION OF A CYSTEINE RESIDUE IN THE RGS BOX IS CRITICAL FOR RGS16 GTPase ACCELERATING ACTIVITY AND REGULATION OF Gi-COUPLED SIGNALING*
The Journal of biological chemistry, 2003Co-Authors: James L. Osterhout, Richard J. Ward, Graeme Milligan, Abdul Waheed, Abel Hiol, Penelope C. Davey, Lylia Nini, Jiun Wang, Teresa L. Z. Jones, Kirk M DrueyAbstract:Palmitoylation is a reversible post-translational modification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. R., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. Biol. Chem. 278, 19301-19308), we determined that mutation of NH2-terminal cysteine residues in RGS16 (Cys-2 and Cys-12) reduced GTPase accelerating (GAP) activity toward a 5-hydroxytryptamine (5-HT1A)/G alpha o1 receptor fusion protein in cell membranes. NH2-terminal acylation also permitted palmitoylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal palmitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys-98 or RGS4 Cys-95 to alanine reduced GAP activity on the 5-HT1A/G alpha o1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein alpha subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmitoylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT1a/G alpha o1 displayed increased GAP activity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate Gi-coupled signaling in mammalian cells.
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rgs4 and rgs2 bind coatomer and inhibit copi association with golgi membranes and intracellular transport
Molecular Biology of the Cell, 2000Co-Authors: Brandon M Sullivan, Kimberly J Harrisonlavoie, John H Kehrl, Vladimir Marshansky, Dennis A. Ausiello, Dennis Brown, Kirk M DrueyAbstract:COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit β′-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant β′-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of β′-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on Giα. In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi–plasma membrane or intra-Golgi transport.
Javier Garzon - One of the best experts on this subject based on the ideXlab platform.
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Expression of Neural RGS-R7 and Gβ5 Proteins in Response to Acute and Chronic Morphine
Neuropsychopharmacology, 2005Co-Authors: Almudena López-fando, Pilar Sánchez-blázquez, María Rodríguez-muñoz, Javier GarzonAbstract:The R7 subfamily of regulators of G-protein signaling (RGS) proteins (RGS6, RGS7, RGS9-2, and RGS11), and its binding protein G β 5, are found in neural structures of mouse brain. A single intracerebroventricular priming dose of 10 nmol morphine gave rise to acute tolerance to the analgesic effects of successive identical test doses of the opioid. At 2 h after administering the acute opioid, RGS7 mRNA levels in the striatum plus those of RGS9-2 in the striatum and thalamus were increased, whereas RGS9-2 and RGS11 mRNA were reduced in the cortex. Similar but attenuated RGS-R7 mRNA changes persisted 24 h after acute morphine administration. No changes in G β 5 mRNA levels were observed. At 2 days after commencing sustained morphine treatment, the levels of mRNA for RGS7, RGS9-2, RGS11, and G β 5 increased in most of the brain structures studied (striatum, thalamus, periaqueductal gray matter (PAG), and cortex). In these morphine tolerant-dependent mice, the greater changes were found for RGS9-2 in the thalamus (>500%) and PAG (>200%). In post-dependent mice, the increases in RGS-R7 and G β 5 mRNA still persisted in the PAG and striatum at 8 and 16 days after starting the chronic opioid treatment. The raised mRNA levels promoted by chronic, but not by acute, morphine, were accompanied by increases in the encoded proteins. This is probably a result of the costabilization of the RGS-R7 and G β 5 proteins forming heterodimers. Opioid-induced adaptations of RGS-R7 and G β 5 genes may regulate the severity of morphine-induced tolerance/dependence and the duration of the post-dependent period, helping to recover the normal response.
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the r7 subfamily of rgs proteins assists tachyphylaxis and acute tolerance at μ opioid receptors
Neuropsychopharmacology, 2003Co-Authors: Javier Garzon, Almudena Lopezfando, Pilar SanchezblazquezAbstract:Members of the R7 subfamily of regulators of G-protein signaling (RGS) proteins (RGS6, RGS7, RGS9-2, and RGS11) are found in the mouse CNS. The expression of these proteins was effectively reduced in different neural structures by blocking their mRNA with antisense oligodeoxynucleotides (ODNs). This was achieved without noticeable changes in the binding characteristics of labeled β-endorphin to opioid receptors. Knockdown of R7 proteins enhanced the potency of antinociception promoted by morphine and [D-Ala2, N-MePhe4, Gly-ol5]-enkephalin (DAMGO)—both agonists at μ-opioid receptors. The duration of morphine analgesia was greatly increased in RGS9-2 and in RGS11 knockdown mice. The impairment of R7 proteins brought about different changes in the analgesic activity of selective δ agonists. Knockdown of RGS11 reduced [D-Ala2]deltorphin II analgesic effects. Those of RGS6 and RGS9-2 proteins caused [D-Ala2]deltorphin II to produce a smoothened time-course curve—the peak effect blunted and analgesia extended during the declining phase. RGS9-2 impairment also promoted a similar pattern of change for [D-Pen2,5]-enkephalin (DPDPE). RGS7-deficient mice showed an increased response to both [D-Ala2]deltorphin II and DPDPE analgesic effects. A single intracerebroventricular (i.c.v.) ED80 analgesic dose of morphine gave rise to acute tolerance in control mice, but did not promote tolerance in RGS6, RGS7, RGS9-2, or RGS11 knockdown animals. Thus, R7 proteins play a critical role in agonist tachyphylaxis and acute tolerance at μ-opioid receptors, and show differences in their modulation of δ-opioid receptors.
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The R7 Subfamily of RGS Proteins Assists Tachyphylaxis and Acute Tolerance at μ -Opioid Receptors
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology, 2003Co-Authors: Javier Garzon, Almudena López-fando, Pilar Sánchez-blázquezAbstract:Members of the R7 subfamily of regulators of G-protein signaling (RGS) proteins (RGS6, RGS7, RGS9-2, and RGS11) are found in the mouse CNS. The expression of these proteins was effectively reduced in different neural structures by blocking their mRNA with antisense oligodeoxynucleotides (ODNs). This was achieved without noticeable changes in the binding characteristics of labeled beta-endorphin to opioid receptors. Knockdown of R7 proteins enhanced the potency of antinociception promoted by morphine and [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO)-both agonists at mu-opioid receptors. The duration of morphine analgesia was greatly increased in RGS9-2 and in RGS11 knockdown mice. The impairment of R7 proteins brought about different changes in the analgesic activity of selective delta agonists. Knockdown of RGS11 reduced [D-Ala(2)]deltorphin II analgesic effects. Those of RGS6 and RGS9-2 proteins caused [D-Ala(2)]deltorphin II to produce a smoothened time-course curve-the peak effect blunted and analgesia extended during the declining phase. RGS9-2 impairment also promoted a similar pattern of change for [D-Pen(2,5)]-enkephalin (DPDPE). RGS7-deficient mice showed an increased response to both [D-Ala(2)]deltorphin II and DPDPE analgesic effects. A single intracerebroventricular (i.c.v.) ED(80) analgesic dose of morphine gave rise to acute tolerance in control mice, but did not promote tolerance in RGS6, RGS7, RGS9-2, or RGS11 knockdown animals. Thus, R7 proteins play a critical role in agonist tachyphylaxis and acute tolerance at mu-opioid receptors, and show differences in their modulation of delta-opioid receptors.
Rory A. Fisher - One of the best experts on this subject based on the ideXlab platform.
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RGS proteins as critical regulators of motor function and their implications in Parkinson's disease.
Molecular pharmacology, 2020Co-Authors: Katelin E Ahlers-dannen, Mackenzie M. Spicer, Rory A. FisherAbstract:Parkinson's disease (PD) is a devastating, largely non-familial age-related disorder caused by the progressive loss of dopamine (DA) neurons in the SNc. Release of DA from these neurons into the dorsal striatum is crucial for regulating movement and their loss causes PD. Unfortunately, the mechanisms underlying SNc neurodegeneration remain unclear, and currently there is no cure for PD, only symptomatic treatments. Recently, several regulator of G protein signaling (RGS) proteins have emerged as critical modulators of PD pathogenesis and/or motor dysfunction and dyskinesia: RGSs 4, 6, 9 and 10. Striatal RGS4 has been shown to exacerbate motor symptoms of DA loss by suppressing M4 autoreceptor-Gαi/o signaling in striatal cholinergic interneurons. RGS6 and RGS9 are key regulators of D2R-Gαi/o signaling in SNc DA neurons and striatal medium spiny neurons (MSNs), respectively. RGS6, expressed in human and mouse SNc DA neurons, suppresses characteristic PD hallmarks in aged mice, including SNc DA neuron loss, motor deficits, and α-synuclein accumulation. Following DA depletion, RGS9, through its inhibition of MSN D2R signaling, suppresses motor dysfunction induced by L-DOPA or D2R-selective agonists. RGS10 is highly expressed in microglia, the brain's resident immune cells. Within the SNc, RGS10 may promote DA neuron survival through the upregulation of pro-survival genes and inhibition of microglial inflammatory factor expression. Thus, RGSs 4, 6, 9, and 10 are critical modulators of cell signaling pathways that promote SNc DA neuron survival and/or proper motor control. Accordingly, these RGS proteins represent novel therapeutic targets for the treatment of PD pathology. SIGNIFICANCE STATEMENT: Parkinsons (PD), the most common movement disorder, is a progressive neurodegenerative disease characterized by SNc dopamine neuron loss and subsequent motor deficits. Current PD therapies only target disease motor symptomology and are fraught with side effects. Therefore, researchers have begun to explore alternative therapeutic options. Regulator of G protein signaling (RGS) proteins, whether primarily expressed in SNc DA neurons (RGS6), striatal neurons (RGSs 4 and 9), or microglia (RGS10), modulate key signaling pathways important for SNc dopamine neuron survival and/or proper motor control. As such, RGS proteins represent novel therapeutic targets in PD.
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Defective Retinal Depolarizing Bipolar Cells in Regulators of G Protein Signaling (RGS) 7 and 11 Double Null Mice
The Journal of biological chemistry, 2012Co-Authors: Hoon Shim, Rory A. Fisher, Chih-ting Wang, Yen-lin Chen, Viet Q. Chau, Jianqi Yang, A. Rory Mcquiston, Ching-kang ChenAbstract:Two members of the R7 subfamily of regulators of G protein signaling, RGS7 and RGS11, are present at dendritic tips of retinal depolarizing bipolar cells (DBCs). Their involvement in the mGluR6/Gα(o)/TRPM1 pathway that mediates DBC light responses has been implicated. However, previous genetic studies employed an RGS7 mutant mouse that is hypomorphic, and hence the exact role of RGS7 in DBCs remains unclear. We have made a true RGS7-null mouse line with exons 6-8 deleted. The RGS7(-/-) mouse is viable and fertile but smaller in body size. Electroretinogram (ERG) b-wave implicit time in young RGS7(-/-) mice is prolonged at eye opening, but the phenotype disappears at 2 months of age. Expression levels of RGS6 and RGS11 are unchanged in RGS7(-/-) retina, but the Gβ5S level is significantly reduced. By characterizing a complete RGS7 and RGS11 double knock-out (711dKO) mouse line, we found that Gβ5S expression in the retinal outer plexiform layer is eliminated, as is the ERG b-wave. Ultrastructural defects akin to those of Gβ5(-/-) mice are evident in 711dKO mice. In retinas of mice lacking RGS6, RGS7, and RGS11, Gβ5S is undetectable, whereas levels of the photoreceptor-specific Gβ5L remain unchanged. Whereas RGS6 alone sustains a significant amount of Gβ5S expression in retina, the DBC-related defects in Gβ5(-/-) mice are caused solely by a combined loss of RGS7 and RGS11. Our data support the notion that the role of Gβ5 in the retina, and likely in the entire nervous system, is mediated exclusively by R7 RGS proteins.
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A functional polymorphism in RGS6 modulates the risk of bladder cancer.
Cancer research, 2004Co-Authors: David M. Berman, Zhengyu Liu, Yunfei Wang, Qiong Dong, Lorri Anne Burke, Lance A. Liotta, Rory A. FisherAbstract:RGS proteins negatively r egulate heterotrimeric G protein s ignaling. Recent reports have shown that RGS proteins modulate neuronal, cardiovascular, and lymphocytic activity, yet their role in carcinogenesis has not been explored. In an epidemiologic study of 477 bladder cancer patients and 446 matched controls, three noncoding single-nucleotide polymorphisms (SNPs) in RGS2 and RGS6 were each associated with a statistically significant reduction in bladder cancer risk. The risk of bladder cancer was reduced by 74% in those individuals with the variant genotype at all three SNPs (odds ratio, 0.26; 95% confidence interval, 0.09–0.71). When the SNPs were analyzed separately, the RGS6-rs2074647 (C→T) polymorphism conferred the greatest overall reduction in risk of bladder cancer (odds ratio, 0.66; 95% confidence interval, 0.46–0.95). These reductions in risk were more pronounced in ever smokers, suggesting a gene-environment interaction. In transfection assays, the RGS6-rs2074647 (C→T) polymorphism increased the activity of a luciferase-RGS fusion protein by 2.9-fold, suggesting that this SNP is functionally significant. Finally, we demonstrate that RGS2 transcripts and several splice variants of RGS6 are expressed in bladder cancer cells. These data provide the first evidence that RGS proteins may be important modulators of cancer risk and validate RGS6 as a target for further study.
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RGS6 interacts with DMAP1 and DNMT1 and inhibits DMAP1 transcriptional repressor activity.
The Journal of biological chemistry, 2004Co-Authors: Zhengyu Liu, Rory A. FisherAbstract:Abstract RGS6 is a member of a subfamily of mammalian RGS proteins that possess DEP (disheveled, Egl-10, pleckstrin) and GGL (G protein γ subunit-like) domains in addition to the hallmark RGS domain. RGS proteins negatively regulate heterotrimeric G protein signaling by virtue of the GTPase-activating protein activity of their RGS domains. RGS6 exists in multiple splice forms with a long (6L) or short (6S) N terminus, a complete or incomplete GGL domain, in combination with various C-terminal domains. Green fluorescent protein-tagged RGS6L and RGS6S forms exhibit predominantly cytoplasmic and nuclear patterns of distribution in COS-7 cells, respectively, and traffic from these sites to nucleoli in response to stress signaling. We undertook a yeast two-hybrid screen for nuclear RGS6-binding proteins and here identify DMAP1 as an RGS6-interacting protein. DMAP1 is a component of the Dnmt1 complex involved in repression of newly replicated genes. The domains of interaction were mapped to the N-terminal region of the GGL domain of RGS6, a region distinct from its Gβ5 binding region, and the C-terminal domain of DMAP1. Gβ5 and DMAP1 did not compete for each other's interaction with RGS6. Co-immunoprecipitation studies in COS-7 cells showed that RGS6L and RGS6S, but not RGS6LΔ258-293 deletion mutant lacking a DMAP1-binding module, co-immunoprecipitate DMAP1 as well as Dnmt1 in a DMAP1-dependent manner. A recombinant GGL domain of RGS6 precipitated endogenous DMAP1 and Dnmt1 in neuroblastoma cell lysates and endogenous DMAP1 co-immunoprecipitated with RGS6L from mouse brain. Co-expression of DMAP1 with RGS6L promoted nuclear migration of RGS6L and its co-localization with DMAP1, a response not observed with RGS6LΔ258-293. RGS6 inhibited the transcriptional repressor activity of DMAP1. RGS6 is the first member of the RGS protein family shown to interact with proteins involved in transcriptional regulation.