Galpha protein dependent and independent effects of human RGS1 expression in yeast

Regulators of G-protein Signalling (RGS) regulate the functional lifetime of G-Protein Coupled Receptor (GPCR)-activated heterotrimeric G-protein by serving as GTPase Accelerating Proteins (GAPs) for the G(alpha) subunit. A number of mammalian RGSs can functionally replace the yeast RGS containing SST2 gene and inhibit GPCR signalling. Using yeast strains harbouring a G(betagamma)-responsive FUS1-LacZ reporter gene, we demonstrate that heterologously expressed mammalian RGS1 also serves to decrease basal signalling in the absence of agonist. Although this effect was dependent on the expression of a GPA1-encoded functional G(alpha) protein, the GPCR itself was nevertheless not required. Using the GAL1 inducible promoter to express RGS1, we further demonstrate that in addition to serving as a GAP for Gpa1p in yeast, RGS1 is a dosage-dependent inhibitor of growth. This effect is specific to RGS1 since growth is not altered in cells expressing either mammalian RGS2 or RGS5. We further demonstrate that neither of the two yeast G(alpha) proteins is responsible for mediating this growth inhibitory effect of RGS1. Taken together, our results indicate that RGS1 can function in both G-protein-dependent and -independent manners in yeast.     

Mechanism of isoproterenol-induced RGS2 up-regulation in astrocytes

Regulators of G protein signaling (RGSs) are inducibly expressed in response to various stimuli and the up-regulation of RGSs leads to significant decreases in GPCR responsiveness. Isoproterenol, an adrenergic receptor agonist, stimulated RGS2 mRNA in C6 rat astrocytoma cells. The up-regulation of RGS2 mRNA was abrogated by genistein, a protein tyrosine kinase inhibitor (PTK), and by broad-spectrum protein kinase C (PKC) inhibitors (staurosporine and GF109203X). alpha-Adrenergic antagonist (prazocin), beta-adrenergic antagonist (prazocin), and pertussis toxin only partially blocked the RGS2 up-regulation, suggesting that the RGS2 up-regulation is concomitantly mediated by Galphai, Galphas, and Galphaq. It is interesting to note that SB203580, a potent p38 mitogen-activated protein kinase (MAPK) inhibitor, completely inhibited the isoproterenol-mediated RGS2 expression. In addition, isoproterenol also markedly stimulated RGS2 mRNA in rat primary astrocytes, which were sensitive to SB203580 and staurosporine. Therefore, our data suggest that adrenergic receptor-mediated signaling (induced by isoproterenol) may be involved in the regulation of RGS2 expression in astrocytes via activating PTK, PKC, and p38 MAPK.     

Acute stress responsive RGS proteins in the mouse brain

Regulator of G-protein signaling (RGS) proteins play an important role in G-protein coupled receptor (GPCR) signaling and the activity of some GPCRs is modulated via RGS protein levels during stress response. The aim of this study was to investigate changes in RGS protein mRNA expressions in the mouse brain after 2h restraint stress. The mRNA level of 19 RGS proteins was analyzed using real-time PCR in six brain regions, which included the prefrontal cortex, amygdala, hippocampus, hypothalamus, striatum, and pituitary gland, from control and stressed mouse. We found that the level of mRNA of each RGS varied according to brain region and that two to eight RGS proteins exhibited changes in mRNA levels in each brain region by restraint stress. It was also revealed that RGS4 protein amount was consistent with mRNA level, indicating RGS4 protein may have regulatory roles in the acute stress response.     

Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors

Signalling by G proteins is controlled by the regulator of G-protein signalling (RGS) proteins that accelerate the GTPase activity of Galpha subunits and act in a G-protein-coupled receptor (GPCR)-specific manner. The conserved RGS domain accelerates the G subunit GTPase activity, whereas the variable amino-terminal domain participates in GPCR recognition. How receptor recognition is achieved is not known. Here, we show that the scaffold protein spinophilin (SPL), which binds the third intracellular loop (3iL) of several GPCRs, binds the N-terminal domain of RGS2. SPL also binds RGS1, RGS4, RGS16 and GAIP. When expressed in Xenopus laevis oocytes, SPL markedly increased inhibition of alpha-adrenergic receptor (alphaAR) Ca2+ signalling by RGS2. Notably, the constitutively active mutant alphaAR(A293E) (the mutation being in the 3iL) did not bind SPL and was relatively resistant to inhibition by RGS2. Use of betaAR-alphaAR chimaeras identified the 288REKKAA293 sequence as essential for the binding of SPL and inhibition of Ca2+ signalling by RGS2. Furthermore, alphaAR-evoked Ca2+ signalling is less sensitive to inhibition by SPL in rgs2-/- cells and less sensitive to inhibition by RGS2 in spl-/- cells. These findings provide a general mechanism by which RGS proteins recognize GPCRs to confer signalling specificity.     

Regulator of G Protein Signaling 2 (RGS2) and RGS4 Form Distinct G Protein-Dependent Complexes with Protease Activated-Receptor 1 (PAR1) in Live Cells

Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor (GPCR) that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to G_i, G_q and G₁₂ to activate linked signaling pathways. Regulators of G protein signaling (RGS) proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET), we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven) and either RGS2-Luciferase (RGS2-Luc) or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gα_q/11, and PAR1-RGS4 by Gα_o, but not by other Gα subunits. Gα_q/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR) stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A) that eliminates PAR1-G_q/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.     

RGS mRNA expression in rat striatum: modulation by dopamine receptors and effects of repeated amphetamine administration

Single injections of cocaine, amphetamine, or methamphetamine increased RGS2 mRNA levels in rat striatum by two- to fourfold. The D1 dopamine receptor-selective antagonist SCH-23390 had no effect by itself but strongly attenuated RGS2 mRNA induction by amphetamine. In contrast, the D2 receptor-selective antagonist raclopride induced RGS2 mRNA when administered alone and greatly enhanced stimulation by amphetamine. To examine the effects of repeated amphetamine on RGS2 expression, rats were treated with escalating doses of amphetamine (1.0-7.5 mg/kg) for 4 days, followed by 8 days of multiple daily injections (7.5 mg/kg/2 h x four injections). Twenty hours after the last injection the animals were challenged with amphetamine (7.5 mg/kg) or vehicle and killed 1 h later. In drug-naive animals, acute amphetamine induced the expression of RGS2, 3, and 5 and the immediate early genes c-fos and zif/268. RGS4 mRNA levels were not affected. Prior repeated treatment with amphetamine strongly suppressed induction of immediate early genes and RGS5 to a challenge dose of amphetamine. In sharp contrast, prior exposure to amphetamine did not reduce the induction of RGS2 and RGS3 mRNAs to a challenge dose of amphetamine, indicating that control of these genes is resistant to amphetamine-induced tolerance. These data establish a role for dopamine receptors in the regulation of RGS2 expression and suggest that RGS2 and 3 might mediate some aspects of amphetamine-induced tolerance.     

Opioid-induced down-regulation of RGS4: role of ubiquitination and implications for receptor cross-talk

Regulator of G protein signaling protein 4 (RGS4) acts as a GTPase accelerating protein to modulate μ- and δ- opioid receptor (MOR and DOR, respectively) signaling. In turn, exposure to MOR agonists leads to changes in RGS4 at the mRNA and/or protein level. Here we have used human neuroblastoma SH-SY5Y cells that endogenously express MOR, DOR, and RGS4 to study opioid-mediated down-regulation of RGS4. Overnight treatment of SH-SY5Y cells with the MOR agonist DAMGO or the DOR agonist DPDPE decreased RGS4 protein by ～60% accompanied by a profound loss of opioid receptors but with no change in RGS4 mRNA. The decrease in RGS4 protein was prevented by the pretreatment with pertussis toxin or the opioid antagonist naloxone. The agonist-induced down-regulation of RGS4 proteins was completely blocked by treatment with the proteasome inhibitors MG132 or lactacystin or high concentrations of leupeptin, indicating involvement of ubiquitin-proteasome and lysosomal degradation. Polyubiquitinated RGS4 protein was observed in the presence of MG132 or the specific proteasome inhibitor lactacystin and promoted by opioid agonist. The loss of opioid receptors was not prevented by MG132, demonstrating a different degradation pathway. RGS4 is a GTPase accelerating protein for both Gα(i/o) and Gα(q) proteins. After overnight treatment with DAMGO to reduce RGS4 protein, signaling at the Gα(i/o)-coupled DOR and the Gα(q)-coupled M(3) muscarinic receptor (M(3)R) was increased but not signaling of the α(2) adrenergic receptor or bradykinin BK(2) receptor, suggesting the development of cross-talk between the DOR and M(3)R involving RGS4.     

Inhibition of somatostatin receptor 5-signaling by mammalian regulators of G-protein signaling (RGS) in yeast

Regulators of G-protein signaling (RGSs) are negative regulators of G-protein coupled receptor (GPCR)-mediated signaling that function to limit the lifetime of receptor-activated G(alpha)-proteins. Here we show that four mammalian RGSs differentially inhibit the activation of a FUS1--LacZ reporter gene by the STE2 encoded GPCR in yeast. In order to examine the role of the GPCR in modulating RGS function, we functionally expressed the human somatostatin receptor 5 (SST(5)) in yeast. In the absence of RGSs, FUS1--LacZ activation in response to somatostatin increased in a dose-dependent manner in cells expressing SST(5). In contrast to the results obtained with Ste2p, all RGSs completely inhibited SST(5)-mediated signaling even at concentrations of agonist as high as 10(minus sign5) M. The ability of RGSs to inhibit SST(5) signaling was further assessed in cells expressing modified Gpa1 proteins. Even though SST(5)-mediated FUS1--LacZ activation was 5-fold more efficient with a Gpa1p/G(i3alpha) chimera, response to somatostatin was completely abolished by all four RGSs. Furthermore, we demonstrate that RGS1, RGS2 and RGS5 have reduced ability to inhibit SST(5)-mediated activation of the RGS-resistant Gpa1p(Gly302Ser) mutant suggesting that the ability to interact with the G(alpha)-protein is required for the inhibition of signaling. Taken together, our results indicate that RGSs serve as better GAPs for Gpa1p when activated by SST(5) than when this G-protein is activated by Ste2p.     

GIPC recruits GAIP (RGS19) to attenuate dopamine D2 receptor signaling

Pleiotropic G proteins are essential for the action of hormones and neurotransmitters and are activated by stimulation of G protein-coupled receptors (GPCR), which initiates heterotrimer dissociation of the G protein, exchange of GDP for GTP on its Galpha subunit and activation of effector proteins. Regulator of G protein signaling (RGS) proteins regulate this cascade and can be recruited to the membrane upon GPCR activation. Direct functional interaction between RGS and GPCR has been hypothesized. We show that recruitment of GAIP (RGS19) by the dopamine D2 receptor (D2R), a GPCR, required the scaffold protein GIPC (GAIP-interacting protein, C terminus) and that all three were coexpressed in neurons and neuroendocrine cells. Dynamic translocation of GAIP to the plasma membrane and coassembly in a protein complex in which GIPC was a required component was dictated by D2R activation and physical interactions. In addition, two different D2R-mediated responses were regulated by the GTPase activity of GAIP at the level of the G protein coupling in a GIPC-dependent manner. Since GIPC exclusively interacted with GAIP and selectively with subsets of GPCR, this mechanism may serve to sort GPCR signaling in cells that usually express a large repertoire of GPCRs, G proteins, and RGS.     

RGS2 inhibits β-adrenergic receptor-induced cardiomyocyte hypertrophy

The chronic stimulation of certain G protein-coupled receptors promotes cardiomyocyte hypertrophy and thus plays a pivotal role in the development of human heart failure. The beta-adrenergic receptors (β-AR) are unique among these in that they signal via Gs, whereas others, such as the alpha1-adrenergic (α1-AR) and endothelin-1 (ET-1) receptors, predominantly act through Gq. In this study, we investigated the potential role of regulator of G protein signalling 2 (RGS2) in modulating the hypertrophic effects of the β-AR agonist isoproterenol (ISO) in rat neonatal ventricular cardiomyocytes. We found that ISO-induced hypertrophy in rat neonatal ventricular myocytes was accompanied by the selective upregulation of RGS2 mRNA, with little or no change in RGS1, RGS3, RGS4 or RGS5. The adenylyl cyclase activator forskolin had a similar effect suggesting that it was mediated through cAMP production. To study the role of RGS2 upregulation in β-AR-dependent hypertrophy, cardiomyocytes were infected with adenovirus encoding RGS2 and assayed for cell growth, markers of hypertrophy, and β-AR signalling. ISO-induced increases in cell surface area were virtually eliminated by the overexpression of RGS2, as were increases in α-skeletal actin and atrial natriuretic peptide. RGS2 overexpression also significantly attenuated ISO-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and Akt activation, which may account for, or contribute to, its observed antihypertrophic effects. In contrast, RGS2 overexpression significantly activated JNK MAP kinase, while decreasing the potency but not the maximal effect of ISO on cAMP accumulation. In conclusion, the present results suggest that RGS2 negatively regulates hypertrophy induced by β-AR activation and thus may play a protective role in cardiac hypertrophy.     

Structure-based design, synthesis, and pharmacologic evaluation of peptide RGS4 inhibitors.

Regulators of G-protein signaling (RGS) proteins form a multifunctional signaling family. A key role of RGS proteins is binding to the G-protein Galpha-subunit and acting as GTPase-activating proteins (GAPs), thereby rapidly terminating G protein-coupled receptor (GPCR) signaling. Using the published RGS4-Gialpha1 X-ray structure we have designed and synthesized a series of cyclic peptides, modeled on the Gialpha Switch I region, that inhibit RGS4 GAP activity. These compounds should prove useful for elucidating RGS-mediated activity and serve as a starting point for the development of a novel class of therapeutic agent.     

Interferon beta-1a counteracts effects of activation on the expression of G-protein-coupled receptor kinases 2 and 3, beta-arrestin-1, and regulators of G-protein signalling 2 and 16 in human mononuclear leukocytes

Activation regulates the responsiveness of G-protein-coupled receptors (GPCRs) on T cells, and modifications in the activity of GPCRs characterize lymphocytes from some immune disorders such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Some lines of evidence suggest that such an effect is connected with the altered expression of some GPCRs regulatory proteins. Herein we demonstrate that phitoemagglutinin (PHA)-induced activation leads to differential expression of G-protein-coupled receptor kinase (GRK) 2, GRK3, beta-arrestin-1, regulators of G-protein signalling (RGS) 2, and RGS16 and decreases responsiveness of mononuclear leukocytes (MNL) to the beta-adrenergic agonist isoproterenol. Interferon beta-1a (IFN beta-1a), which is known to ameliorate the course of MS, counteracts the activation-induced effects on the expression of these GPCR regulatory proteins in MNL. Furthermore, IFN beta-1a quenches the effects of PHA on the isoproterenol-induced accumulation of cyclic AMP (cAMP). We suggest that regulation of GPCRs responsiveness may be a relevant property of IFN beta-1a in MS.     

The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Galpha-interaction face

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thus crucial to the timing of G protein-coupled receptor (GPCR) signaling. Small molecule inhibition of RGS proteins is an attractive therapeutic approach to diseases involving dysregulated GPCR signaling. Methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986) was reported as a selective RGS4 inhibitor, but with an unknown mechanism of action [D.L. Roman, J.N. Talbot, R.A. Roof, R.K. Sunahara, J.R. Traynor, R.R. Neubig, Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay, Mol. Pharmacol. 71 (2007) 169-75]. Here, we describe its mechanism of action as covalent modification of RGS4. Mutant RGS4 proteins devoid of surface-exposed cysteine residues were characterized using surface plasmon resonance and FRET assays of Galpha binding, as well as single-turnover GTP hydrolysis assays of RGS4 GAP activity, demonstrating that cysteine-132 within RGS4 is required for sensitivity to CCG-4986 inhibition. Sensitivity to CCG-4986 can be engendered within RGS8 by replacing the wildtype residue found in this position to cysteine. Mass spectrometry analysis identified a 153-Dalton fragment of CCG-4986 as being covalently attached to the surface-exposed cysteines of the RGS4 RGS domain. We conclude that the mechanism of action of the RGS protein inhibitor CCG-4986 is via covalent modification of Cys-132 of RGS4, likely causing steric hindrance with the all-helical domain of the Galpha substrate.     

Modulation of subfamily B/R4 RGS protein function by 14-3-3 proteins

Regulator of G protein signalling (RGS) proteins are primarily known for their ability to act as GTPase activating proteins (GAPs) and thus attenuate G protein function within G protein-coupled receptor (GPCR) signalling pathways. However, RGS proteins have been found to interact with additional binding partners, and this has introduced more complexity to our understanding of their potential role in vivo. Here, we identify a novel interaction between RGS proteins (RGS4, RGS5, RGS16) and the multifunctional protein 14-3-3. Two isoforms, 14-3-3β and 14-3-3ε, directly interact with all three purified RGS proteins and data from in vitro steady state GTP hydrolysis assays show that 14-3-3 inhibits the GTPase activity of RGS4 and RGS16, but has limited effects on RGS5 under comparable conditions. Moreover in a competitive pull-down experiment, 14-3-3ε competes with Go for RGS4, but not for RGS5. This mechanism is further reinforced in living cells, where 14-3-3ε sequesters RGS4 in the cytoplasm and impedes its recruitment to the plasma membrane by G protein. Thus, 14-3-3 might act as a molecular chelator, preventing RGS proteins from interacting with G, and ultimately prolonging the signal transduction pathway. In conclusion, our findings suggest that 14-3-3 proteins may indirectly promote GPCR signalling via their inhibitory effects on RGS GAP function.     

Acute amphetamine down-regulates RGS4 mRNA and protein expression in rat forebrain: distinct roles of D1 and D2 dopamine receptors

Administration of psychostimulants modulates mRNA of several regulators of guanine nucleotide-binding protein signaling (RGSs) proteins in the brain. In the present study, the regulation of amphetamine-induced decrease of RGS4 expression in the rat forebrain was evaluated. RGS4 mRNA was reduced by amphetamine in an inverse, dose-dependent manner. The lowest dose (2.5 mg/kg) decreased RGS4 mRNA in caudate putamen for up to 6 h after injection whereas the decrease in several frontal cortical areas was detected at 3 h only. Analysis of RGS4 immunoreactivity by western blotting revealed a decrease 3 h after amphetamine solely in the caudate putamen. Systemic administration of D(1) (SCH23390) or D(2) (eticlopride) receptor antagonists blocked amphetamine-induced locomotion but amphetamine augmented both the SCH23390-induced increase and the eticlopride-induced decrease in RGS4 mRNA in the caudate putamen. Further, the down-regulation of RGS4 immunoreactivity by eticlopride was robust whereas the effect of SCH23390 was blunted as compared with its effect on mRNA. These data suggest that, by decreasing RGS4 expression in the caudate putamen via D(1) receptors, acute amphetamine could disinhibit RGS4-sensitive guanine nucleotide-binding protein alpha-subunit i- and/or q-coupled signaling pathways and favor mechanisms that counterbalance D(1) receptor stimulation.     

RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling

RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate Galpha signaling events. In live cells, members of the B/R4 subfamily of RGS proteins selectively modulate G protein signaling depending on the associated receptor (GPCR). Here we examine whether GPCRs selectively recruit RGS proteins to modulate linked G protein signaling. We report the novel finding that RGS2 binds directly to the third intracellular (i3) loop of the G(q/11)-coupled M1 muscarinic cholinergic receptor (M1 mAChR; M1i3). This interaction is selective because closely related RGS16 does not bind M1i3, and neither RGS2 nor RGS16 binds to the G(i/o)-coupled M2i3 loop. When expressed in cells, RGS2 and M1 mAChR co-localize to the plasma membrane whereas RGS16 does not. The N-terminal region of RGS2 is both necessary and sufficient for binding to M1i3, and RGS2 forms a stable heterotrimeric complex with both activated G(q)alpha and M1i3. RGS2 potently inhibits M1 mAChR-mediated phosphoinositide hydrolysis in cell membranes by acting as an effector antagonist. Deletion of the N terminus abolishes this effector antagonist activity of RGS2 but not its GTPase-activating protein activity toward G(11)alpha in membranes. These findings predict a model where the i3 loops of GPCRs selectively recruit specific RGS protein(s) via their N termini to regulate the linked G protein. Consistent with this model, we find that the i3 loops of the mAChR subtypes (M1-M5) exhibit differential profiles for binding distinct B/R4 RGS family members, indicating that this novel mechanism for GPCR modulation of RGS signaling may generally extend to other receptors and RGS proteins.     

RGS9 modulates dopamine signaling in the basal ganglia

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.     

Differential effects of RGS proteins on G alpha(q) and G alpha(11) activity

Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human G alpha(q) and G alpha(11) spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of G alpha-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both G alpha(q) and G alpha(11) but, in contrast, RGS5 and RGS16 were much less effective against G alpha(11) than G alpha(q). Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active G alpha(q)QL/G alpha(11)QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-G alpha specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of G alpha(q)/G alpha(11) enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both G alpha subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential G alpha activity, further highlighting that RGS proteins can discriminate between two very closely related G alpha subunits.     

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

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.     

Mammalian RGS proteins: multifunctional regulators of cellular signalling

Regulators of G-protein signalling (RGS) proteins are a large and diverse family initially identified as GTPase activating proteins (GAPs) of heterotrimeric G-protein Galpha-subunits. At least some can also influence Galpha activity through either effector antagonism or by acting as guanine nucleotide dissociation inhibitors (GDIs). As our understanding of RGS protein structure and function has developed, so has the realisation that they play roles beyond G-protein regulation. Such diversity of function is enabled by the variety of RGS protein structure and their ability to interact with other cellular molecules including phospholipids, receptors, effectors and scaffolds. The activity, sub-cellular distribution and expression levels of RGS proteins are dynamically regulated, providing a layer of complexity that has yet to be fully elucidated.