RGS17/RGSZ2, a novel regulator of Gi/o, Gz, and Gq signaling

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.     

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.     

G protein selectivity is a determinant of RGS2 function

RGS (regulator of G protein signaling) proteins are GTPase-activating proteins that attenuate signaling by heterotrimeric G proteins. Whether the biological functions of RGS proteins are governed by quantitative differences in GTPase-activating protein activity toward various classes of Galpha subunits and how G protein selectivity is achieved by differences in RGS protein structure are largely unknown. Here we provide evidence indicating that the function of RGS2 is determined in part by differences in potency toward G(q) versus G(i) family members. RGS2 was 5-fold more potent than RGS4 as an inhibitor of G(q)-stimulated phosphoinositide hydrolysis in vivo. In contrast, RGS4 was 8-fold more potent than RGS2 as an inhibitor of G(i)-mediated signaling. RGS2 mutants were identified that display increased potency toward G(i) family members without affecting potency toward G(q). These mutations and the structure of RGS4-G(i)alpha(1) complexes suggest that RGS2-G(i)alpha interaction is unfavorable in part because of the geometry of the switch I binding pocket of RGS2 and a potential interaction between the alpha8-alpha9 loop of RGS2 and alphaA of G(i) class alpha subunits. The results suggest that the function of RGS2 relative to other RGS family members is governed in part by quantitative differences in activity toward different classes of Galpha subunits.     

Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins

The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (G(q), G(s), and G(i)) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic alphaT3-1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca(2+) mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the G(i)-coupled m2 muscarinic receptor interacted with a chimeric G(s) protein (G(s)i5) containing the C-terminal five amino acids of Galpha(i2), the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of alphaT3-1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive element-driven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH-elicited rises in [Ca(2+)](i). In alphaT3-1 and CHO#3 cell membranes, addition of [alpha-(32)P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of Galpha(q/11) but not of Galpha(i), Galpha(s) or Galpha(12/13) proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the G(q/11) family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor.     

Polarity exchange at the interface of regulators of G protein signaling with G protein alpha-subunits

RGS proteins are GTPase-activating proteins (GAPs) for G protein alpha-subunits. This GAP activity is mediated by the interaction of conserved residues on regulator of G protein signaling (RGS) proteins and Galpha-subunits. We mutated the important contact sites Glu-89, Asn-90, and Asn-130 in RGS16 to lysine, aspartate, and alanine, respectively. The interaction of RGS16 and its mutants with Galpha(t) and Galpha(i1) was studied. The GAP activities of RGS16N90D and RGS16N130A were strongly attenuated. RGS16E89K increased GTP hydrolysis of Galpha(i1) by a similar extent, but with an about 100-fold reduced affinity compared with non-mutated RGS16. As Glu-89 in RGS16 is interacting with Lys-210 in Galpha(i1), this lysine was changed to glutamate for compensation. Galpha(i1)K210E was insensitive to RGS16 but interacted with RGS16E89K. In rat uterine smooth muscle cells, wild type RGS16 abolished G(i)-mediated alpha(2)-adrenoreceptor signaling, whereas RGS16E89K was without effect. Both Galpha(i1) and Galpha(i1)K210E mimicked the effect of alpha(2)-adrenoreceptor stimulation. Galpha(i1)K210E was sensitive to RGS16E89K and 10-fold more potent than Galpha(i1). Analogous mutants of Galpha(q) (Galpha(q)K215E) and RGS4 (RGS4E87K) were created and studied in COS-7 cells. The activity of wild type Galpha(q) was counteracted by wild type RGS4 but not by RGS4E87K. The activity of Galpha(q)K215E was inhibited by RGS4E87K, whereas non-mutated RGS4 was ineffective. We conclude that mutation of a conserved lysine residue to glutamate in Galpha(i) and Galpha(q) family members renders these proteins insensitive to wild type RGS proteins. Nevertheless, they are sensitive to glutamate to lysine mutants of RGS proteins. Such mutant pairs will be helpful tools in analyzing Galpha-RGS specificities in living cells.     

The M3 muscarinic acetylcholine receptor expressed in HEK-293 cells signals to phospholipase D via G12 but not Gq-type G proteins: regulators of G proteins as tools to dissect pertussis toxin-resistant G proteins in receptor-effector coupling

The M(3) muscarinic acetylcholine receptor (mAChR) expressed in HEK-293 cells couples to G(q) and G(12) proteins and stimulates phospholipase C (PLC) and phospholipase D (PLD) in a pertussis toxin-insensitive manner. To determine the type of G protein mediating M(3) mAChR-PLD coupling in comparison to M(3) mAChR-PLC coupling, we expressed various Galpha proteins and regulators of the G protein signaling (RGS), which act as GTPase-activating proteins for G(q)- or G(12)-type G proteins. PLD stimulation by the M(3) mAChR was enhanced by the overexpression of Galpha(12) and Galpha(13), whereas the overexpression of Galpha(q) strongly increased PLC activity without affecting PLD activity. Expression of the RGS homology domain of Lsc, which acts specifically on Galpha(12) and Galpha(13), blunted the M(3) mAChR-induced PLD stimulation without affecting PLC stimulation. On the other hand, overexpression of RGS4, which acts on Galpha(q)- but not Galpha(12)-type G proteins, suppressed the M(3) mAChR-induced PLC stimulation without altering PLD stimulation. We conclude that the M(3) mAChR in HEK-293 cells apparently signals to PLD via G(12)- but not G(q)-type G proteins and that G protein subtype-selective RGS proteins can be used as powerful tools to dissect the pertussis toxin-resistant G proteins and their role in receptor-effector coupling.     

Potent activation of RhoA by Galpha q and Gq-coupled receptors

Heterotrimeric G proteins of the G(i), G(s), and G(q) family control a wide array of physiological functions primarily by regulating the activity of key intracellular second messenger-generating systems. alpha subunits of the G(12) family, Galpha(12) and Galpha(13), however, can promote cellular responses that are independent of conventional second messengers but that result from the activation of small GTP-binding proteins of the Rho family and their downstream targets. These findings led to the identification of a novel family of guanine-nucleotide exchange factors (GEFs) that provides a direct link between Galpha(12/13) and Rho stimulation. Recent observations suggest that many cellular responses elicited by Galpha(q) and its coupled receptors also require the functional activity of Rho. However, available evidence suggests that Galpha(q) may act on pathways downstream from Rho rather than by promoting Rho activation. These seemingly conflicting observations and the recent development of sensitive assays to assess the in vivo levels of active Rho prompted us to ask whether Galpha(q) and its coupled receptors can stimulate endogenous Rho. Here we show that the expression of activated forms of Galpha(q) and the stimulation of G(q)-coupled receptors or chimeric Galpha(q) molecules that respond to G(i)-linked receptors can promote a robust activation of endogenous Rho in HEK-293T cells. Interestingly, this response was not prevented by molecules interfering with the ability of Galpha(13) to stimulate its linked RhoGEFs, together suggesting the existence of a novel molecular mechanism by which Galpha(q) and the large family of G(q)-coupled receptors can regulate the activity of Rho and its downstream signaling pathways.     

Regulators of G protein signaling 6 and 7. Purification of complexes with gbeta5 and assessment of their effects on g protein-mediated signaling pathways.

Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein gamma subunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein beta subunits, and certain biochemical properties of RGS6/beta5 and RGS7/beta5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the Gbeta5 subunit (but not Gbetas 1-4) are found in the cytosol. When purified, these complexes are similar to RGS11/beta5 in that they act as GTPase-activating proteins specifically toward Galpha(o). Unlike conventional G(betagamma) complexes, RGS6/beta5 and RGS7/beta5 do not form heterotrimeric complexes with either Galpha(o)-GDP or Galpha(q)-GDP. Neither RGS6/beta5 nor RGS7/beta5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-beta1 or -beta2. However, the RGS/beta5 complexes inhibited beta(1)gamma(2)-mediated activation of phospholipase C-beta2. RGS/beta5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to G(i) and G(o) by binding to phospholipase C and stimulating the GTPase activity of Galpha(o).     

Mechanisms for reversible regulation between G13 and Rho exchange factors.

The heterotrimeric G proteins, G(12) and G(13), mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Galpha(13) of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Galpha(12) and Galpha(13) is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for G(i) and G(q). Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted "RGS box" region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Galpha(13) and activation of RhoA exchange activity by Galpha(13). In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Galpha(13) but was not stimulated by the alpha subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Galpha(13) also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Galpha(13) did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Galpha(13). These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Galpha(13) to both its RGS and DH domains.     

RGS proteins: more than just GAPs for heterotrimeric G proteins

Members of the newly described RGS family of proteins have a common RGS domain that contains GTPase-activating activity for many Galpha subunits of heterotrimeric G proteins. Their ability to dampen signalling via Galphai-, Galphaq- and Galpha12/13-coupled pathways makes them crucial players in mediating the multitude of cellular processes controlled by heterotrimeric G proteins. Some RGS proteins also contain additional motifs that link them to other signalling networks, where they constitute effector-type molecules. This review summarizes recent findings on RGS proteins, especially those that implicate RGS proteins in more than just enhancing the GTPase activity of their Galpha subunit targets.     

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.     

Up-regulation of endogenous RGS2 mediates cross-desensitization between Gs and Gq signaling in osteoblasts

Regulator of G protein signaling (RGS) proteins limit G protein signals. In this study, we investigated the role of RGS2 in the control of G protein signaling cascades in osteoblasts, the cells responsible for bone formation. Expression of RGS2 was up-regulated in primary cultures of mouse calvarial osteoblasts by parathyroid hormone-related peptide (PTHrP)-(1-34), which stimulates G(s) signaling. RGS2 was also up-regulated by extracellular ATP, which selectively activates G(q), as well as by forskolin and phorbol myristate acetate, which activate targets downstream of G(s) and G(q), respectively. To assess the role of endogenous RGS2, we characterized G(s) and G(q) signaling in osteoblasts derived from wild type and rgs2(-/-) mice. Under control conditions, nucleotide-stimulated calcium release, endothelin-stimulated accumulation of inositol phosphates, and PTHrP-stimulated cAMP accumulation were equivalent in osteoblasts isolated from wild type and rgs2(-/-) mice. Thus, basal levels of endogenous RGS2 do not appear to regulate G(s) or G(q) signaling in osteoblasts. Interestingly, forskolin treatment of wild type but not rgs2(-/-) osteoblasts suppressed both endothelin-stimulated accumulation of inositol phosphates and nucleotide-stimulated calcium release, indicating that up-regulation of RGS2 by G(s) signaling desensitizes G(q) signals. Furthermore, pretreatment with ATP suppressed PTHrP-dependent cAMP accumulation in wild type but not rgs2(-/-) osteoblasts, implying that up-regulation of RGS2 by G(q) signaling desensitizes G(s) signals. Our findings demonstrate that endogenously expressed RGS2 can limit G(s) signaling. Moreover, up-regulation of RGS2 contributes to cross-desensitization of G(s)- and G(q)-coupled signals.     

Ca2+/Calmodulin reverses phosphatidylinositol 3,4, 5-trisphosphate-dependent inhibition of regulators of G protein-signaling GTPase-activating protein activity

Regulators of G protein signaling (RGS proteins) are GTPase-activating proteins (GAPs) for G(i) and/or G(q) class G protein alpha subunits. RGS GAP activity is inhibited by phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) but not by other lipid phosphoinositides or diacylglycerol. Both the negatively charged head group and long chain fatty acids (C16) are required for binding and inhibition of GAP activity. Amino acid substitutions in helix 5 within the RGS domain of RGS4 reduce binding affinity and inhibition by PIP(3) but do not affect inhibition of GAP activity by palmitoylation. Conversely, the GAP activity of a palmitoylation-resistant mutant RGS4 is inhibited by PIP(3). Calmodulin binds all RGS proteins we tested in a Ca(2+)-dependent manner but does not directly affect GAP activity. Indeed, Ca(2+)/calmodulin binds a complex of RGS4 and a transition state analog of Galpha(i1)-GDP-AlF(4)(-). Ca(2+)/calmodulin reverses PIP(3)-mediated but not palmitoylation-mediated inhibition of GAP activity. Ca(2+)/calmodulin competition with PIP(3) may provide an intracellular mechanism for feedback regulation of Ca(2+) signaling evoked by G protein-coupled agonists.     

Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regulation of Galpha 11 signaling

RGS proteins (regulators of G protein signaling) attenuate heterotrimeric G protein signaling by functioning as both GTPase-activating proteins (GAPs) and inhibitors of G protein/effector interaction. RGS2 has been shown to regulate Galpha(q)-mediated inositol lipid signaling. Although purified RGS2 blocks PLC-beta activation by the nonhydrolyzable GTP analog guanosine 5'-O-thiophosphate (GTPgammaS), its capacity to regulate inositol lipid signaling under conditions where GTPase-promoted hydrolysis of GTP is operative has not been fully explored. Utilizing the turkey erythrocyte membrane model of inositol lipid signaling, we investigated regulation by RGS2 of both GTP and GTPgammaS-stimulated Galpha(11) signaling. Different inhibitory potencies of RGS2 were observed under conditions assessing its activity as a GAP versus as an effector antagonist; i.e. RGS2 was a 10-20-fold more potent inhibitor of aluminum fluoride and GTP-stimulated PLC-betat activity than of GTPgammaS-promoted PLC-betat activity. We also examined whether RGS2 was regulated by downstream components of the inositol lipid signaling pathway. RGS2 was phosphorylated by PKC in vitro to a stoichiometry of approximately unity by both a mixture of PKC isozymes and individual calcium and phospholipid-dependent PKC isoforms. Moreover, RGS2 was phosphorylated in intact COS7 cells in response to PKC activation by 4beta-phorbol 12beta-myristate 13alpha-acetate and, to a lesser extent, by the P2Y(2) receptor agonist UTP. In vitro phosphorylation of RGS2 by PKC decreased its capacity to attenuate both GTP and GTPgammaS-stimulated PLC-betat activation, with the extent of attenuation correlating with the level of RGS2 phosphorylation. A phosphorylation-dependent inhibition of RGS2 GAP activity was also observed in proteoliposomes reconstituted with purified P2Y(1) receptor and Galpha(q)betagamma. These results identify for the first time a phosphorylation-induced change in the activity of an RGS protein and suggest a mechanism for potentiation of inositol lipid signaling by PKC.     

Endogenous RGS proteins modulate SA and AV nodal functions in isolated heart: implications for sick sinus syndrome and AV block

G protein-coupled receptors play a pivotal role in regulating cardiac automaticity. Their function is controlled by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for Galpha subunits to suppress Galpha(i) and Galpha(q) signaling. Using knock-in mice in which Galpha(i2)-RGS binding and negative regulation are disrupted by a genomic Galpha(i2)G184S (GS) point mutation, we recently (Fu Y, Huang X, Zhong H, Mortensen RM, D'Alecy LG, Neubig RR. Circ Res 98: 659-666, 2006) showed that endogenous RGS proteins suppress muscarinic receptor-mediated bradycardia. To determine whether this was due to direct regulation of cardiac pacemakers or to alterations in the central nervous system or vascular responses, we examined isolated, perfused hearts. Isoproterenol-stimulated beating rates of heterozygote (+/GS) and homozygote (GS/GS) hearts were significantly more sensitive to inhibition by carbachol than were those of wild type (+/+). Even greater effects were seen in the absence of isoproterenol; the potency of muscarinic-mediated bradycardia was enhanced fivefold in GS/GS and twofold in +/GS hearts compared with +/+. A(1)-adenosine receptor-mediated bradycardia was unaffected. In addition to effects on the sinoatrial node, +/GS and GS/GS hearts show significantly increased carbachol-induced third-degree atrioventricular (AV) block. Atrial pacing studies demonstrated an increased PR interval and AV effective refractory period in GS/GS hearts compared with +/+. Thus loss of the inhibitory action of endogenous RGS proteins on Galpha(i2) potentiates muscarinic inhibition of cardiac automaticity and conduction. The severe carbachol-induced sinus bradycardia in Galpha(i2)G184S mice suggests a possible role for alterations of Galpha(i2) or RGS proteins in sick sinus syndrome and pathological AV block.     

Non-canonical functions of RGS proteins

Regulators of G protein signalling (RGS) proteins are united into a family by the presence of the RGS domain which serves as a GTPase-activating protein (GAP) for various Galpha subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate signalling of numerous G protein-coupled receptors. In addition to the RGS domains, RGS proteins contain diverse regions of various lengths that regulate intracellular localization, GAP activity or receptor selectivity of RGS proteins, often through interaction with other partners. However, it is becoming increasingly appreciated that through these non-RGS regions, RGS proteins can serve non-canonical functions distinct from inactivation of Galpha subunits. This review summarizes the data implicating RGS proteins in the (i) regulation of G protein signalling by non-canonical mechanisms, (ii) regulation of non-G protein signalling, (iii) signal transduction from receptors not coupled to G proteins, (iv) activation of mitogen-activated protein kinases, and (v) non-canonical functions in the nucleus.     

Palmitoylation of a conserved cysteine in the regulator of G protein signaling (RGS) domain modulates the GTPase-activating activity of RGS4 and RGS10

RGS4 and RGS10 expressed in Sf9 cells are palmitoylated at a conserved Cys residue (Cys(95) in RGS4, Cys(66) in RGS10) in the regulator of G protein signaling (RGS) domain that is also autopalmitoylated when the purified proteins are incubated with palmitoyl-CoA. RGS4 also autopalmitoylates at a previously identified cellular palmitoylation site, either Cys(2) or Cys(12). The C2A/C12A mutation essentially eliminates both autopalmitoylation and cellular [(3)H]palmitate labeling of Cys(95). Membrane-bound RGS4 is palmitoylated both at Cys(95) and Cys(2/12), but cytosolic RGS4 is not palmitoylated. RGS4 and RGS10 are GTPase-activating proteins (GAPs) for the G(i) and G(q) families of G proteins. Palmitoylation of Cys(95) on RGS4 or Cys(66) on RGS10 inhibits GAP activity 80-100% toward either Galpha(i) or Galpha(z) in a single-turnover, solution-based assay. In contrast, when GAP activity was assayed as acceleration of steady-state GTPase in receptor-G protein proteoliposomes, palmitoylation of RGS10 potentiated GAP activity >/=20-fold. Palmitoylation near the N terminus of C95V RGS4 did not alter GAP activity toward soluble Galpha(z) and increased G(z) GAP activity about 2-fold in the vesicle-based assay. Dual palmitoylation of wild-type RGS4 remained inhibitory. RGS protein palmitoylation is thus multi-site, complex in its control, and either inhibitory or stimulatory depending on the RGS protein and its sites of palmitoylation.     

Endogenous RGS protein action modulates mu-opioid signaling through Galphao. Effects on adenylyl cyclase,extracellular signal-regulated kinases,and intracellular calcium pathways

RGS (regulators of G protein signaling) proteins are GTPase-activating proteins for the Galpha subunits of heterotrimeric G proteins and act to regulate signaling by rapidly cycling G protein. RGS proteins may integrate receptors and signaling pathways by physical or kinetic scaffolding mechanisms. To determine whether this results in enhancement and/or selectivity of agonist signaling, we have prepared C6 cells stably expressing the mu-opioid receptor and either pertussis toxin-insensitive or RGS- and pertussis toxin-insensitive Galpha(o). We have compared the activation of G protein, inhibition of adenylyl cyclase, stimulation of intracellular calcium release, and activation of the ERK1/2 MAPK pathway between cells expressing mutant Galpha(o) that is either RGS-insensitive or RGS-sensitive. The mu-receptor agonist [d-Ala(2),MePhe(4),Gly(5)-ol]enkephalin and partial agonist morphine were much more potent and/or had an increased maximal effect in inhibiting adenylyl cyclase and in activating MAPK in cells expressing RGS-insensitive Galpha(o). In contrast, mu-opioid agonist increases in intracellular calcium were less affected. The results are consistent with the hypothesis that the GTPase-activating protein activity of RGS proteins provides a control that limits agonist action through effector pathways and may contribute to selectivity of activation of intracellular signaling pathways.     

Differential contribution of GTPase activation and effector antagonism to the inhibitory effect of RGS proteins on Gq-mediated signaling in vivo

RGS proteins act as negative regulators of G protein signaling by serving as GTPase-activating proteins (GAP) for alpha subunits of heterotrimeric G proteins (Galpha), thereby accelerating G protein inactivation. RGS proteins can also block Galpha-mediated signal production by competing with downstream effectors for Galpha binding. Little is known about the relative contribution of GAP and effector antagonism to the inhibitory effect of RGS proteins on G protein-mediated signaling. By comparing the inhibitory effect of RGS2, RGS3, RGS5, and RGS16 on Galpha(q)-mediated phospholipase Cbeta (PLCbeta) activation under conditions where GTPase activation is possible versus nonexistent, we demonstrate that members of the R4 RGS subfamily differ significantly in their dependence on GTPase acceleration. COS-7 cells were transiently transfected with either muscarinic M3 receptors, which couple to endogenous Gq protein and mediate a stimulatory effect of carbachol on PLCbeta, or constitutively active Galphaq*, which is inert to GTP hydrolysis and activates PLCbeta independent of receptor activation. In M3-expressing cells, all of the RGS proteins significantly blunted the efficacy and potency of carbachol. In contrast, Galphaq* -induced PLCbeta activation was inhibited by RGS2 and RGS3 but not RGS5 and RGS16. The observed differential effects were not due to changes in M3, Galphaq/Galphaq*, PLCbeta, or RGS expression, as shown by receptor binding assays and Western blots. We conclude that closely related R4 RGS family members differ in their mechanism of action. RGS5 and RGS16 appear to depend on G protein inactivation, whereas GAP-independent mechanisms (such as effector antagonism) are sufficient to mediate the inhibitory effect of RGS2 and RGS3.