G protein selectivity is a determinant of RGS2 function.
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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. (+info)
Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2.
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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. (+info)
Feedback phosphorylation of an RGS protein by MAP kinase in yeast.
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Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity in vitro and to promote G protein desensitization in vivo. Less is known about how RGS proteins are themselves regulated. To address this question we purified the RGS in yeast, Sst2, and used electrospray ionization mass spectrometry to identify post-translational modifications. This analysis revealed that Sst2 is phosphorylated at Ser-539 and that phosphorylation occurs in response to pheromone stimulation. Ser-539 lies within a consensus mitogen-activated protein (MAP) kinase phosphorylation site, Pro-X-Ser-Pro. Phosphorylation is blocked by mutations in the MAP kinase genes (FUS3, KSS1), as well as by mutations in components needed for MAP kinase activation (STE11, STE7, STE4, STE18). Phosphorylation is also blocked by replacing Ser-539 with Ala, Asp, or Glu (but not Thr). These point mutations do not alter pheromone sensitivity, as determined by growth arrest and reporter transcription assays. However, phosphorylation appears to slow the rate of Sst2 degradation. These findings indicate that the G protein-regulated MAP kinase in yeast can act as a feedback regulator of Sst2, itself a regulator of G protein signaling. (+info)
PKC translocation without changes in Galphaq and PLC-beta protein abundance in cardiac hypertrophy and failure.
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Activation of protein kinase C (PKC) has been implicated as playing a key role in the pathogenesis of cardiac hypertrophy. This study investigates the response of several signal transduction proteins responsible for PKC activation during the transition from compensated pressure-overload hypertrophy (POH) to congestive heart failure (CHF). Pressure overload was produced on male, adult, Hartley strain guinea pigs using a ligature around the descending thoracic aorta. Sham-operated controls, POH, and CHF groups were identified based on left ventricular hypertrophy, pulmonary congestion, and isolated heart Langendorff mechanics. Quantitative immunoblotting revealed phospholipase C (PLC)-betaI and Galphaq were unchanged during POH and CHF, as were RGS2, RGS3, and RGS4 (regulators of G protein signaling, which are activators of intrinsic GTPase activity). Translocation of PKC-alpha, -epsilon, and -gamma from cytosolic to membranous fractions were significantly increased during POH and CHF. Cytosolic PKC activity was also elevated during POH. We conclude that differential PKC activation may be mediated by increases in Galphaq and PLC-betaI activity rather than upregulation of expression. (+info)
Palmitoylation of a conserved cysteine in the regulator of G protein signaling (RGS) domain modulates the GTPase-activating activity of RGS4 and RGS10.
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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. (+info)
Copurification of brain G-protein beta5 with RGS6 and RGS7.
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A structurally divergent G-protein beta subunit expressed in brain and retina, Gbeta5, exhibits functional specialization in its protein-protein interactions in vitro. In retina, Gbeta5 has been isolated in a soluble complex with regulator of G-protein signaling RGS7. The function and molecular associations of Gbeta5 in brain are unknown. To identify tightly bound proteins associated with Gbeta5 in the brain, it was immunoaffinity-purified from a nonionic detergent extract of washed mouse brain membranes using an antibody directed against its N terminus. Elution with cognate peptide revealed a broad band of 55 kDa that coeluted with Gbeta5 on SDS-PAGE. The copurifying 55 kDa band was identified as an approximately 1:1 mixture of RGS6 and RGS7 by matrix-assisted laser desorption ionization mass spectroscopic analysis of tryptic peptides. Gbeta5 and RGS7 could be reciprocally coimmunoprecipitated from unfractionated brain membrane extracts confirming the tight association of native proteins. In contrast, immunoblotting of the peptide eluate revealed no copurifying Galphaq/11, Galphai1/2, Ggamma2, Ggamma3, or Ggamma7. These findings implicate RGS6 and RGS7 in the function of Gbeta5 in the brain and suggest that a large fraction of membrane-targeted Gbeta5 has no associated G subunit and therefore functions outside the canonical framework of G(beta)(gamma) interactions. (+info)
Regulator of G protein signaling 1 (RGS1) markedly impairs Gi alpha signaling responses of B lymphocytes.
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Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-alpha. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various G alpha subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Gi alpha and Gq alpha GTPase-activating protein and a potential G12 alpha effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12 alpha. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells. (+info)
A regulator of G protein signaling interaction surface linked to effector specificity.
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Proteins of the regulator of G protein signaling (RGS) family accelerate GTP hydrolysis by the alpha subunits (G(alpha)) of G proteins, leading to rapid recovery of signaling cascades. Many different RGS proteins can accelerate GTP hydrolysis by an individual G(alpha), and GTP hydrolysis rates of different G(alpha)s can be enhanced by the same RGS protein. Consequently, the mechanisms for specificity in RGS regulation and the residues involved remain unclear. Using the evolutionary trace (ET) method, we have identified a cluster of residues in the RGS domain that includes the RGS-G(alpha) binding interface and extends to include additional functionally important residues on the surface. One of these is within helix alpha3, two are in alpha5, and three are in the loop connecting alpha5 and alpha6. A cluster of surface residues on G(alpha) previously identified by ET, and composed predominantly of residues from the switch III region and helix alpha3, is spatially contiguous with the ET-identified residues in the RGS domain. This cluster includes residues proposed to interact with the gamma subunit of G(talpha)'s effector, cGMP phosphodiesterase (PDEgamma). The proximity of these clusters suggests that they form part of an interface between the effector and the RGS-G(alpha) complex. Sequence variations in these residues correlate with PDEgamma effects on GTPase acceleration. Because ET identifies residues important for all members of a protein family, these residues likely form a general site for regulation of G protein-coupled signaling cascades, possibly by means of effector interactions. (+info)