Signaling pathways using heterotrimeric guanine-nucleotide-binding-proteins (G proteins) trigger physiological responses elicited by hormones, neurotransmitters and sensory stimuli. GTP binding activates G proteins by dissociating G alpha from G beta gamma subunits, and GTP hydrolysis by G alpha subunits deactivates G proteins by allowing heterotrimers to reform. However, deactivation of G-protein signalling pathways in vivo can occur 10- to 100-fold faster than the rate of GTP hydrolysis of G alpha subunits in vitro, suggesting that GTPase-activating proteins (GAPs) deactivate G alpha subunits. Here we report that RGS (for regulator of G-protein signalling) proteins are GAPs for G alpha subunits. RGS1, RGS4 and GAIP (for G alpha-interacting protein) bind specifically and tightly to G alphai and G alpha0 in cell membranes treated with GDP and AlF4(-), and are GAPs for G alphai, G alpha0 and transducin alpha-subunits, but not for G alphas. Thus, these RGS proteins are likely to regulate a subset of the G-protein signalling pathways in mammalian cells. Our results provide insight into the mechanisms that govern the duration and specificity of physiological responses elicited by G-protein-mediated signalling pathways.

RGS (regulators of G protein signaling) proteins are GTPase activating proteins that inhibit signaling by heterotrimeric G proteins. All RGS proteins studied to date act on members of the Gi␣ family, but not Gs␣ or G12␣. RGS4 regulates Gi␣ family members and Gq␣. RGS2 (G0S8) is exceptional because the G proteins it regulates have not been identified. We report that RGS2 is a selective and potent inhibitor of Gq␣ function. RGS2 selectively binds Gq␣, but not other G␣ proteins (Gi, Go, Gs, G12͞13) in brain membranes; RGS4 binds Gq␣ and Gi␣ family members. RGS2 binds purified recombinant Gq␣, but not Go␣, whereas RGS4 binds either. RGS2 does not stimulate the GTPase activities of Gs␣ or Gi␣ family members, even at a protein concentration 3000-fold higher than is sufficient to observe effects of RGS4 on Gi␣ family members. In contrast, RGS2 and RGS4 completely inhibit Gq-directed activation of phospholipase C in cell membranes. When reconstituted with phospholipid vesicles, RGS2 is 10-fold more potent than RGS4 in blocking Gq␣-directed activation of phospholipase C␤1. These results identify a clear physiological role for RGS2, and describe the first example of an RGS protein that is a selective inhibitor of Gq␣ function.

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GTP hydrolysis by guanine nucleotide-binding proteins, an essential step in many biological processes, is stimulated by GTPase-activating proteins (GAPs). The mechanisms whereby GAPs stimulate GTP hydrolysis are unknown. We have used mutational, biochemical, and structural data to investigate how RGS4, a GAP for heterotrimeric G protein alpha subunits, stimulates GTP hydrolysis. Many of the residues of RGS4 that interact with Gi alpha 1 are important for GAP activity. Furthermore, optimal GAP activity appears to require the additive effects of interactions along the RGS4-G alpha interface. GAP-defective RGS4 mutants invariably were defective in binding G alpha subunits in their transition state; furthermore, the apparent strengths of GAP and binding defects were correlated. Thus, none of these residues of RGS4, including asparagine 128, the only residue positioned at the active site of Gi alpha 1, is required exclusively for catalyzing GTP hydrolysis. These results and structural data (Tesmer, J. G. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261) indicate that RGS4 stimulates GTP hydrolysis primarily by stabilizing the transition state conformation of the switch regions of the G protein, favoring the transition state of the reactants. Therefore, although monomeric and heterotrimeric G proteins are related, their GAPs have evolved distinct mechanisms of action.

The members of a recently identified protein family termed regulators of G-protein signaling (RGS) act as GTPase-activating proteins for certain G ␣ subunits in vitro, but their physiological effects in cells are uncertain in the face of similar biochemical activity and overlapping patterns of tissue expression. Consistent with its activity in in vitro GTPase-activating protein assays, RGS4 interacts efficiently with endogenous proteins of the G i and G q subclasses of G ␣ subunits but not with G 12␣ or G s␣ . Unlike other RGS proteins such as RGS9, RGS-GAIP, and Sst2p, which have been reported to be largely membrane-associated, a majority of cellular RGS4 is found as a soluble protein in the cytoplasm. However, the expression of a GTPase-deficient G i␣ subunit (G i␣2 -Q204L) resulted in the translocation of both wild type RGS4 and a non-G i␣ -binding mutant (L159F) to the plasma membrane. These data suggest that RGS4 may be recruited to the plasma membrane indirectly by Gprotein activation and that multiple RGS proteins within a given cell might be differentially localized to determine a physiologic response to a G-protein-linked stimulus.Numerous biological processes such as vision, olfaction, and many hormonal responses generate signals that are transduced through heterotrimeric guanine nucleotide (GTP)-binding proteins (G-proteins) (1). The regulatory mechanisms that control G-protein signaling intracellularly have not been well characterized, but the variability of signal strength and the specificity of a given stimulus among different cell types suggest that cell typespecific interacting proteins participate (2). A newly discovered family of regulators of G-protein signaling (RGS proteins) 1 may play a role in cell type-specific desensitization. RGS proteins were originally identified by genetic complementation of a yeast homologue (Sst2p) and by the identification of a closely related homologue in Caenorhabditis elegans (EGL10) that regulated G-protein signaling (3-6). Subsequently, transfection experiments and biochemical studies with recombinant proteins have shown that RGS family members likely down-regulate signaling through G-protein-coupled receptors (GPCRs) by acting as GTPase-activating proteins (GAPs) for some heterotrimeric G-protein ␣-subunits (7-11).Although several RGS proteins such as RGS1, RGS4, RGS10, and RGS-GAIP accelerate the GTPase activity of G i␣ subunits with apparently similar ability, the precise affinities of the various RGS proteins for individual G ␣ subunits is only slowly emerging, and almost nothing is known about the physiologic regulation of these proteins (8 -11). Although previous studies documented the inability of RGS1, RGS4, and RGS-GAIP to enhance G s␣ or G 12␣ GTPase activity (7-9), recent experiments have demonstrated that either recombinant RGS4 or RGS-GAIP accelerates GTP hydrolysis by G q␣ in reconstituted GPCR-Gprotein phospholipid vesicles and inhibits second messenger generation through a G q␣ -coupled GPCR when added to membranes prepared from the neural c...