RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate G␣ 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 ␣ 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 ␣ 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.Cells rely upon G protein-coupled receptors (GPCRs) 1 to convey signals from extracellular hormones and neurotransmitters to intracellular effectors and linked signaling pathways. Agonist occupancy of the GPCR activates a heterotrimeric G protein (G␣␤␥) by catalyzing the exchange of GDP for GTP on the G␣ subunit (1). This initiates dissociation of the trimer into free G␣ and G␤␥, which independently or in coordinated fashion activate downstream effectors and linked signaling pathways. Members of the regulators of G protein signaling (RGS) family of proteins are direct modulators of G protein activity. RGS proteins are best understood as GTPaseactivating proteins (GAPs), which bind to the activated form of G␣ and accelerate its GTPase activity thereby promoting the termination of G protein signaling (2-5). By virtue of their interactions with activated G␣, RGS proteins also serve as effector antagonists to block activation of downstream effector molecules (6, 7).All RGS proteins share a conserved RGS core domain of ϳ130 amino acids that contains binding sites for G␣ and is responsible for their GAP activity (5,8,9). Outside of the RGS domain, however, the more than 30 family members are widely divergent. Some RGS proteins are quite complex and contain multiple domains for binding a variety of signaling proteins. Other RGS proteins are simple, with relatively short, featureless N...

RGS (regulator of G protein signaling) proteins areGTPase-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 G␣ 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 ␣ 1 complexes suggest that RGS2-G i ␣ interaction is unfavorable in part because of the geometry of the switch I binding pocket of RGS2 and a potential interaction between the ␣8-␣9 loop of RGS2 and ␣A of G i class ␣ 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 G␣ subunits.Many hormones, neurotransmitters, and sensory stimuli exert their effect on target tissues by activating receptors that are coupled to heterotrimeric G proteins 1 (1, 2). Receptor activation results in exchange of GTP for GDP on G␣ subunits, dissociation of GTP-bound G␣ subunits from the G␤␥ heterodimers, and activation of downstream effector pathways. Signals are terminated following G␣-catalyzed hydrolysis of GTP and reformation of G protein heterotrimers. Thus, G proteins are molecular switches that coordinate physiological responses elicited by a variety of stimuli.RGS (regulator of G protein signaling) proteins are a family of more than 20 members that regulate G protein signaling in part by acting as GTPase-activating proteins (GAPs) for several classes of G protein ␣ subunits (3-6). The GAP activity of RGS proteins decreases the lifetime of active, GTP-bound G␣ subunits, thereby attenuating responses or accelerating the kinetics of signal termination (7,8). Binding of RGS proteins to active G␣ subunits can also antagonize effector activation, thereby blocking signal production (9). These activities are mediated by the conserved RGS domain of ϳ120 amino acids that is characteristic of this protein family.Higher eukaryotes express several types of RGS proteins, potentially to provide selective regulation of distinct types of G protein signaling pathways. Consistent with this hypothesis, RGS proteins are structurally diverse, distinguished by various domains that are likely to confer specific functions. For example, the N terminus of RGS4 confers receptor-selective regulation of G q -coupled responses (10, 11), the PDZ domain of RGS12 binds peptides from the C termini of certain G protein coupled r...

RGS4, a mammalian GTPase activating protein for G protein ␣ subunits, was identified by its ability to inhibit the pheromone response pathway in Saccharomyces cerevisiae. To define regions of RGS4 necessary for its function in vivo, we assayed mutants for activity in this system. Deletion of the N-terminal 33 aa of RGS4 (⌬1-33) yielded a nonfunctional protein and loss of plasma membrane localization. These functions were restored by addition of a C-terminal membrane-targeting sequence to RGS4 (⌬1-33). Thus, plasma membrane localization is tightly coupled with the ability of RGS4 to inhibit signaling. Fusion of the N-terminal 33 aa of RGS4 to green f luorescent protein was sufficient to localize an otherwise soluble protein to the plasma membrane, defining this N-terminal region as a plasma membrane anchorage domain. RGS4 is palmitoylated, with Cys-2 and Cys-12 the likely sites of palmitoylation. Surprisingly, mutation of the cysteine residues within the N-terminal domain of RGS4 did not affect plasma membrane localization in yeast or the ability to inhibit signaling. Features of the N-terminal domain other than palmitoylation are responsible for the plasma membrane association of RGS4 and its ability to inhibit pheromone response in yeast.Heterotrimeric G proteins couple receptors for hormones, neurotransmitters, and sensory signals to intracellular effector molecules, thereby eliciting cellular responses (1, 2). Guanine nucleotide exchange and hydrolysis on the G protein ␣ subunit drives the cycle of activation and deactivation of these signaling pathways. The duration of G protein-mediated responses is subject to the intrinsic GTPase rate of the G protein ␣ subunit, but is also modulated by extrinsic factors. A recently appreciated form of regulation has come from the discovery that members of a protein family called regulators of G protein signaling, or RGS proteins, stimulate the rate of GTP hydrolysis by G protein ␣ subunits (3-5). RGS proteins are found in species ranging from yeast to mammals and constitute a family of at least 20 mammalian proteins (6-8).All RGS family members share sequence similarity that extends over approximately 130 aa, separated in some cases by insertions of varying length (9-11). This conserved RGS domain is sufficient to stimulate GTPase activity of G protein ␣ subunits in vitro (12)(13)(14). Expression of the RGS homology domain of RET-RGS1 or RGS4 yields a recombinant protein that is a functional GAP (GTPase-activating protein). In the crystal structure of RGS4 bound to AlF 4 Ϫ -activated G i␣1 , only the core domain is visible (15). The RGS homology domain binds to the switch regions of G i␣1 and appears to catalyze GTP hydrolysis by stabilizing the switch regions of the G protein in a conformation that favors the transition state of the reactants (14, 15). Sequences outside the RGS homology domain exhibit considerable diversity among family members.Although rapid progress has been made in dissecting the biochemical mechanism by which RGS proteins regulate G protein a...

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RGS4, a mammalian GTPase-activating protein for G protein ␣ subunits, requires its N-terminal 33 amino acids for plasma membrane localization and biological activity (Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584 -5589). In this study, we tested the hypothesis that the N-terminal domain mediates membrane binding by forming an amphipathic ␣-helix. RGS4 bound to liposomes containing anionic phospholipids in a manner dependent on the first 33 amino acids. Circular dichroism spectroscopy of a peptide corresponding to amino acids 1-31 of RGS4 revealed that the peptide adopted an ␣-helical conformation in the presence of anionic phospholipids. Point mutations that either neutralized positive charges on the hydrophilic face or substituted polar residues on the hydrophobic face of the model helix disrupted plasma membrane targeting and biological activity of RGS4 expressed in yeast. Recombinant mutant proteins were active as GTPase-activating proteins in solution but exhibited diminished binding to anionic liposomes. Peptides corresponding to mutants with the most pronounced phenotypes were also defective in forming an ␣-helix as measured by circular dichroism spectroscopy. These results support a model for direct interaction of RGS4 with membranes through hydrophobic and electrostatic interactions of an N-terminal ␣-helix. Regulators of G protein signaling (RGS proteins)1 are a recently appreciated family of proteins that participate as negative regulators or effectors in G protein pathways (reviewed in Refs. 1 and 2). RGS proteins catalytically accelerate GTP hydrolysis on ␣ subunits, resulting in faster termination of G protein signaling. The GAP activity of RGS proteins may account for discrepancies between the measured intrinsic rates of GTP hydrolysis of the ␣ subunit and the deactivation rate of physiological effectors. In addition to their functions as GAPs, some RGS proteins may also regulate G protein pathways by serving as effector antagonists (3, 4). As new RGS family members are identified and characterized, it has become clear that RGS proteins can act as effectors, as well as inhibitors, of G protein pathways (5).More than 20 mammalian RGS proteins have been identified to date (1). All RGS family members share sequence similarity that extends over approximately 120 amino acids. In many RGS proteins, this so-called "RGS box" or core domain is sufficient to bind G protein ␣ subunits and catalyze GTPase activity in vitro (6 -9). However in a cellular context, regions outside the RGS domain are necessary for biological activity of the protein (8, 10). Thus, important regulatory information is likely to be contained within these highly divergent flanking regions of RGS proteins.One way in which these RGS flanking regions can modulate protein activity is by determining subcellular localization. For several RGS proteins, regions near the N terminus are responsible for targeting the proteins to particular cellular locations. RGS-GAIP and RGSZ1...