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...

RGS proteins negatively regulate heterotrimeric G proteins at the plasma membrane. RGS2-GFP localizes to the nucleus, plasma membrane, and cytoplasm of HEK293 cells. Expression of activated G q increased RGS2 association with the plasma membrane and decreased accumulation in the nucleus, suggesting that signal-induced redistribution may regulate RGS2 function. Thus, we identified and characterized a conserved N-terminal domain in RGS2 that is necessary and sufficient for plasma membrane localization. Mutational and biophysical analyses indicated that this domain is an amphipathic ␣-helix that binds vesicles containing acidic phospholipids. However, the plasma membrane targeting function of the amphipathic helical domain did not appear to be essential for RGS2 to attenuate signaling by activated G q . Nevertheless, truncation mutants indicated that the N terminus is essential, potentially serving as a scaffold that binds receptors, signaling proteins, or nuclear components. Indeed, the RGS2 N terminus directs nuclear accumulation of GFP. Although RGS2 possesses a nuclear targeting motif, it lacks a nuclear import signal and enters the nucleus by passive diffusion. Nuclear accumulation of RGS2 does not limit its ability to attenuate G q signaling, because excluding RGS2 from the nucleus was without effect. RGS2 may nonetheless regulate signaling or other processes in the nucleus.Many hormones, neurotransmitters, and sensory stimuli elicit specific physiological responses in target tissues by activating receptors that are coupled to heterotrimeric G proteins 1 (1, 2). Activated receptors promote exchange of GTP for GDP on G␣ subunits leading to dissociation of GTP-bound G␣ subunits from 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 act as molecular switches to coordinate the wide range of responses elicited by extracellular stimuli.The regulators of G protein signaling (RGS) proteins are a large family 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 shortens the half-life of active, GTP-bound G␣ subunits and leads to attenuation of a response during prolonged stimulation or accelerated termination of the signal following removal of agonist (7-11). Binding of RGS proteins to active G␣ subunits can also interfere with effector binding, thereby blocking activation and downstream signaling (12). These activities are mediated by the ϳ120-amino acid RGS domain, a conserved feature of this protein family.Genetic studies in budding yeast (13) and Caenorhabditis elegans (14) indicate that eukaryotes express several types of RGS proteins to provide selective regulation of distinct G protein signaling pathways, and a means of differentially regulating a single pathway in response to various physiologic conditions. Similarly, certain mammalian RGS proteins have b...