RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2 is a much more potent attenuator of M1 muscarinic receptor signaling than RGS5. We hypothesize that this difference is mediated by variation in their ability to constitutively associate with the plasma membrane (PM). Compared with full-length RGS2, the RGS-box domains of RGS2 and RGS5 both show reduced PM association and activity. Prenylation of both RGSbox domains increases activity to RGS2 levels, demonstrating that lipid bilayer targeting increases RGS domain function. Amino-terminal domain swaps confirm that key determinants of localization and function are found within this important regulatory domain. An RGS2 amphipathic helix domain mutant deficient for phospholipid binding (L45D) shows reduced PM association and activity despite normal binding to the M1 muscarinic receptor third intracellular loop and activated G␣ q . Replacement of a unique dileucine motif adjacent to the RGS2 helix with corresponding RGS5 residues disrupts both PM localization and function. These data suggest that RGS2 contains a hydrophobic extension of its helical domain that imparts high efficiency binding to the inner leaflet of the lipid bilayer. In support of this model, disruption of membrane phospholipid composition with N-ethylmaleimide reduces PM association of RGS2, without affecting localization of the M1 receptor or G␣ q . Together, these data indicate that novel features within the RGS2 amphipathic ␣ helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.Heterotrimeric G-protein-coupled receptors mediate cellular responses to a variety of extracellular ligands, including hormones, neurotransmitters, and sensory stimuli. Proper coordination of G-protein-coupled receptor signaling at the cellular and tissue level is required to ensure appropriate physiologic responses to rapidly changing environmental conditions. An important component of G-protein-coupled receptors signal coordination in human cells is the RGS (regulator of G-protein signaling) superfamily of proteins. RGS proteins inhibit G-protein signaling via their activities as GTPase-activating proteins for G-protein ␣ subunits (1-4).The human genome contains 37 RGS proteins (5) characterized by a ϳ120-amino acid domain called the "RGS-box." The RGS protein superfamily can be further subclassified based on the architectural organization and RGS-box function of its members (6). Many subfamilies are made up of larger RGS-boxcontaining proteins that include the RGS7-like, RGS12-like, RhoGEF-containing, and G-protein-coupled receptor kinases. These subfamilies are mainly composed of members with multiple modular signaling domains. By contrast, the RGSZ-like and RGS4-like (R4/B) subfamilies are made up of smaller proteins containing a RGS-box domain flanked by short aminoterminal and carboxyl-terminal extensions. These two families are distinguishe...