Abstract

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein α subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the G_i subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the α subunit of transducin, Gα_t. Here we report another unique property of RGS9: high specificity for Gα_t. The core (RGS) domain of RGS9 (RGS9) stimulates Gα_t GTPase activity by 10-fold and Gα_i1 GTPase activity by only 2-fold at a concentration of 10 μm. Using chimeric Gα_t/Gα_i1 subunits we demonstrated that the α-helical domain of Gα_t imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Gα subunits as measured by a change in fluorescence of a mutant Gα_t (Chi6b) selectively labeled at Cys-210.K _d values for RGS9 complexes with Gα_tand Gα_i1 calculated from the direct binding and competition experiments were 185 nm and 2 μm, respectively. The γ subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pγ to increase the affinity of RGS9 for Gα_t. A distinct, nonoverlapping pattern of RGS and Pγ interaction with Gα_t suggests a unique mechanism of effector-mediated GAP function of the RGS9.