The a-Helical Domain of Gat Determines Specific Interaction with Regulator of G Protein Signaling 9*

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein a 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 Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the a subunit of transducin, Gat. Here we report another unique property of RGS9: high specificity for Gat. The core (RGS) domain of RGS9 (RGS9) stimulates Gat GTPase activity by 10-fold and Gai1 GTPase activity by only 2-fold at a concentration of 10 mM. Using chimeric Gat/Gai1 subunits we demonstrated that the a-helical domain of Gat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Ga subunits as measured by a change in fluorescence of a mutant Gat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Gat and Gai1 calculated from the direct binding and competition experiments were 185 nM and 2 mM, respectively. The g subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pg to increase the affinity of RGS9 for Gat. A distinct, nonoverlapping pattern of RGS and Pg interaction with Gat suggests a unique mechanism of effector-mediated GAP function of the RGS9.