The alpha-helical domain of G alpha(t) 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 alpha subunits (GAPs), thus con trolling the response kinetics of a variety of cell signaling processes. Mo st RGS domains that have been studied have relatively little GTPase activat ing specificity especially for G proteins within the G(i) subfamily. Retina l RGS9 is unique in its ability to act synergistically with a downstream ef fector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, G alpha(t). Here we report another unique property of RGS9: high specificity for G alpha(t). The core (RGS) domain of RGS9 (RG S9) stimulates G alpha(t) GTPase activity by 10-fold and G alpha(i1) GTPase activity by only 2-fold at a concentration of 10 mu M. Using chimeric G al pha(t)/G alpha(i1) subunits we demonstrated that the alpha-helical domain o f G alpha(t) imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated G alpha subunits as measure d by a change in fluorescence of a mutant G alpha(t) (Chi6b) selectively la beled at Cys-210, K-d values for RGS9 complexes with G alpha(t) and G alpha (i1) calculated from the direct binding and competition experiments were 18 5 nM and 2 mu M, respectively. The gamma subunit of phosphodiesterase incre ases the GAP activity of RGS9, We demonstrate that this is because of the a bility of P gamma to increase the affinity of RGS9 for G alpha(t). A distin ct, nonoverlapping pattern of RGS and P gamma interaction with G alpha(t) s uggests a unique mechanism of effector-mediated GAP function of the RGS9.