Rapid kinetics of regulator of G-protein signaling (RGS)-mediated Gαi and Gαo deactivation -: Gα specificity of RGS4 and RGS7

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by G alpha subunits speeding deactivation. Ga deactivation kinetics mediated by RGS are too fast to be directly studied using conventional radiochemical methods. We describe a stopped-flow spectroscopic approach to visualize these rapid kinetics by measuring the intrinsic tryptophan fluorescence decrease of G alpha accompanying GTP hydrolysis and G alpha deactivation on the millisecond time scale. Basal k(cat) values for G alpha (o), G alpha (i1), and G alpha (i2) at 20 degreesC were similar (0.025-0.033 s(-1)). Glutathione S-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305-453) enhanced the rate of G alpha deactivation in a manner Linear with RGS concentration. RGS4 stimulated rates could be measured up to 5 s(-1) at 3 muM, giving a catalytic efficiency of 1.7-2.8 x 10(6) M-1 s(-1) for all three Ga subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 x 10(6) M-1 s(-1) toward G alpha (o), G alpha (i2), and G alpha (i1), respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all Ga subunits tested, but it is specific for G alpha (o) over G alpha (i1) or G alpha (i2) Furthermore, the specificity of RGS7 for G alpha (o) does not depend on N- or C-terminal extensions or a G beta (5) subunit but resides in the RGS domain itself.