SUBSTITUTION OF TRANSDUCIN SER(202) BY ASP ABOLISHES G-PROTEIN RGS INTERACTION/

Known RGS proteins stimulate GTPase activity of G(i) and G(q) family m embers, but do not interact with G(s) alpha and G(12)alpha. To determi ne the role of specific G alpha residues for RGS protein recognition, six RGS contact residues of chimeric transducin alpha-subunit (G(t) al pha) corresponding to the residues that differ between G(i) alpha and G(s) alpha have been replaced by G(s) alpha residues, The ability of h uman retinal RGS (hRGSr) to bind mutant G(t) alpha subunits and accele rate their GTPase activity was investigated Substitutions Thr(178) --> Ser, Ile(181) --> Phe, and Lys(205) --> Arg of G(t) alpha did not alt er its interaction with hRGSr. The Lys(176) --> Leu mutant had the sam e affinity for hRGSr as G(t) alpha, but the maximal GTPase stimulation by hRGSr was reduced by similar to 2.5-fold, The substitution His(209 ) --> Gln led to a 3-fold decrease in the affinity of hRGSr for the G( t) alpha mutant without significantly affecting the maximal GTPase enh ancement. The Ser(202) --> Asp mutation abolished G(t) alpha recogniti on by hRGSr. A counteracting replacement of Glu(129) by Ala in hRGSr d id not restore the interaction of hRGSr with the G(t) alpha Ser(202) - -> Asp mutant. Our data suggest that the Ser residue at position 202 o f G(t) alpha is critical for the specificity of RGS proteins toward G( i) and G(q) families of G-proteins. Consequently, the corresponding re sidue, Asp(229) of G(s) alpha, is likely responsible for the inability of RGS proteins to interact with G(s) alpha.