Substitution of transducin Ser202 by Asp abolishes G-protein/RGS interaction

Known RGS proteins stimulate GTPase activity of G(i) and G(q) family members, but do not interact with G(s) alpha and G(12)alpha. To determine the role of specific G alpha residues for RGS protein recognition, six RGS contact residues of chimeric transducin alpha-subunit (G(t) alpha) corresponding to the residues that differ between G(i) alpha and G(s) alpha have been replaced by G(s) alpha residues, The ability of human retinal RGS (hRGSr) to bind mutant G(t) alpha subunits and accelerate their GTPase activity was investigated Substitutions Thr(178) --> Ser, Ile(181) --> Phe, and Lys(205) --> Arg of G(t) alpha did not alter its interaction with hRGSr. The Lys(176) --> Leu mutant had the same 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 enhancement. The Ser(202) --> Asp mutation abolished G(t) alpha recognition by hRGSr. A counteracting replacement of Glu(129) by Ala in hRGSr did 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 of G(t) alpha is critical for the specificity of RGS proteins toward G(i) and G(q) families of G-proteins. Consequently, the corresponding residue, Asp(229) of G(s) alpha, is likely responsible for the inability of RGS proteins to interact with G(s) alpha.