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

Abstract

Known RGS proteins stimulate GTPase activity of G1 and G(q) family members, but do not interact with G(s)α and G12α. To determine the role of specific Gα residues for RGS protein recognition, six RGS contact residues of chimeric transducin α-subunit (G(t)α) corresponding to the residues that differ between G(i)α G(s)α have been replaced by G(s)α residues. The ability of human retinal RGS (hRGSr) to bind mutant G(t)α subunits and accelerate their GTPase activity was investigated. Substitutions Thr178 →Ser, Ile181→ Phe, and Lys205 → Arg of G(t)α did not alter its interaction with hRGSr. The Lys176 → Leu mutant had the same affinity for hRGSr as G(t)α, but the maximal GTPase stimulation by hRGSr was reduced by ~2.5-fold. The substitution His209 → Gln led to a 3-fold decrease in the affinity of hRGSr for the G(t)α mutant without significantly affecting the maximal GTPase enhancement. The Ser202 → Asp mutation abolished G(t)α recognition by hRGSr. A counteracting replacement of Glu129 by Ala in hRGSr did not restore the interaction of hRGSr with the G(t)α Ser202 → Asp mutant. Our data suggest that the Ser residue at position 202 of G(t)α is critical for the specificity of RGS proteins toward G1 and G(q) families of G-proteins. Consequently, the corresponding residue, Asp229 of G(s)α, is likely responsible for the inability of RGS proteins to interact with G(s)α.