MUTATIONAL ANALYSIS OF THE ASN RESIDUE ESSENTIAL FOR RGS PROTEIN-BINDING TO G-PROTEINS

Members of the RGS family serve as GTPase-activating proteins (GAPs) f or heterotrimeric G-proteins and negatively regulate signaling via G-p rotein-coupled receptors, The recently resolved crystal structure of R GS4 bound to G(i) alpha(1) suggests two potential mechanisms for the G AP activity of RGS proteins as follows: stabilization of the G(i) alph a(1) switch regions by RGS4 and the catalytic action of RGS4 residue A sn(128), To elucidate a role of the Asn residue for RGS GAP function, we have investigated effects of the synthetic peptide corresponding to the Ga binding domain of human retinal RGS (hRGSr) containing the key Asn at position 131, and we have carried out mutational analysis of A sn(131), Synthetic peptide hRGSr-(123-140) retained its ability to bin d the AlF4--complexed transducin alpha-subunit, G(t) alpha.AlF4-, but failed to elicit stimulation of Gt alpha GTPase activity, Wild-type hR GSr stimulated G(t) alpha GTPase activity by similar to 10-fold with a n EC50 value of 100 nM. Mutant hRGSr proteins with substitutions of As n(131) by Ser and Gin had a significantly reduced affinity for G(t) al pha but were capable of substantial stimulation of G(t) alpha GTPase a ctivity, 80 and 60% of V-max, respectively, Mutants hRGSr-Leu(131), hR GSr-Ala(131), and hRGSr-Asp(131) were able to accelerate G(t) alpha GT Pase activity only at very high concentrations (>10 mu m which appears to correlate with a further decrease of their affinity for transducin , Two mutants, hRGSr-His(131) and hRGSr-Delta(131), had no detectable binding to transducin, Mutational analysis of Asn(131) suggests that t he stabilization of the G-protein switch regions rather than catalytic action of the Asn residue is a key component for the RGS GAP action.