A SINGLE MUTATION ASP(229)-]SER CONFERS UPON G(S)ALPHA THE ABILITY TOINTERACT WITH REGULATORS OF G-PROTEIN SIGNALING

RGS proteins (regulators of G protein signaling) are GTPase activating proteins (GAPs) for G(i) and G(q) families of heterotrimeric G protei ns but have not been found to interact with G(s)alpha. The G(s)alpha r esidue Asp229 has been suggested to be responsible for the inability o f RGS proteins to interact with G(s)alpha [Natochin, M., and Artemyev, N. O. (1998) J. Biol. Chem. 273, 4300-4303]. To test this hypothesis, we have investigated the possibility of generating an interaction bet ween G(s)alpha and RGS proteins by substituting G(s)alpha Asp229 with Ser and replacing the potential G(s)alpha Asp229 contact residues in R GS16, Glu129 and Asn131, by Ala and Ser, respectively. RCS16 and its m utants failed to interact with G(s)alpha. A single mutation of G(s)alp ha, Asp229Ser, rendered the G(s)alpha subunit with the ability to inte ract with RGS16 and RGS4. Like RCS protein binding to G(i) and G(q) al pha-subunits, RGS16 preferentially recognized the AlF4--bound conforma tion of G(s)alpha Asp229Ser. In a single-turnover assay, RGS16 maximal ly stimulated GTPase activity of G(s)alpha Asp229Ser by similar to 5-f old with an EC50 value of 7.5 mu M. Our findings demonstrate that Asp2 29 of G(s)alpha represents a major barrier for G(s)alpha interaction w ith known RGS proteins.