A Single Mutation Asp229→ Ser Confers upon Gsα the Ability To Interact with Regulators of G Protein Signaling

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Abstract
RGS proteins (regulators of G protein signaling) are GTPase activating proteins (GAPs) for Gi and Gq families of heterotrimeric G proteins but have not been found to interact with Gsα. The Gsα residue Asp229 has been suggested to be responsible for the inability of RGS proteins to interact with Gsα [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 between Gsα and RGS proteins by substituting Gsα Asp229 with Ser and replacing the potential Gsα Asp229 contact residues in RGS16, Glu129 and Asn131, by Ala and Ser, respectively. RGS16 and its mutants failed to interact with Gsα. A single mutation of Gsα, Asp229Ser, rendered the Gsα subunit with the ability to interact with RGS16 and RGS4. Like RGS protein binding to Gi and Gq α-subunits, RGS16 preferentially recognized the AlF4--bound conformation of Gsα Asp229Ser. In a single-turnover assay, RGS16 maximally stimulated GTPase activity of Gsα Asp229Ser by ∼5-fold with an EC50 value of 7.5 μM. Our findings demonstrate that Asp229 of Gsα represents a major barrier for Gsα interaction with known RGS proteins.