A dominant negative G alpha s mutant is rescued by secondary mutation of the alpha chain amino terminus.

The Gs protein alpha subunit, alpha s, stimulates the activity of adenylyl cyclase. The sequence 223Asp-Val-Gly-Gly-Gln227 in the alpha s polypeptide is predicted to interact with the gamma-phosphate of GTP and mediate the conformational change involved in alpha s activation. Mutation of the alpha s polypeptide within this region at Gly225----Thr had two demonstrative phenotypic effects when expressed in COS-1 cells: the mutant alpha s chain was ineffective in activating adenylyl cyclase and inhibited in a concentration-dependent manner the beta-adrenergic receptor stimulation of cAMP synthesis. Thus, the Gly225----Thr mutation alters the ability of GTP to activate the alpha s chain and when overexpressed the mutant polypeptide exerts a dominant negative phenotype. Mutation at the amino terminus which creates a constitutively active alpha s rescued the inhibited state of the Gly225----Thr mutant when both mutations were encoded in the same polypeptide. This finding defines the amino terminus as a functional regulatory domain controlling the properties of the GTP/GDP binding site of G protein alpha subunit polypeptide chains.