Mutations at the domain interface of GSalpha impair receptor-mediated activation by altering receptor and guanine nucleotide binding.

G protein alpha subunits consist of two domains, a GTPase domain and a helical domain. Receptors activate G proteins by catalyzing replacement of GDP, which is buried between these two domains, with GTP. Substitution of the homologous alphai2 residues for four alphas residues in switch III, a region that changes conformation upon GTP binding, or of one nearby helical domain residue decreases the ability of alphas to be activated by the beta-adrenergic receptor and by aluminum fluoride. Both sets of mutations increase the affinity of alphas for the beta-adrenergic receptor, based on an increased amount of high affinity binding of the beta-adrenergic agonist, isoproterenol. The mutations also decrease the rate of receptor-mediated activation and disrupt the ability of the beta-adrenergic receptor to increase the apparent affinity of alphas for the GTP analog, guanosine 5'-O-(3-thiotriphosphate). Simultaneous replacement of the helical domain residue and one of the four switch III residues with the homologous alphai2 residues restores normal receptor-mediated activation, suggesting that the defects caused by mutations at the domain interface are due to altered interdomain interactions. These results suggest that interactions between residues across the domain interface are involved in two key steps of receptor-mediated activation, promotion of GTP binding and subsequent receptor-G protein dissociation.