Hydrophobicity of residue351 of the G protein Gi1α determines the extent of activation by the α2A-adrenoceptor

Cysteine(351) is the site for pertussis toxin-catalyzed ADP-ribosylation in the G protein G(il)alpha, Alteration of this residue, or the equivalent cysteine in other G(i)-family G proteins, has been used to examine specific interactions between receptors and these G proteins. However, no systematic analysis has been performed to determine the quantitative effect of such alterations. To address this we mutated cysteine(351) of G(il)alpha to all other possible amino acids. Each of the G protein mutants was transiently coexpressed along with the porcine alpha(2A)-adrenoceptor in HEK 293/T cells. Following pertussis toxin treatment of the cells, membranes were prepared and the capacity of the agonist UK14304 to stimulate the binding of [S-35]GTP gamma S to the modified G proteins was measured. A spectrum of function was observed. The presence of either a charged amino acid or a proline at this position essentially attenuated agonist regulation. The wild-type G protein did not result in maximal stimulation by agonist. The presence of certain branched chain aliphatic amino acids or bulky aromatic R groups at amino acid(351) resulted in substantially greater maximal stimulation by the alpha(2A)-adrenoceptor than that achieved with the wild-type sequence. The degree of activation of the forms of G(il)alpha correlated strongly with the octanol/water partition coefficient of the amino acid at residue(351). Variation in EC50 values for agonist-induced stimulation of binding of [S-35]GTP gamma S to the mutant G proteins also correlated with the octanol/water partition coefficient. These results define a central role for hydrophobicity of this residue in defining productive receptor-G protein interactions.