The G226A mutant of G_Sα highlights the requirement for dissociation of G protein subunits

Adenylylcyclase cannot be activated by hormones or guanine nucleotide analogs in membranes from cells that express the G226A mutant form of G_Sα, instead of the wild-type protein. The mutant G_Sα protein appears incapable of undergoing the conformational change necessary for guanine nucleotide-induced dissociation of the G protein α subunit from the βγ subunit complex (Miller, R. T., Masters, S. B., Sullivan, K. A., Beiderman, B., and Bourne, H. R. (1988) Nature 334, 712-715). G226A G_Sα, was synthesized in Escherichia coli, purified, and characterized. Examination of the kinetics of dissociation of guanosine 5′-3-O-(thio)triphosphate (GTPγS) suggests that G226A G_Sα is incapable of assuming the conformation necessary for high affinity binding of Mg²⁺ to the α subunit-GTPγS complex. Associated changes include the failure of Mg²⁺ and GTPγ8 to confer resistance to tryptic proteolysis upon the protein, to enhance intrinsic tryptophan fluorescence, or to cause dissociation of α from βγ. However, the GTPase activity of the mutant protein is near normal (at high Mg²⁺ concentrations), and the protein is capable of activating adenylylcyclase. A similar defect is present in G49 V G_Sα. Failure of G protein subunit dissociation appears to be the explanation for the phenotypic properties of cells that express G226A G_Sα, and this mutation thus highlights the crucial nature of this reaction as a component of G protein action.