Conditional activation defect of a human G(sα )mutant

Hormonal signals activate trimeric G proteins by promoting exchange of GTP for GDP bound to the G protein’s α subunit (Gα). Here we describe a point mutation that impairs this activation mechanism in the α subunit of G(s), producing an inherited disorder of hormone responsiveness. Biochemical analysis reveals an activation defect that is paradoxically intensified by hormonal and other stimuli. By substituting histidine for a conserved arginine residue, the mutation removes an internal salt bridge (to a conserved glutamate) that normally acts as an intramolecular hasp to maintain tight binding of the γ-phosphate of GTP. In its basal, unperturbed state, the mutant α(s) binds guanosine 5′-[γ-thio]triphosphate (GTP[γS]), a GTP analog, slightly less tightly than does normal α(s), but (in the GTP[γS]-bound form) can stimulate adenylyl cyclase. The activation defect becomes prominent only under conditions that destabilize binding of guanine nucleotide (receptor stimulation) or impair the ability of α(s) to bind the γ-phosphate of GTP (cholera toxin, AlF(4)(−) ion). Although GDP release is usually the rate-limiting step in nucleotide exchange, the biochemical phenotype of this mutant α(s) indicates that efficient G protein activation by receptors and other stimuli depends on the ability of Gα to clasp tightly the GTP molecule that enters the binding site.