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, AlF₄^- 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 Ga to clasp tightly the GTP molecule that enters the binding site.