CONDITIONAL ACTIVATION DEFECT OF A HUMAN G(S-ALPHA) MUTANT

Hormonal signals activate trimeric G proteins by promoting exchange of GTP for GDP bound to the G protein's LY subunit (G alpha\), Here we d escribe a point mutation that impairs this activation mechanism in the alpha subunit of G(s), producing an inherited disorder of hormone res ponsiveness, Biochemical analysis reveals an activation defect that is paradoxically intensified by hormonal and other stimuli, By substitut ing histidine for a conserved arginine residue, the mutation removes a n internal salt bridge (to a conserved glutamate) that normally acts a s an intramolecular hasp to maintain tight binding of the gamma-phosph ate of GTP, In its basal, unperturbed state, the mutant alpha(s) binds guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a GTP analog, s lightly less tightly than does normal alpha(s), but (in the GTP[gamma S]-bound form) can stimulate adenylyl cyclase, The activation defect b ecomes prominent only under conditions that destabilize binding of gua nine nucleotide (receptor stimulation) or impair the ability of alpha( s) to bind the gamma-phosphate of GTP (cholera toxin, AlFJ(4)(-) ion), Although GDP release is usually the rate-limiting step in nucleotide exchange, the biochemical phenotype of this mutant cu, 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.