Mutations in the GTP-binding site of GSα alter stimulation of adenylyl cyclase

Mutational replacements of specific residues in the GTP-binding pocket of the 21-kDa ras proteins (p21ras) reduce their GTPase activity. To test the possibility that the cognate regions of G protein α chains participate in GTP binding and hydrolysis, we compared signaling functions of normal and mutated α chains (termed αs) of Gs, the stimulatory regulator of adenylyl cyclase. αs chains were expressed in an αs-deficient S49 mouse lymphoma cell line, cyc-. αs in which leucine replaces glutamine 227 (corresponding to glutamine 61 of p21ras) constitutively activates adenylyl cyclase and reduces the kcat for GTP hydrolysis more than 100-fold. There is a smaller reduction in GTPase activity in another mutant in which valine replaces glycine 49 (corresponding to glycine 12 of p21ras). This mutant αs is a poor activator of adenylyl cyclase. Moreover, the glycine 49 protein, unlike normal αs, is not protected against tryptic cleavage by hydrolysis resistant GTP analogs; this finding suggests impairment of the mutant protein's ability to attain the active (GTP-bound) conformation. We conclude that αs residues near glutamine 227 and glycine 49 participate in binding and hydrolysis of GTP, although the GTP binding regions of αs and p21ras are not identical.