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 alpha chains participate in GTP binding and hydrolysis, we compared signaling functions of normal and mutated alpha chains (termed alpha s) of Gs, the stimulatory regulator of adenylyl cyclase. alpha s chains were expressed in an alpha s-deficient S49 mouse lymphoma cell line, cyc-. alpha s 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 alpha s is a poor activator of adenylyl cyclase. Moreover, the glycine 49 protein, unlike normal alpha s, 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 alpha s residues near glutamine 227 and glycine 49 participate in binding and hydrolysis of GTP, although the GTP binding regions of alpha s and p21ras are not identical.

The molecular genetic approach has just begun to provide hints of answers to some of the questions posed at the outset of this paper. We have some idea of which portions of the alpha chain of Gs interact with receptors and effectors, and we guess that the same is true of corresponding regions of other alpha chains. We have a tantalizing hint that points to a key region of alpha s that is necessary for the conformational change induced by binding GTP, and the vague outline of a hypothesis regarding the mechanism by which receptors release GDP from its binding site on the alpha chain. The alpha chain region (domain) that interacts with beta gamma remains unknown. The tenuous quality of all these hints and hypotheses is obvious, and at least in the short term, frustrating. Even the present level of our understanding, however, is impressive in comparison with what was known of G-protein function only 5 years ago. Now we can pose much more precise questions and hope that a combination of the molecular genetic approach with biophysical probes of structure will provide satisfying answers.