Conformational changes of adenylate cyclase regulatory proteins mediated by guanine nucleotides.

Cholera toxin, in the presence of r3’P]NAD+, catalyzes the specific radiolabeling of a M, = 42,000 subunit of a protein (G protein) which mediates guanine nucleotide regulation of hormone-sensitive adenylate cyclase in pigeon erythrocyte membranes. Treatment of labeled membranes with GMP and isoproterenol followed by extensive washing presumably releases tightly bound GDP from the G-protein regulatory site. In the absence of added guanine nucleotide, treatment of these membranes with trypsin results in the partial digestion of the M, = 42,000 radiolabeled peptide and production of several minor trypsin-specific fragments which remain associated with the membrane. Incubation of membranes with guanosine-5’-0-(3-thiotriphosphate) (GTPyS) alters the partial tryptic digestion of the M, = 42,000 radiolabeled peptide, resulting in a loss of the minor fragments and the quantitative generation of a M, = 41,000 peptide. GTPyS exposes a new site to tryptic cleavage which releases from the membrane an inactive fragment of the G-protein containing the Mr = 41,000 labeled peptide. Guanosine-5’-0-(2-thiodiphosphate) (GDPBS) fails to activate adenylate cyclase and does not cause the generation of the M, = 41,000 tryptic fragment. Incubation of membranes with G D P P does, however, decrease tryptic digestion of the M, = 42,000 labeled peptide. The effects of GTPyS and GDPBS on the partial tryptic digestion of the G-protein are competitive suggesting their action is mediated by a common binding site. Generation of the GTPyS-specific M, = 41,000 tryptic fragment from the M, = 42,000 toxin labeled peptide exhibits a time-dependent increase upon incubation of membranes with G T P y S which is similar to that for cyclase activation. Addition of isoproterenol to the GTPyS incubation diminishes the lag time for cyclase activation and the generation of the M, = 41,000 fragment. Human erythrocyte membranes possess the G-protein but functionally lack both the P-adrenergic receptor and catalytic adenylate cyclase. Incubation of cholera toxin-labeled human erythrocyte membranes with GTPyS followed by exposure to trypsin also generates a specific M, = 41,000 fragment.