Exchange of guanine nucleotide between GTP-binding proteins that regulate neuronal adenylate cyclase.
GTP-binding proteins have been demonstrated to stimulate and inhibit rat brain adenylate cyclase without the prior addition of hormone. Exposure of rat cerebral cortex membranes to hydrolysis-resistant GTP analogs results in inhibition (or stimulation) of adenylate cyclase, which persists subsequent to buffer washing. The hydrolysis-resistant GTP photoaffinity probe P3-(4-azidoanilido)-P1-5' GTP (AAGTP) can promote a similar persistent inhibition of adenylate cyclase, and, after removal of unbound AAGTP and subsequent UV photolysis, AAGTP is covalently linked to the 40-kDa inhibitory GTP binding protein, GNi (inhibitory guanine nucleotide binding regulatory subunit of adenylate cyclase). Under conditions where the persistent inhibition of adenylate cyclase is overcome by subsequent incubation with 5'-guanylyl imidodiphosphate or NaF, AAGTP bound to the 40-kDa GNi protein is diminished while that bound to the 42-kDa stimulatory GTP-binding protein (GNs) is increased. Additionally, we have identified a 32-kDa protein that binds AAGTP with an affinity similar to that of GNs. This protein does not appear to be a byproduct of proteolysis as demonstrated by Staphylococcus aureus V8 protease digestion experiments, and it is not a substrate for ADP-ribosylation by bacterial toxins. The sum of the AAGTP bound by the GNi and GNs proteins is constant, and the transfer of nonphotoactivated AAGTP to GNs from GNi is stable to buffer washing. Furthermore, this alteration in the AAGTP-labeling pattern corresponds to the shift in adenylate cyclase from inhibition to stimulation. These data raise the possibility that hydrolysis-resistant GTP analogs might be exchanged directly between the GNi and GNs and that there exists some interaction between those proteins in the regulation of adenylate cyclase activity.
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism*; D002540:Cerebral Cortex / Q000201:enzymology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010777:Photochemistry; D051381:Rats; D013570:Synaptic Membranes / Q000201:enzymology
D000067956:Adenylyl Cyclase Inhibitors; D000345:Affinity Labels; D001386:Azides; C031907:GTP gamma-4-azidoanilide; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases