Guanosine 5′-triphosphate and guanosine 5′-[βγ-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase

Abstract

GTP, but not p[NH]ppG (guanosine 5''-[.beta..gamma.-imido]triphosphate), abolished the sensitivity of glucagon-stimulated adenylate cyclase to the lipid-phase separations occurring in the outer half of the bilayer in liver plasma membranes from rat. When either GTP or p[NH]ppG alone stimulated adenylate cyclase, the enzyme sensed only those lipid-phase separations occurring in the inner half of the bilayer. Trypsin treatment of intact hepatocytes had no effect on the basal, F-, GTP- or p[NH]ppG-stimulated adenylate cyclase activity. However, 125I-labeled-glucagon specific binding decayed with a half-life matching that of the decay of glucagon-stimulated adenylate cyclase activity. When GTP or p[NH]ppG were added to assays of glucagon-stimulated activity, the half-life of the trypsin-mediated decay of activity was substantially increased and the decay plots were no longer 1st-order. Trypsin treatment of purified rat liver plasma membranes abolished basal and all ligand-stimulated adenylate cyclase activity, and 125I-labeled-glucagon specific binding. Benzyl alcohol activated the GTP- and p[NH]ppG-stimulated activities in an identical fashion, whereas these activities were affected differently when glucagon was present in the assays. Guanine nucleotides may alter the mode of coupling between the receptor and catalytic unit. In the presence of glucagon and GTP, a complex of receptor, catalytic unit and nucleotide regulatory protein occurs as a transient intermediate, releasing a free unstable active catalytic unit. In the presence of p[NH]ppG and glucagon, the transient complex yields a relatively stable complex of the catalytic unit associated with a p[NH]ppG-bound nucleotide-regulatory protein.