Relation of glucagon-specific binding sites to glucagon-dependent stimulation of adenylyl cyclase activity in plasma membranes of rat liver.

Under conditions where both glucagon and hormonal stimulation of adenylyl cyclase activity increase with increasing concentrations of glucagon, it was found that hormonal stimulation was complete when occupation of the glucagon-specific binding sites was only 10 to 20% of that achieved upon prolonged incubation; thus, under these conditions, 80 to 90% of the glucagon which binds to liver plasma membranes does so without affecting adenylyl cyclase activity. Des-1-histidine glucagon, a competitive inhibitor of glucagon binding, when added 5 min after glucagon, completely inhibited hormonal stimulation, while at the same time only displacing 10 to 20% of glucagon from its binding sites; thus, under these conditions, there is glucagon bound to liver membranes which does not lead to hormonal stimulation, although bound labeled glucagon is in its biologically active form and the assay conditions used are optimal for determination of hormonal stimulation of adenylyl cyclase activity. More labeled glucagon was found to dissociate from liver membranes after 5 to 10 min of incubation in the presence of excess native unlabeled glucagon than in that of excess des-lhistidine glucagon, suggesting the existence of cooperative interactions between glucagon-specific binding sites. GTP stimulated dissociation of bound labeled glucagon from liver membranes under conditions which do not selectively affect the glucagon-stimulated adenylyl cyclase activity, suggesting that previously discovered effects of purine nucleotides on hormone binding and on hormonal stimulation may be unrelated phenomena. It was concluded that a large proportion (at least 80 to 90%) of the glucagon-specific sites in liver plasma membranes do not participate in the activation of adenylyl cyclase system by glucagon and that these sites cannot be called receptors until it is demonstrated that they produce an effect on cellular