Characterization of the beta 2-adrenoceptor-dependent adenylate cyclase of A431 epidermoid carcinoma cells

Abstract

In this study we characterize the beta 2 adrenergic dependent adenylate cyclase system of epidermoid carcinoma cells (A431). We show that the cells synthesize up to 130,000 [125I]-cyanopindolol binding sites per cell when freshly plated, a value which decreased to 40,000-50,000 receptors/cell within 24 hr. Production of this high number of receptors can be strongly inhibited by actinomycin D. We confirm and extend the fact that these beta-adrenoceptors are of the beta 2-subtype, using selective ligands, photoaffinity labeling with [125I]CYP-diazirine identified two protein subunits: p59 and p72, the beta 2-adrenoceptor dependent adenylate cyclase desensitizes with half-life of 2.2 +/- 0.3 min whereas the loss of [125I]CYP binding from the cell surface requires longer exposure times to the agonist, phorbol-12-myristate-13-acetate (PMA) has no effect on the desensitization process nor does it have any effect on the modulation of beta-agonist affinity by guanyl nucleotides. Rather, PMA was found to stimulate adenylate cyclase activation by forskolin. We conclude that protein kinase C is probably not involved in the beta-adrenoceptor desensitization in this cell line.