Functional Alterations in Components of Pigeon Erythrocyte Adenylate Cyclase following Desensitization to Isoproterenol

In comparison with controls, membranes isolated from pigeon erythrocytes exposed to isoproterenol exhibit decreased adenylate cyclase activity. Fluoride and guanosine-5’-O-(3-thiotriphosphate) (GTPyS) activations are lowered in desensitized membranes and in Lubrol PX extracts of membranes previously treated with isoproterenol and GMP to remove GDP bound to the guanine nucleotide regulatory protein (G-protein). The decreases are not due to changes in Kact for either GTPyS or isoproterenol. The affinity and number of [3H]dihydroalprenolol binding sites in desensitized membranes are similar to those found in control membranes. No functional differences are found between G-proteins solubiized from control and desensitized pigeon erythrocyte membranes as determined in reconstitution experiments with cyc membranes. This laboratory recently has described [J. Biol. Chem. 256:1459-1465 (1981)] techniques to monitor conforma-tional changes in pigeon erythrocyte G-protein mediated by guanine nucleotides and hormone-receptor interactions. These involve partial tryptic digestion and peptide map-ping of the cholera toxin-labeled Mr 42,000 subunit of the G-protein. This procedure, carried out on control and desensitized membranes, demonstrates (a) that isoproterenol has a diminished ability to alter the conformation of the G-protein in desensitized membranes, (b) that a lag in the conformational change induced by GTPyS in the presence or absence of isoproterenol is observed in desensitized preparations, and (c) that the fraction of G-proteins found in the GTPyS-specific conformation is significantly decreased in desensitized versus control membranes incubated with GTP-yS with or without isoproterenol. These findings indicate that alterations occur during desensitiza-tion which affect coupling of hormone receptor to G-protein and the GDP exchange reaction of adenylate cyclase