Molecular Pharmacology, Vol 20, 694-703, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

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

¹ Section of Physiological Chemistry, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

In comparison with controls, membranes isolated from pigeon erythrocytes exposed to isoproterenol exhibit decreased adenylate cyclase activity. Fluoride and guanosine-5'-O-(3-thiotriphosphate) (GTPS) 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 K_act for either GTPS 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 solubilized 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 conformational changes in pigeon erythrocyte G-protein mediated by guanine nucleotides and hormone-receptor interactions. These involve partial tryptic digestion and peptide mapping of the cholera toxin-labeled M_r = 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 GTPS in the presence or absence of isoproterenol is observed in desensitized preparations, and (c) that the fraction of G-proteins found in the GTPS-specific conformation is significantly decreased in desensitized versus control membranes incubated with GTPS with or without isoproterenol. These findings indicate that alterations occur during desensitization which affect coupling of hormone receptor to G-protein and the GDP exchange reaction of adenylate cyclase.

Note:
ACKNOWLEDGMENT We thank Ms. Nancy Johnson for assistance in preparing the manuscript.

This article has been cited by other articles:

				B Strulovici, R. Cerione, B. Kilpatrick, M. Caron, and R. Lefkowitz Direct demonstration of impaired functionality of a purified desensitized beta-adrenergic receptor in a reconstituted system Science, August 24, 1984; 225(4664): 837 - 840. [Abstract] [PDF]