Molecular Pharmacology, Vol 20, 435-441, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Hormone-Sensitive Adenylate Cyclase

Mutant Phenotype with Normally Regulated Beta-Adrenergic Receptors Uncoupled from Catalytic Adenylate Cyclase

¹ Division of Clinical Pharmacology, Departments of Medicine and Pharmacology and the Cardiovascular Research Institute, University of California School of Medicine, San Francisco, California 94143

Adenylate cyclase in H21a, a recently isolated S49 mutant cell line, fails to synthesize cyclic AMP in response to hormones, cholera toxin, guanine nucleotides, and NaF. H21a membranes contain M_r = 42,000 and 52,000 peptide subunits of the guanine nucleotide-binding regulatory component (N protein) of adenylate cyclase. These peptides are ADP-ribosylated by cholera toxin in H21a, as in wild-type S49. Nonetheless, extracts of H21a membranes do not complement the functional defect of N-deficient cyc^- S49 membranes in vitro [M. R. Salomon and H. R. Bourne, Mol. Pharmacol. 19:109-116 (1981)]. We asked whether N in H21a can perform functions observed in wild-type S49 but absent or deficient in cyc^- and in another S49 mutant, unc. The N lesion in unc is thought to prevent interaction with receptors and allow normal interaction with the catalytic unit. Like wild-type, but unlike cyc^- and unc, membranes of H21a contained beta-adrenergic receptors that exhibited high affinity for binding an agonist (isoproterenol) in the absence, but not in the presence, of GTP. In addition, exposure of H21a cells to isoproterenol for 16 hr caused an 85% decrease in density of beta-adrenergic receptors. Agonist-induced down-regulation of beta-adrenergic receptors occurred to a similar extent in wild-type, but not in unc. These results suggest that the N protein in H21a can interact with hormone receptors, but not with catalytic adenylate cyclase. Two-dimensional gel electrophoresis showed that the size and charge of the M_r = 42,000 and 52,000 cholera toxin substrates are similar in wild-type and H21a membranes. The H21a and unc lesions appear to affect the same protein, because extracts of each type of mutant membrane failed to complement the adenylate cyclase defect of the other in vitro.

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ACKNOWLEDGMENT We thank Naomi Walker for technical assistance.

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