Molecular Pharmacology, Vol 15, 16-27, Copyright © 1979 by the American Society for Pharmacology and Experimental Therapeutics

Isolation and Characterization of S49 Lymphoma Cells Deficient in -Adrenergic Receptors: Relation of Receptor Number to Activation of Adenylate Cyclase

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

Variant S49 lymphoma cells that have a diminished response to beta-adrenergic amines were selected from wild-type clones, using the beta-adrenergic agonist terbutaline in the presence of phosphodiesterase inhibitors. Variants (termed beta_p variants) having approximately 50% of wild-type beta-adrenergic receptors and 50% catecholamine-stimulated adenylate cyclase activity relative to wild-type were isolated in a single step selection. When these beta_p variants were mutagenized and subjected to a second round of selection with terbutaline and phosphodiesterase inhibitors, receptor deficient variants (termed beta_d) expressing 10-30% of wild-type beta-adrenergic receptors and responsiveness to isoproterenol were isolated. Two independent beta_p clones and the respective beta_d clones selected from them were characterized with respect to their adenylate cyclase and receptor properties. Both beta_p and beta_d variant cells demonstrated a diminished response to isoproterenol and an increased response to PGE₁ relative to wild-type. Membranes from all four receptor-deficient clones responded normally to Gpp(NH)p, NaF, and PGE₁.

The density of beta-adrenergic receptors in membranes measured by the specific binding of [¹²⁵I]IHYP was between 40 and 65%, relative to wild-type, for clones beta_p1 and beta_p2 and from 10 to 30% in beta_d1 and beta_d2. The dissociation constant for agonist and antagonist binding was the same for receptor-deficient and wild-type membranes. A direct relationship was obtained between beta-adrenergic receptor number and maximal activation of adenylate cyclase, indicating that beta-adrenergic receptor number limits the maximal activation of adenylate cyclase by catecholamines. These results conflict with the finding that isoproterenol stimulates S49 adenylate cyclase at concentrations well below those required to produce an equivalent fractional occupancy of receptors. The beta_p and beta_d phenotypes rule out an explanation of this discrepancy that depends on a number of receptors in excess of that required for maximal stimulation.

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