Epinephrine-induced sequestration of the beta-adrenergic receptor in cultured S49 WT and cyc- lymphoma cells.

Abstract

Pretreatment of either intact wild type S49 lymphoma cells (WT) or the uncoupled variants, cyc-, H21a, or UNC with epinephrine results in the redistribution of 20-30% of the beta-adrenergic receptors into a light vesicle fraction in sucrose gradients. Since the variants are uncoupled with respect to hormonal stimulation of adenylate cyclase, it appears that productive interaction with Gs is not required for the sequestration of beta-adrenergic receptors. Characterization of the epinephrine-induced redistribution of the beta-adrenergic receptor has revealed the following: The EC50 for the redistribution in WT cells was between 100 and 200 nM. Pretreatment of WT cells with 50 nM epinephrine for 30 min induced only a slight redistribution of receptors in sucrose gradients but produced a significant desensitization of adenylate cyclase. The desensitization was characterized by an increase in the Kact of epinephrine stimulation of adenylate cyclase while the Vmax was unaltered. Pretreatment with 10 microM epinephrine resulted in a significant decrease in the Vmax (50%) of epinephrine stimulation of adenylate cyclase and a 3-fold increase in Kact in the heavy vesicles. The beta-receptors in the light vesicle fraction of WT were uncoupled from adenylate cyclase and displayed low affinity for epinephrine binding, comparable to the cyc-. The "desensitized" receptor in the light vesicle fractions of cyc- was capable of stimulating adenylate following reconstitution with cholate extracts of WT membranes containing Gs. The molecular weight of the photolabeled beta-receptor in the light vesicle fractions (65,000 +/- 2,000) was not significantly different from the Mr 65,000 polypeptide photolabeled in the heavy fractions. The Mr 55,000 beta-receptor polypeptide was not detected in the light vesicles. Our results suggest first that the redistribution of the beta-receptor into light vesicles may follow an earlier stage of desensitization, and second that the beta-receptor in light vesicles while sequestered from Gs is capable of activating adenylate cyclase.