Genetic analysis of beta-adrenergic receptor internalization and down-regulation.

Abstract

Wild-type and variants of the [mouse] T-lymphoma cell line S49 were used to explore internalization and down-regulation of adenylate cyclase-linked .beta.-adrenergic receptors. Internalization was defined by the loss in surface receptors detected at 4.degree. C on intact cells by the antagonists [3H]CGP-12177 [4-(3-tert-butylamino-2-hydroxy propoxy) benzimidazol-2-one] or [125I]iodocyanopindolol, whereas down-regulation was defined as the loss in total cellular content of receptors ([125I]iodocyanopindolol binding assayed at 37.degree. C). In wild-type cells, the .beta.-adrenergic agonist isoproterenol induced a rapid (t1/2, .apprxeq. 1 min) and reversible loss in surface receptors. The surface sites were lost at a rate similar to the rate of desensitization of .beta.-adrenergic receptor-mediated cAMP generation of S49 cells. A series of S49 variants (cyc-, UNC, H21a) having lesions in Ns (the guanine nucleotide binding protein that couples .beta.-receptors to adenylate cyclase) or with absent cAMP-dependent protein kinase activity (kin-), has a loss in surface sites that was equivalent to that of wild-type cells. S49 variant cells having lesions in Ns showed variable rates and extents of down-regulation of .beta.-adrenergic receptors. In wild-type and kin- S49 cells, .beta.-receptors down-regulated with a t1/2 of .apprxeq. 4 h. Down-regulation was blunted in the cyc- and UNC variants that have altered coupling of receptors to Ns, but it was faster in the H21a variant that retains receptor-Ns interaction. Recovery of receptors after down-regulation occurred at a similar rate (t1/2 .apprxeq. 6 h) in wild-type, UNC and H21a cells. Internalization of .beta.-adrenergic receptors may be necessary, but is not sufficent, to explain agonist-induced receptor down-regulation in S49 cells. The variable expression in the development of down-regulation in S49 variants implies that receptor-Ns interaction regulates the fate of receptors linked to the stimulation of adenylate cyclase.