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

We have used wild-type and variants of the T-lymphoma cell line S49 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 degrees C on intact cells by the antagonists [3H]CGP-12177 or [125I]iodocyanopindolol, whereas down-regulation was defined as the loss in total cellular content of receptors [( 125I]iodocyanopindolol binding assayed at 37 degrees C). In wild-type cells, the beta-adrenergic agonist isoproterenol induced a rapid (t 1/2, approximately equal to 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 cyclic AMP 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-), had a loss in surface sites that was equivalent to that of wild-type cells. By contrast, 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 t 1/2 of approximately equal to 4 hr. 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 (t 1/2, approximately equal to 6 hr) in wild-type, UNC, and H21a cells. These results demonstrate that internalization of beta-adrenergic receptors may be necessary, but is not sufficient, 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.