Phosphorylation of the mammalian β-adrenergic receptor by cyclic AMP-dependent protein kinase. Regulation of the rate of receptor phosphorylation and dephosphorylation by agonist occupancy and effects on coupling of the receptor to the stimulatory guanine nucleotide regulatory protein

J. L. Benovic, L. J. Pike, R. A. Cerione, C. Staniszewski, T. Yoshimasa, J. Codina, M. G. Caron, R. J. Lefkowitz

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Abstract

In some systems, such as the turkey erythrocyte, agonist-promoted phosphorylation of the β-adrenergic receptor appears to be associated with desensitization of the adenylate cyclase system. This process can be partially mimicked by cyclic AMP analogs. Accordingly, we have investigated the phosphorylation of the pure mammalian β-adrenergic receptor by the pure catalytic subunit of the cyclic AMP-dependent protein kinase. The β-adrenergic receptor, purified from hamster lung to apparent homogeneity, contains a single polypeptide of M(r) ~ 64,000. The receptor can be phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase (~ 2 mol of phosphate (on serine residues) per mol). Isoproterenol, a β-agonist, promoted a 2-3-fold increase in the rate of receptor phosphorylation which was blocked by the β-antagonists propranolol and alprenolol. High performance liquid chromatographic tryptic peptide mapping reveals two major phosphorylation sites. Phosphorylated receptor can be completely dephosphorylated by a high molecular weight phosphorylation phosphatase. The rate of receptor dephosphorylation is enhanced 2-3-fold by isoproterenol and this effect is blocked by alprenolol. The functional significance of receptor phosphorylation was examined using ligand binding and reconstitution techniques. While the binding of isoproterenol and alprenolol to the receptor was unaffected by phosphorylation, the ability of the receptor to interact with the stimulatory guanine nucleotide regulatory protein, as assessed by isoproterenol-promoted-GTPase activity, was decreased 24 ± 1% (mean ± S.E., p < 0.001, n = 17). The quantitative extent of receptor phosphorylation and functional impairment are virtually identical to those previously observed when intact turkey erythrocytes were incubated with cyclic AMP. These data provide a direct demonstration of regulation of the function of the isolated β-adrenergic receptor by cyclic AMP-dependent protein kinase.

Original languageEnglish
Pages (from-to)7094-7101
Number of pages8
JournalJournal of Biological Chemistry
Volume260
Issue number11
StatePublished - Dec 1 1985

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