Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of alpha 1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells.
Continuous exposure of DDT1 MF-2 smooth muscle cells to 10-100 microM norepinephrine results in a dramatic attenuation of the ability of norepinephrine to stimulate inositol phospholipid hydrolysis via alpha 1-adrenergic receptors (alpha 1-AR). In addition to the functional desensitization, norepinephrine exposure also reduces the number of accessible cell surface alpha 1-AR as assayed by [3H]prazosin binding at 4 degrees C. Desensitization of the cells with norepinephrine results in an increase in the phosphorylation of the Mr 80,000 alpha 1-AR ligand binding peptide (2.4 +/- 0.2 mol of 32P per mol of alpha 1-AR; n = 5) when compared to control cells (1.1 +/- 0.1 mol of 32P per mol of alpha 1-AR; n = 5). The time courses of these three processes are all comparable being half-maximal within 1-2 min. These norepinephrine-promoted effects can be prevented by the alpha 1-AR receptor antagonist phentolamine indicating that they are mediated via the alpha 1-AR. Treatment of cells with the vasoactive peptide bradykinin (10 microM) induces desensitization of alpha 1-AR function similar to that induced by tumor-promoting phorbol ester treatment (Leeb-Lundberg, L. M. F., Cotecchia, S., Lomasney, J. W., DeBernardis, J. F., Lefkowitz, R. J., and Caron, M. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5651-5655). Both treatments also result in phosphorylation of the alpha 1-AR, with stoichiometries of 1.7 +/- 0.1 (bradykinin; n = 5) and 3.6 +/- 0.1 (PMA; n = 5) mol of 32P/mol of alpha 1-AR. However, neither phorbol esters nor bradykinin reduce the number of accessible cell surface alpha 1-AR. Similar phosphopeptide maps are obtained from tryptic phosphopeptides generated from phosphorylated alpha 1-AR derived from cells treated with norepinephrine, phorbol 12-myristate 13-acetate, and bradykinin. Phosphoamino acid analysis reveals that the various agents induce phosphorylation on both serine and threonine residues. Thus, phosphorylation of receptors linked to the inositol phospholipid/Ca2+ signaling pathway may represent an important mechanism of regulation of receptor responsiveness.
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002460:Cell Line; D004361:Drug Tolerance; D007700:Kinetics; D009130:Muscle, Smooth / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation; D011224:Prazosin / Q000378:metabolism; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism
D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D011942:Receptors, Adrenergic, alpha; D013912:Threonine; D012694:Serine; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin; D009638:Norepinephrine; D011224:Prazosin