Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of α1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells
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Continuous exposure of DDT1 MF-2 smooth muscle cells to 10-100 μM norepinephrine results in a dramatic attenuation of the ability of norepinephrine to stimulate inositol phospholipid hydrolysis via α1-adrenergic receptors (α1-AR). In addition to the functional desensitization, norepinephrine exposure also reduces the number of accessible cell surface α1-AR as assayed by [3H]prazosin binding at 4° C. Desensitization of the cells with norepinephrine results in an increase in the phosphorylation of the M(r) 80,000 α1-AR ligand binding peptide (2.4 ± 0.2 mol of 32P per mol of α1-AR; n = 5) when compared to control cells (1.1 ± 0.1 mol of 32P per mol of α1-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 α1-AR receptor antagonist phentolamine indicating that they are mediated via the α1-AR. Treatment of cells with the vasoactive peptide bradykinin (10 μM) induces desensitization of α1-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 α1-AR, with stoichiometries of 1.7 ± 0.1 (bradykinin; n = 5) and 3.6 ± 0.1 (PMA; n = 5) mol of 32P/mol of α1-AR. However, neither phorbol esters nor bradykinin reduce the number of accessible cell surface α1-AR. Similar phosphopeptide maps are obtained from tryptic phosphopeptides generated from phosphorylated α1-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.
|Research areas and keywords||
Subject classification (UKÄ) – MANDATORY
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1987 Nov 4|