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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

Leeb-Lundberg, L. M.F. LU ; Cotecchia, S. ; DeBlasi, A. ; Caron, M. G. and Lefkowitz, R. J. (1987) In Journal of Biological Chemistry 262(7). p.3098-3105
Abstract

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... (More)

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.

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; ; ; and
publishing date
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Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
262
issue
7
pages
3098 - 3105
publisher
American Society for Biochemistry and Molecular Biology
external identifiers
  • pmid:3029100
  • scopus:0023225641
ISSN
0021-9258
language
English
LU publication?
no
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cdb60558-f91d-4155-bb53-ae4f3f66df2c
date added to LUP
2019-06-04 14:22:18
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2024-01-01 09:17:30
@article{cdb60558-f91d-4155-bb53-ae4f3f66df2c,
  abstract     = {{<p>Continuous exposure of DDT<sub>1</sub> 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 α<sub>1</sub>-adrenergic receptors (α<sub>1</sub>-AR). In addition to the functional desensitization, norepinephrine exposure also reduces the number of accessible cell surface α<sub>1</sub>-AR as assayed by [<sup>3</sup>H]prazosin binding at 4° C. Desensitization of the cells with norepinephrine results in an increase in the phosphorylation of the M(r) 80,000 α<sub>1</sub>-AR ligand binding peptide (2.4 ± 0.2 mol of <sup>32</sup>P per mol of α<sub>1</sub>-AR; n = 5) when compared to control cells (1.1 ± 0.1 mol of <sup>32</sup>P per mol of α<sub>1</sub>-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 α<sub>1</sub>-AR receptor antagonist phentolamine indicating that they are mediated via the α<sub>1</sub>-AR. Treatment of cells with the vasoactive peptide bradykinin (10 μM) induces desensitization of α<sub>1</sub>-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 α<sub>1</sub>-AR, with stoichiometries of 1.7 ± 0.1 (bradykinin; n = 5) and 3.6 ± 0.1 (PMA; n = 5) mol of <sup>32</sup>P/mol of α<sub>1</sub>-AR. However, neither phorbol esters nor bradykinin reduce the number of accessible cell surface α<sub>1</sub>-AR. Similar phosphopeptide maps are obtained from tryptic phosphopeptides generated from phosphorylated α<sub>1</sub>-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/Ca<sup>2+</sup> signaling pathway may represent an important mechanism of regulation of receptor responsiveness.</p>}},
  author       = {{Leeb-Lundberg, L. M.F. and Cotecchia, S. and DeBlasi, A. and Caron, M. G. and Lefkowitz, R. J.}},
  issn         = {{0021-9258}},
  language     = {{eng}},
  month        = {{11}},
  number       = {{7}},
  pages        = {{3098--3105}},
  publisher    = {{American Society for Biochemistry and Molecular Biology}},
  series       = {{Journal of Biological Chemistry}},
  title        = {{Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of α<sub>1</sub>-adrenergic receptors coupled to inositol phospholipid metabolism in DDT<sub>1</sub> MF-2 smooth muscle cells}},
  volume       = {{262}},
  year         = {{1987}},
}