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Phorbol esters promote α1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism

Leeb-Lundberg, L. M. LU ; Cotecchia, S. ; Lomasney, J. W. ; DeBernardis, J. F. ; Lefkowitz, R. J. and Caron, M. G. (1985) In Proceedings of the National Academy of Sciences of the United States of America 82(17). p.5651-5655
Abstract

DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain α1-adrenergic receptors (54,800 ± 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of α1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32P(i). These phorbol ester-treated cells also... (More)

DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain α1-adrenergic receptors (54,800 ± 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of α1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32P(i). These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular α1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the α1 receptors were purified ~300-fold from control and phorbol ester-treated 32P(i)-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the M(r) 80,000 α1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of α1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of α1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and α1-receptor phosphorylation showed the same rapid time course with a t( 1/2 )<min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.

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author
publishing date
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Contribution to journal
publication status
published
subject
in
Proceedings of the National Academy of Sciences of the United States of America
volume
82
issue
17
pages
5651 - 5655
publisher
National Acad Sciences
external identifiers
  • scopus:0000726422
  • pmid:2994039
ISSN
0027-8424
DOI
10.1073/pnas.82.17.5651
language
English
LU publication?
no
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c1133958-de2c-483a-b4bc-80a1650254f7
date added to LUP
2019-06-04 14:45:26
date last changed
2019-08-04 05:30:32
@article{c1133958-de2c-483a-b4bc-80a1650254f7,
  abstract     = {<p>DDT<sub>1</sub> MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain α<sub>1</sub>-adrenergic receptors (54,800 ± 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of α<sub>1</sub>-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the <sup>32</sup>P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with <sup>32</sup>P(i). These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular α<sub>1</sub> receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the α<sub>1</sub> receptors were purified ~300-fold from control and phorbol ester-treated <sup>32</sup>P(i)-prelabeled cells. As assessed by NaDodSO<sub>4</sub>/polyacrylamide gel electrophoresis, the M(r) 80,000 α<sub>1</sub>-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of α<sub>1</sub> receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of α<sub>1</sub> receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and α<sub>1</sub>-receptor phosphorylation showed the same rapid time course with a t( 1/2 )&lt;min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.</p>},
  author       = {Leeb-Lundberg, L. M. and Cotecchia, S. and Lomasney, J. W. and DeBernardis, J. F. and Lefkowitz, R. J. and Caron, M. G.},
  issn         = {0027-8424},
  language     = {eng},
  month        = {12},
  number       = {17},
  pages        = {5651--5655},
  publisher    = {National Acad Sciences},
  series       = {Proceedings of the National Academy of Sciences of the United States of America},
  title        = {Phorbol esters promote α<sub>1</sub>-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism},
  url          = {http://dx.doi.org/10.1073/pnas.82.17.5651},
  doi          = {10.1073/pnas.82.17.5651},
  volume       = {82},
  year         = {1985},
}