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

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.