Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.

Abstract

DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain .alpha.1-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 .alpha.1-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 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular .alpha.1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the .alpha.1 receptors were purified .apprxeq. 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 .alpha.1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of .alpha.1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of .alpha.1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and .alpha.1-receptor phosphorylation showed the same rapid time course with a t1/2 < 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.