Evidence for alpha 1-adrenergic receptor internalization in DDT1 MF-2 cells following exposure to agonists plus protein kinase C activators

MS Cowlen and ML Toews

Department of Pharmacology, School of Medicine, University of Missouri- Columbia 65212.

Agonist-induced sequestration and internalization of alpha 1-adrenergic receptors were examined in DDT1 MF-2 cells. Pretreatment of cells with epinephrine or norepinephrine alone, but not with phorbol 12-myristate 13-acetate alone, resulted in a marked decrease in [3H]prazosin binding to intact cells at 4 degrees. These pretreatments resulted in little or no change in the fraction of alpha 1-adrenergic receptors exhibiting limited accessibility to the hydrophilic competing ligand epinephrine in short-time competition binding assays with intact cells and little or no change in the subcellular distribution of alpha 1-adrenergic receptors between the plasma membrane and light vesicle compartments as assessed by sucrose density gradient centrifugation assays. Pretreatment with a combination of agonist plus phorbol 12-myristate 13- acetate resulted in a greater decrease in [3H]prazosin binding at 4 degrees than was observed when cells were pretreated with agonist alone, induced the conversion of about half of cell surface alpha 1- adrenergic receptors to a form exhibiting limited accessibility to epinephrine in short-time assays, and induced a shift of about half of alpha 1-adrenergic receptors from the plasma membrane fraction to a light vesicle fraction on sucrose density gradients. A similar shift of alpha 1-adrenergic receptors was observed on sucrose density gradients after exposure to agonist plus either mezerein or beta-phorbol didecanoate, but not with agonist plus alpha-phorbol didecanoate, indicating involvement of protein kinase C. These results suggest that pretreatment with agonist alone induces the sequestration of alpha 1- adrenergic receptors into a compartment that is inaccessible to [3H]prazosin at 4 degrees but that is accessible to hydrophilic ligands at 37 degrees and remains associated with the plasma membrane. In contrast, alpha 1-adrenergic receptors are apparently internalized from the plasma membrane to a separate compartment, presumably an intracellular vesicle, when cells are pretreated simultaneously with a combination of agonist plus a protein kinase C activator.

Volume 34, Issue 3, pp. 340-346, 09/01/1988
Copyright © 1988 by American Society for Pharmacology and Experimental Therapeutics

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