title: Evidence for alpha 1-adrenergic receptor internalization in DDT1 MF-2 cells following exposure to agonists plus protein kinase C activators.
abstract: 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.
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D011224:Prazosin / Q000378:metabolism; D011493:Protein Kinase C / Q000032:analysis*; D011942:Receptors, Adrenergic, alpha / Q000032:analysis*; D011943:Receptors, Adrenergic, beta / Q000032:analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D000316:Adrenergic alpha-Agonists; D011942:Receptors, Adrenergic, alpha; D011943:Receptors, Adrenergic, beta; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D011224:Prazosin