Evidence for α₁-adrenergic receptor internalization in DDT₁ MF-2 cells following exposure to agonists plus protein kinase C activators

Agonist-induced sequestration and internalization of α₁-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°. These pretreatments resulted in little or no change in the fraction of α₁-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 α₁-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° than was observed when cells were pretreated with agonist alone, induced the conversion of about half of cell surface α₁-adrenergic receptors to a form exhibiting limited accessibility to epinephrine in short-term assays, and induced a shift of about half of α₁-adrenergic receptors from the plasma membrane fraction to a light vesicle fraction on sucrose density gradients. A similar shift of α₁-adrenergic receptors was observed on sucrose density gradients after exposure to agonist plus either mezerein or β-phorbol didecanoate, but not with agonist plus α-phorbol didecanoate, indicating involvement of protein kinase C. These results suggest that pretreatment with agonist alone induces the sequestration of α₁-adrenergic receptors into a compartment that is inaccessible to [³H]prazosin at 4° but that is acccessible to hydrophilic ligands at 37° and remains associated with the plasma membrane. In contrast, α₁-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.