AGONIST REGULATION OF ALPHA 1B-ADRENERGIC RECEPTOR SUBCELLULAR DISTRIBUTION AND FUNCTION

Abstract We have monitored agonist-induced α1B-adrenergic receptor (α1BAR) redistribution by immunocytochemical procedures in concert with functional measurements of agonist-elicited [3H]inositol phosphate (InsP) production in human embryonal kidney 293 cells stably expressing α1BAR cDNA (HEK293/α1B). Anti-peptide antibodies directed against the carboxyl-terminal decapeptide of the α1BAR were prepared and shown to react specifically with α1BAR on immunoblots and in situ in HEK293/α1Btransfectants. Treatment of HEK293/α1Bcells with norepinephrine (10 μ M) results in a rapid (5-15 min) and striking internalization of cell surface receptor as visualized by confocal immunofluorescence microscopy. Receptor redistribution is sustained in the presence of agonist, rapidly reversed upon agonist removal, and prevented by the α1antagonist prazosin. Receptor internalizes to endosomes, as shown by colocalization with transferrin receptor, an endosomal marker. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (50 n M) causes receptor endocytosis similar to agonist; agonist-induced internalization is blocked by the PKC inhibitor staurosporine (0.5 μ M). In parallel experiments, agonist-induced [3H]InsP production is abolished by phorbol 12-myristate 13-acetate but potentiated by staurosporine. Inhibition of receptor internalization with hypertonic sucrose attenuates agonist-induced [3H]InsP formation; this effect is reversed by concomitant inhibition of PKC with staurosporine. These results suggest that PKC-dependent phosphorylation occurring as a consequence of α1AR stimulation induces receptor desensitization and internalization. Internalized receptor is reactivated and continuously recycled to the cell surface during agonist exposure.