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

We have monitored agonist-induced alpha(1B)-adrenergic receptor (alpha(1B)AR) redistribution by immunocytochemical procedures in concert with functional measurements of agonist-elicited [H-3]inositol phosphate (InsP) production in human embryonal kidney 293 cells stably expressing alpha(1B)AR cDNA (HEK293/alpha(1B)). Anti-peptide antibodies directed against the carboxyl-terminal decapeptide of the alpha(1B)AR were prepared and shown to react specifically with alpha(1B)AR on immunoblots and in situ in HEK293/alpha(1B) transfectants. Treatment of HEK293/alpha(1B) cells with norepinephrine (10 mu 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 alpha(1) antagonist 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 nM) causes receptor endocytosis similar to agonist; agonist-induced internalization is blocked by the PKC inhibitor staurosporine (0.5 mu M). In parallel experiments, agonist-induced [H-3]InsP production is abolished by phorbol 12-myristate 13-acetate but potentiated by staurosporine. Inhibition of receptor internalization with hypertonic sucrose attenuates agonist-induced [H-3]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 alpha(1)AR stimulation induces receptor desensitization and internalization. Internalized receptor is reactivated and continuously recycled to the cell surface during agonist exposure.