Abstract

α_1a-Adrenergic receptors (α_1aARs) are present intracellularly and at the cell surface in cultured and natural cell models, where they are subject to agonist-mediated desensitization and internalization. To explore α_1aAR trafficking, a hemagglutinin (HA)-tagged α_1aAR/enhanced green fluorescent protein (EGFP) fusion protein was expressed in rat-1 fibroblasts and tracked by EGFP fluorescence and antibody labeling of surface receptors. Confocal analysis of antibody-labeled surface receptors revealed unexpected constitutive internalization in the absence of agonist stimulation. In partial agreement, the inverse agonist prazosin also caused a modest 20 ± 2% increase in surface receptor levels, suggesting a partial block of constitutive internalization caused by decreased basal activation. However, prazosin was unable to prevent internalization of antibody-tagged surface receptors observed by confocal microscopy or cause obvious redistribution of intracellular receptor to the surface, suggesting that the α_1aAR is internalizing even in a basal-inactive state. In contrast to the α_1aAR, surface labeling of an HA-tagged α_1b-EGFP fusion protein did not result in any apparent constitutive internalization. Constitutive internalization of the α_1aAR seems to occur alongside reversible agonist-induced internalization, and both seem to involve clathrin-mediated endocytosis but not degradation in lysozymes. Surface receptor density must be maintained by recycling, because the protein synthesis inhibitor cycloheximide has no effect on total or surface receptor density in agonist-treated or untreated cells for 6 h. Constitutive agonist-independent trafficking of α_1aARs may provide a novel mechanism by which an internal pool of α_1aARs are maintained and recycled to allow continuous agonist-induced signaling.