title: Cellular trafficking of human alpha1a-adrenergic receptors is continuous and primarily agonist-independent.
abstract: Alpha1a-adrenergic receptors (alpha1aARs) 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 alpha1aAR trafficking, a hemagglutinin (HA)-tagged alpha1aAR/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 alpha1aAR is internalizing even in a basal-inactive state. In contrast to the alpha1aAR, surface labeling of an HA-tagged alpha1b-EGFP fusion protein did not result in any apparent constitutive internalization. Constitutive internalization of the alpha1aAR 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 alpha1aARs may provide a novel mechanism by which an internal pool of alpha1aARs are maintained and recycled to allow continuous agonist-induced signaling.
authors.affiliation: Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Morris
authors.forename: R Reyn
authors.initials: RR
authors.lastname: Price
authors.forename: Michael P
authors.initials: MP
authors.lastname: Smith
authors.forename: Beilei
authors.initials: B
authors.lastname: Lei
authors.forename: Debra A
authors.initials: DA
authors.lastname: Schwinn
mesh_terms: D000322:Adrenergic Agonists / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D003247:Conservation of Natural Resources; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D009638:Norepinephrine / Q000494:pharmacology; D011224:Prazosin / Q000494:pharmacology; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions
chemical_list: C547713:ADRA1A protein, human; C547715:Adra1a protein, rat; D000322:Adrenergic Agonists; D018340:Receptors, Adrenergic, alpha-1; D009638:Norepinephrine; D011224:Prazosin