title: Complexity of agonist- and cyclic AMP-mediated downregulation of the human beta 1-adrenergic receptor: role of internalization, degradation, and mRNA destabilization.
abstract: Prolonged agonist exposure often induces downregulation of G protein-coupled receptors (GPCRs). Although downregulation of the prototypical beta(2)-adrenergic receptor (beta(2)AR) has been extensively studied, the underlying mechanisms have yet to be resolved. As even less is known about the beta(1)-subtype, we investigated the downregulation of human beta(1)AR stably expressed in Chinese hamster fibroblasts in response to the agonist isoproterenol or the cell-permeable, chlorophenylthio-cAMP (CPT-cAMP). While either effector mediated decreases in both beta(1)AR binding activity and steady-state beta(1)AR mRNA levels, there were significant differences in their actions. Whereas agonist-mediated downregulation of beta(1)AR followed first-order kinetics, that induced by CPT-cAMP was delayed for several hours and approximately 50% of the former. Furthermore, agonist but not CPT-cAMP induced beta(1)AR internalization, and inhibiting internalization also suppressed agonist-mediated downregulation. The latter, however, was more sensitive than the former to agonist concentration (EC(50) of 0.3 vs 48 nM). Thus, at < or =1 nM agonist, downregulation occurred without internalization and with a pattern similar to that mediated by CPT-cAMP. The amounts of beta(1)AR downregulated or internalized were proportional to initial receptor levels but reached saturation at approximately 2 and 3 pmol/mg of protein, respectively. The fate of beta(1)AR protein during downregulation was determined by immunoblotting with anti-C-terminal antibodies. In agonist-treated cells, beta(1)AR protein disappeared with time and without any immunoreactive degradation products. Agonist-mediated downregulation of the human beta(1)AR appears to be a complex process that consists of both agonist- and cAMP-specific components. The former involves both receptor internalization and degradation whereas the latter involves a reduction in receptor mRNA.
authors.affiliation: Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 49 Convent Drive, MSC 4440, Bethesda, MD 20892, USA.
authors.forename: Cheryl D
authors.initials: CD
authors.lastname: Dunigan
authors.forename: Quang
authors.initials: Q
authors.lastname: Hoang
authors.forename: Patricia K
authors.initials: PK
authors.lastname: Curran
authors.forename: Peter H
authors.initials: PH
authors.lastname: Fishman
mesh_terms: D058671:Adrenergic beta-1 Receptor Antagonists; D058672:Adrenergic beta-2 Receptor Antagonists; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000502:physiology*; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D020871:RNA Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D058671:Adrenergic beta-1 Receptor Antagonists; D058672:Adrenergic beta-2 Receptor Antagonists; D000318:Adrenergic beta-Agonists; D007976:Leupeptins; D010436:Pepstatins; D011480:Protease Inhibitors; D012333:RNA, Messenger; D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; C032854:leupeptin; D007545:Isoproterenol; C031375:pepstatin