Complexity of agonist- and cyclic AMP-mediated downregulation of the human beta 1-adrenergic receptor: role of internalization, degradation, and mRNA destabilization.
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.
Dunigan
Cheryl D
CD
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.
Hoang
Quang
Q
Curran
Patricia K
PK
Fishman
Peter H
PH
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
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