Involvement of b-arrestin ubiquitination in agonist-induced down regulation of the m2 mAChRs), 2004
Ford, Knatokie M.
2004-05-01
Muscarinic acetylcholine receptors (mAChRs) belong to a superfamily of G- protein coupled receptors that are characterized by seven transmembrane spanning domains. The M2 mAChR subtype plays a key role in regulating cholinergic transmission in the central and peripheral nervous system. Therefore, analyzing factors that regulate the expression, function, and trafficking of this receptor is vital. The focus of this study was to elucidate mechanisms responsible for mediating agonist-induced down-regulation of the M2 mAChR. To clarify whether down-regulation of the M2 mAChR involved j3- arrestin, mouse embryonic fibroblasts (MEFs) derived from /3-arrestin knockouts that lack expression of either one (KOI or K02) or both isoforms (KOl/2) of /3-arrestin 1 and 2 were utilized. Saturation radioligand binding assays were performed with the tritium- labeled membrane permeable muscarinic antagonist, [3H]-QNB, to examine agonist- induced down-regulation of FLAG-tagged porcine M2 mAChRs transiently expressed in MEF KOI, K02 and KOl/2. The data indicates that M2 mAChR down-regulation is (3- arrestin dependent. Furthennore, the receptor showed no selectivity between the two /3- arrestin isoforms in mediating agonist-induced down-regulation. Agonist-induced down- regulation was both dose and time-dependent in MEF KOI and K02. The M2 mAChR was maximally down-regulated (30-50 %) six hours following stimulation. The researcher conducted experiments in Chinese hamster ovary (CHO) cells stably expressing human M2 mAChRs to determine whether /3-arrestin molecules undergo ubiquitination as a result of agonist stimulation. Immunoprecipitation combined with immunoblot analysis revealed that there was an increase in ubiquitination of exogenously expressed FLAG-tagged /3-arrestin 2 following agonist stimulation. This increase in ubiquitination appeared to be maximal after three minutes of agonist stimulation. Expression of a chimeric form of /3-arrestin 2 that is constitutively mono- ubiquitinated resulted in down-regulated M2 mAChRs in both stimulated and non- stimulated cells in MEF KOl/2. These results indicate that agonist-induced down- regulation of M2 mAChRs in MEFs is /3-arrestin mediated and that ubiquitination of /3- arrestin may serve as a sorting signal for receptor down-regulation. Pretreatment with lactacystin, a proteasomal inhibitor, attenuated agonist-induced down-regulation of M2 mAChRs transiently expressed in MEF KOI, indicating that lactacystin may either block ubiquitination of /3-arrestin, or inhibit trafficking of the receptor to late endosomes.
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Master of Science (MS)
Atlanta University
Department of Chemistry
Jackson, Darrell A.
Clark Atlanta University
Georgia--Atlanta
http://hdl.handle.net/20.500.12322/cau.td:2004_ford_knatokie_m
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