Regulation of Neuronal Muscarinic Acetylcholine Receptor Number by Protein Glycosylation |
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Authors: | W Conrad Liles Neil M Nathanson |
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Institution: | Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington, U.S.A. |
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Abstract: | Tunicamycin, a potent inhibitor of protein glycosylation, was used to study the role of protein glycosylation in the regulation of muscarinic acetylcholine receptor (mAChR) number in cultures of N1E-115, a murine neuroblastoma cell line. At a concentration of 0.35 microgram/ml, tunicamycin inhibited macromolecular incorporation of 3H]mannose by 75-80%, whereas incorporation of 3H]leucine was reduced by only 10%. Treatment with tunicamycin caused a 30% decrease in total membrane mAChR number within 48 h as determined by a filter-binding assay using 3H]quinuclidinyl benzilate (3H]QNB), a highly specific muscarinic antagonist. Tunicamycin also inhibited the recovery of total membrane mAChR by 70% following carbachol-induced down-regulation. The rate of mAChR degradation (control t1/2 12-14 h) was unaffected by incubation with tunicamycin. Intact cell binding studies using 3H]QNB (a membrane-permeable ligand) to measure total cellular (internal plus cell surface) mAChR and 3H]N-methylscopolamine (3H]NMS, a membrane-impermeable ligand) to measure cell surface mAChR were conducted to determine whether tunicamycin selectively depleted cell surface mAChR. With 12 h of treatment with tunicamycin, cell surface mAChR number declined by 35%, whereas total cellular mAChR fell by only 10%. The ratio of cell surface receptor to total receptor decreased by 45% after 24 h. These results indicate that protein glycosylation is required for the maintenance of cell surface mAChR number. Incubation with tunicamycin causes a selective depletion of cell surface mAChR, implying that protein glycosylation plays a critical role in transport and/or incorporation of mAChR into the plasma membrane. |
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Keywords: | Muscarinic acetylcholine receptor Neuroblastoma Protein glycosylation Receptor regulation |
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