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Regulation of Neuronal Muscarinic Acetylcholine Receptor Number by Protein Glycosylation
Author(s) -
Liles W. Conrad,
Nathanson Neil M.
Publication year - 1986
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1986.tb12929.x
Subject(s) - tunicamycin , muscarinic acetylcholine receptor , glycosylation , receptor , muscarinic acetylcholine receptor m3 , acetylcholine receptor , chemistry , cell surface receptor , muscarinic acetylcholine receptor m5 , biochemistry , biology , microbiology and biotechnology , endocrinology , apoptosis , unfolded protein response
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 μg/ml, tunicamycin inhibited macromolecular incorporation of [ 3 H]mannose by 75–80%, whereas incorporation of [ 3 H] 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 [ 3 H]quinuclidinyl benzilate ([ 3 H]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 t 1/2 12–14 h) was unaffected by incubation with tunicamycin. Intact cell binding studies using [ 3 H]QNB (a membrane‐permeable ligand) to measure total cellular (internal plus cell surface) mAChR and [ 3 H] N ‐methylscopolamine ([ 3 H]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.