Regulation of neuronal muscarinic acetylcholine receptor number by protein glycosylation.
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.
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D050260:Carbohydrate Metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D007930:Leucine / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D014415:Tunicamycin / Q000494:pharmacology
D011976:Receptors, Muscarinic; D014415:Tunicamycin; D011813:Quinuclidinyl Benzilate; D002217:Carbachol; D007930:Leucine; D008358:Mannose