Post-translational glycosylation-induced activation of aglycoinsulin receptor accumulated during tunicamycin treatment.
Tunicamycin, which inhibits N-linked oligosaccharide chain addition to nascent polypeptides, interrupts glycosylation of the insulin receptor in 3T3-L1 adipocytes giving rise to inactive receptors. Chronic exposure of cells to low levels (100 ng/ml) of high performance liquid chromatography-purified tunicamycin causes a greater than or equal to 90% depletion of insulin binding to cell surface and Triton X-100-extractable receptors and a 93% inhibition of [3H]glucosamine incorporation into protein in alkali-stable form. Under identical conditions, protein synthesis was inhibited less than 10%. Recovery of insulin binding activity after the removal of tunicamycin achieves 70-80% of control activity within 36 h. Concomitant with the withdrawal of tunicamycin, cells were shifted to medium containing heavy (greater than 95% 15N, 13C, 2H) amino acids after which Triton X-100-solubilized "light" and "heavy" insulin receptors were separated isopycnically on CsCl density gradients. A kinetic analysis of the recovery of functional receptors revealed that the initial appearance of previously synthesized light receptor was followed, after a short lag, by newly synthesized heavy receptor. Similar levels of light receptor, but no new heavy receptor, accrue in the presence of cycloheximide. This strongly suggests that inactive aglycoinsulin receptor accumulated post-translationally during chronic treatment with tunicamycin and then re-entered the glycosylation pathway when the inhibitor was removed giving rise to a functional insulin receptor.
D000818:Animals; D002247:Carbon Isotopes; D002478:Cells, Cultured; D003903:Deuterium; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009587:Nitrogen Isotopes; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology*
D002247:Carbon Isotopes; D006027:Glycosides; D009587:Nitrogen Isotopes; C029957:aglycoinsulin receptor; D014415:Tunicamycin; D003903:Deuterium; D011972:Receptor, Insulin; D005944:Glucosamine