Glycosylation of the epidermal growth factor receptor in A-431 cells. The contribution of carbohydrate to receptor function.
A-431 cells were treated with inhibitors of either N-linked glycosylation (tunicamycin or glucosamine) or of N-linked oligosaccharide processing (swainsonine or monensin) to examine the glycosylation of epidermal growth factor (EGF) receptors and to determine the effect of glycosylation modification on receptor function. The receptor was found to be an Mr = 130,000 polypeptide to which a relatively large amount of carbohydrate is added co-translationally in the form of N-linked oligosaccharides. Processing of these oligosaccharides accounts for the 10,000-dalton difference in electrophoretic migration between the Mr = 160,000 precursor and Mr = 170,000 mature forms of the receptor. No evidence was found for O-linked oligosaccharides on the receptor. Mr = 160,000 receptors resulting from swainsonine or monensin treatment were present on the cell surface and retained full function, as judged by 125I-EGF binding to intact cells and detergent-solubilized extracts and by in vitro phosphorylation in the absence or presence of EGF. On the other hand, when cells were treated with tunicamycin or glucosamine, ligand binding was reduced by more than 50% in either intact cells or solubilized cell extracts. The Mr = 130,000 receptors synthesized in the presence of these inhibitors were not found on the cell surface. In addition, no Mr = 130,000 phosphoprotein was detected in the in vitro phosphorylation of tunicamycin or glucosamine-treated cells. It appears, therefore, that although terminal processing of N-linked oligosaccharides is not necessary for proper translocation or function of the EGF receptor, the addition of N-linked oligosaccharides is required.
D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors; D006026:Glycoside Hydrolases; D006027:Glycosides / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D006027:Glycosides; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D006026:Glycoside Hydrolases