title: Glycosylation defects alter insulin but not insulin-like growth factor I binding to Chinese hamster ovary cells.
abstract: Insulin binding to two Chinese hamster ovary cell lines with well-defined defects in their glycosylation pathway has been characterized and compared to insulin-like growth factor I (IGF-I) binding in the same cell lines. Insulin competition curves indicate that B4-2-1 cells, which transfer co-translationally to proteins an endoglycosidase H insensitive, truncated lipid-linked oligosaccharide, bind insulin with higher than normal affinity. Lec 1 cells, which fail to process oligosaccharide side chains to complex types, bind with a reduced affinity. The potencies of chicken and guinea pig insulins are appropriate for an insulin receptor in the control (WTB) and both mutant cell lines, whereas rat IGF-II is 3 times more potent than expected in the Lec 1 cells and human IGF-I is less potent than anticipated. Insulin bound to Lec 1 cells dissociates more quickly upon dilution than does insulin bound to either WTB or B4-2-1 cells. The Lec 1 insulin receptor is insensitive to pH change, whereas the other lines show the usual optimum of 8. 125I-IGF-I binds well to all three cell lines and is equally pH-sensitive in all three. Serum from a patient with circulating autoantibodies to the insulin receptor competes for insulin but not IGF-I binding, whereas alpha IR3, a monoclonal antibody directed toward the human IGF-I receptor inhibits IGF-I but not insulin binding. Cross-linking of either 125I-insulin or 125I-IGF-I reveals a typical alpha-subunit in the WTB and B4-2-1 cells but a band with faster mobility in the Lec 1 cells. Insulin (10(-8) M) stimulates autophosphorylation of a beta-subunit in all three lines, but again the Lec 1 subunit demonstrates an anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data demonstrate the differential effect of glycosylation on two closely related receptor molecules.
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D010053:Ovary / Q000378:metabolism*; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000276:immunology; D013002:Somatomedins / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D007328:Insulin; D013002:Somatomedins; D011972:Receptor, Insulin