Lectins as Probes of Insulin Receptor Carbohydrate Composition: Studies in Glycosylation Mutants of Chinese Hamster Ovarian Cells with Altered Insulin Binding

Abstract

We recently reported marked differences in insulin binding properties in Chinese hamster ovarian cell mutants with genetic defects in protein N-glycosylation. To further characterize the role of insulin receptor carbohydrates, we have now studied the effect of lectins on [125I]insulin binding to wild type (WT) Chinese hamster ovarian cells and to two mutant cell lines: B4-2-1, to which insulin was previously shown to bind with higher affinity than normal, and Lec 1, to which insulin binds with much lower affinity. The results show that of four lectins that bound to WT cells, only wheat germ agglutinin and phytohemagglutinin-E competed with insulin binding to these cells, while Concanavalin A (ConA) and Erythrina cristagalli agglutinin (ECA) did not. After solubilization of the cells, however, a potent inhibition of insulin binding was also seen with ConA and ECA. This suggests that sugar determinants for ConA and ECA are present on the insulin receptor, but are not accessible at the surface of the cells. Mutant B4-2-1 cells, which are deficient in mannosylphosphoryldolichol synthase and .beta.-galactosidase, differed from WT cells in that ECA and ConA potently inhibited insulin binding in intact cells. This suggests that these lectin binding sites of or near the insulin receptor are more accessible at the cell surface in this mutant cell line. Mutant Lec 1 cells, deficient in N-acetylglucosaminyltransferase 1, cannot process N-linked carbohydrates form their oligomannose to their complex forms. In these cells, marked differences in the pattern of lectin inhibition were observed compared to that in WT or B4-2-1 cells. ConA exerted a strong inhibition of insulin binding to solubilized cell preparations. Its effect on intact cells was modest however, suggesting that in this mutant line exposure of the insulin receptor at the cell surface is not different from that in the WT cells. Neither ECA nor PHA inhibited [125I]insulin binding to either intact or solubilized Lec 1 cells, suggesting that the absence of sugar determinants for these two lectins may play a role in the very low insulin binding affinity previously reported in this cell line. In conclusion, these indirect studies with lectins suggest that the carbohydrate units of the insulin receptor are heterogeneous. While some may be important for proper exposure of the receptor at the cell surface, others may play a role in more intrinsic receptor properties.