Direct demonstration of glycosylation of insulin receptor subunits by biosynthetic and external labeling: evidence for heterogeneity.
Insulin receptors of human lymphocytes (IM-9 line) were biosynthetically labeled with [3H]glucosamine, [3H]galactose, [3H]fucose, or [3H]mannose. After solubilization in Triton X-100, cell extracts were immunoprecipitated with serum from a patient containing autoantibodies to the insulin receptor. Na-DodSO4/polyacrylamide gel electrophoresis of the immunoprecipitates under reducing conditions showed the presence of major labeled subunits of apparent Mr 134,000 and 98,000 and a minor component of Mr 206,000. The ratio of activity in the 134,000 versus 98,000 Mr bands varied from 2:1 for mannose to 1.2:1 for galactose. In addition, the receptor subunits could be demonstrated when the cell surface of intact lymphocytes was labeled with NaB3H4 by using either the galactose oxidase (acts on nonreducing terminal galactose and N-acetylgalactosamine) technique or the periodate (oxidizes sialic acid) technique. With the periodate treatment, NaB3H4 labeled preferentially the Mr 98,000 band. With the galactose oxidase procedure, on the other hand, NaB3H4 labeled only the Mr 134,000 band; prior treatment with neuraminidase increased the labeling of this band and also revealed the Mr 98,000 subunit. These data demonstrate that the major subunits of the insulin receptor are complex glycoproteins that have differences in the nonreducing ends of the carbohydrate chains. In the Mr 134,000 subunit, there appear to be more exposed galactosyl or N-acetylgalactosaminyl (or both) residues, whereas the Mr 98,000 subunit appears to have a higher degree of sialylation. These labeling techniques provide new tools to examine the role of the carbohydrate moiety in insulin receptor function and turnover.
D002478:Cells, Cultured; D005643:Fucose / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D046911:Macromolecular Substances; D008358:Mannose / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D011972:Receptor, Insulin / Q000378:metabolism*
D006023:Glycoproteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D005643:Fucose; D011972:Receptor, Insulin; D005944:Glucosamine; D008358:Mannose; D005690:Galactose