O-linked oligosaccharides on insulin receptor.
The insulin receptor, an integral membrane glycoprotein, is synthesized as a single-chain precursor that is cleaved to produce two mature subunits, both of which contain N-linked oligosaccharide chains and covalently linked fatty acids. We report that the beta-subunit also contains O-linked oligosaccharides. The proreceptor, alpha-subunit, and beta-subunit were labeled with [3H]mannose and [3H]galactose in the presence or absence of an inhibitor of O-linked glycosylation. Tryptic peptides from each component were separated by reverse-phase high-performance liquid chromatography. N- and O-linked oligosaccharide chains were identified on these peptides by specific enzymatic digestions. The proreceptor and alpha-subunit contained only N-linked oligosaccharides, whereas the beta-subunit contained both N- and O-linked oligosaccharides. The O-linked oligosaccharide chains were attached to a single tryptic fraction of the beta-subunit, which also contained N-linked chains. This fraction was further localized to the NH2-terminal tryptic peptide of the beta-subunit by specific immunoprecipitation with an anti-peptide antibody with specificity for this region. Binding of insulin and autophosphorylation of the beta-subunit were not dependent on O-linked glycosylation, because cells grown in the presence of the inhibitor exhibited a normal dose response to insulin. Therefore, the insulin receptor contains O-linked oligosaccharides on the NH2-terminal tryptic peptide of the beta-subunit, and these O-linked oligosaccharides are not necessary to the binding or autophosphorylation function of the receptor.
Collier
E
E
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Gorden
P
P
D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000581:Amidohydrolases / Q000494:pharmacology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000906:Antibodies; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D005690:Galactose / Q000378:metabolism; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis* / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010766:Phosphorylation; D011233:Precipitin Tests; D011972:Receptor, Insulin / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D014316:Tritium; D014357:Trypsin / Q000494:pharmacology
D000596:Amino Acids; D000906:Antibodies; D007328:Insulin; D009844:Oligosaccharides; D014316:Tritium; D011972:Receptor, Insulin; D006596:Hexosaminidases; C060638:glycopeptide alpha-N-acetylgalactosaminidase; D014357:Trypsin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000116:Acetylgalactosamine; D008358:Mannose; D005690:Galactose