CARBOHYDRATE OF THE INSULIN RECEPTOR

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The binding of insulin to its receptor initiates a series of responses which are critical to the metabolism of the target cell. The insulin receptor is known to be a cell surface glycoprotein and according to the current structural model consists of four disulfide linked subunits (alpha 2 beta 2) with the alpha-subunit containing the insulin binding sites while the transmembrane beta- subunit is responsible for signal transduction possibly via its tyrosine kinase activity. The complete amino acid sequence of the receptor protein has been reported based on a DNA clone of the insulin receptor precursor gene, but despite the fact the carbohydrate forms a substantial part of the molecule, little is known in regard to the structure and biological function of the attached saccharide units. Several studies have suggested, however, that carbohydrate may play an important role in the biological activity of the insulin receptor and its transport to the cell surface. The experiments proposed will examine the nature of the carbohydrate units present on the isolated alpha- and beta-subunits of the receptor as well as on its intracellular biosynthetic precursors. The receptor subunits will be obtained from human lymphocytes after metabolic radiolabeling ((3H) glucosamine, (3H) mannose) and will be treated with pronase to prepare glycopeptides. The detail structure of the carbohydrate units will be determined by specific glycosidase digestions, methylation analysis and periodate oxidation subsequent to release and isolation of the N- and O-linked saccharides as well as by lectin affinity chromatography. With the resulting structural information it will be possible to investigate the role of the carbohydrate units by selective removal of the sugars and evaluation of the modified receptor for insulin binding activity and for its ability to undergo insulin-stimulated autophosphorylation; moreover the involvement of carbohydrate in the control of alpha- and beta- subunit formation will be examined. These studies will result in a better understanding of the role of carbohydrate in the biogenesis of our understanding of Type II diabetes and insulin resistant states in which the number of active cell surface receptors is decreased. Furthermore, the information from these studies will represent a significant addition to the current model for the insulin receptor and will allow a logical approach to the study of how insulin action is dependent on receptor structure.