This grant requests continuing support for an on-going study of the structure and turnover of the insulin receptor. Since insulin receptors are altered in a variety of diseases, including diabetes, this information is of utmost importance to human disease. Six major and related aspects of work are presented. Insulin receptors will be structurally characterized in human erythrocytes and fibroblasts, as well as in culture lymphocytes and hepatoma cells. The structure will be examined after biosynthetic and surface labeling techniques under reducing and non-reducing conditions using SDS poly-acrylamide gel electrophoresis. The stoichiometry, turnover, and biological significance of the multiple redox forms will be evaluated. The structure and degree of glycosylation and subcellular localization of the receptor during various stages of biosynthesis will be determined in pulse-chase labeling studies. The effect of hormones, drugs, and other agents which are known to alter receptor turnover will be studied. The nature and effect of biosynthetic processing of the pro-receptor will be determined. These same processes will be evaluated in cells obtained from individuals with a variety of insulin resistant states and mutant cell lines in tissue culture. Peptides will be isolated from specific, important functional domains of the receptor and sequenced. This will be compared to sequence data of possible related proteins. A large panel of monoclonal and polyclonal antibodies will be prepared. The antigenic domain recognized by each antibody will be determined, and the antibodies will be used to study receptor structure lines and cells from patients with insulin resistant states. Attempts will be made to utilize the Xenopus oocyte system for translation of insulin receptor mRNA. This will allow the quantitation of messenger RNA levels and the study of the early steps in receptor biosysthesis. Finally, attempts will be made to identify and clone the insulin receptor gene. Several strategies will be explored including preparation of oligonucleotide probes, antibody screening of cDNA library in a Lambda gt II expression vector, and transfection of the receptor gene into cells which can then be isolated by flourescence-activated cell sorting.
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