Considerable progress has been made in characterization of the insulin receptor, the receptor gene, and its mRNA species. Methods were developed to assess insulin receptor expression and function in vitro and in vivo. The receptor gene was analyzed by restriction fragment length polymorphisms and partial genomic cloning, and the structure of the multiple species of receptor mRNA determined. An in vitro translation system was developed to study the early steps of receptor biosynthesis. These techniques were then used to study the expression and function of the insulin receptor and its gene in physiologic and pathologic states. During the next 5 years we wish to continue these studies and apply a combination of biochemical and molecular biologic techniques to gain a more complete understanding of the structure of the insulin receptor, its biosynthesis and its regulation in disease states. Specifically we will continue to characterize the factors regulating expression of the insulin receptor mRNA and explore the possibility of new sites of alternative splicing. We will identify the domains of the receptor involved in the 3- dimensional insulin binding site by affinity labeling with a family of novel insulin analogues and characterize the role of specificity domains of the receptor in this process by in vitro mutagenesis. We will identify the specific cysteine residues involved in inter-subunit disulfide bonding and in high affinity insulin binding and study the effect of their modification of mutagenesis. We will characterize the insulin receptor in humans with syndromes of insulin resistance by cDNA and genomic cloning supplemented by amplification with the polymerase chain reaction, and screen a large population of Type II diabetics for receptor mutations using an RNAase protection assay and denaturing gradient gel electrophoresis. Taken together these data will help expand our understanding of the structure, synthesis and functional domains of the insulin receptor and their alterations in disease states.
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