The cloning of the human insulin receptor, with deliniation of its primary amino acid sequence, opens up new techniques for understanding the structural basis for insulin receptor function. While the binding domain has been assigned to the alpha subunit and the kinase domain to the proposed intracellular portion of the beta subunit, the location of these functional domains within the subunits cannot be predicted from the amino acid sequence or by homology to other proteins. In preliminary studies, we have demonstrated that it is possible to use peptides representing specific sequences of the insulin receptor to raise antibodies that react with the native insulin receptor. We propose to develop a panel of """"""""sequence specific antibodies"""""""" and use these antibodies, in combination with their peptides, to define the functional domains of the insulin receptor. Antibody affinity columns will be used to rapidily purify native, functional insulin receptor by gently and specifically eluting the receptor with low concentrations of peptide. The purified receptor, the peptide and the antibody would be used to develop a radioimmunoassay with an easily standardizable peptide tracer. The topology of the insulin receptor will be defined. The orientation of the insulin receptor in the plasma membrane will be determined by sandwich immunoassays. Immunoprecipitation and peptide mapping of receptor fragments produced by proteolysis will be used to identify the insulin binding site and determine which amino acids are glycosylated. The antibodies will be used to explain the structural basis for the immunological similarities and differences between the insulin receptor and the IGF-I receptor. The sites of phosphorylation, ATP binding, and kinase activity will be determined by a combination of peptide mapping, identification using antibodies, and sequencing. Antibodies that inhibit the insulin receptor kinase and/or receptor autophosphorylation will be used to explore the role of insulin induced phosphorylation in the biological activity of insulin. Antibodies against the external face of the insulin receptor will be tested for their ability to inhibit insulin binding, mimic insulin action, and induce insulin resistance as a model for the type B syndrome of insulin resistance and acanthosis nigricans.
