The overall objectives of this research proposal are to develop a detailed understanding of the intramolecular subunit interactions responsible for mediating the ligand-dependent transmembrane activation of the insulin and IGF-1 receptor tyrosine kinase activities. The mature cell surface receptors function as alpha2/beta2 heterotetrameric complexes composed of two extracellular ligand binding alpha subunits which are disulfide-linked to two transmembrane beta subunits which also contain intracellular tyrosine kinase domains. In addition, to the homologous insulin and IGF-1 alpha2/beta2 holoreceptors, several studies have documented the presence of insulin/IGF-1 hybrid receptors composed of two non-identical alpha beta half-receptor species. In addition, in vitro assembly of wild type and mutant hybrid receptors have been used to characterize several of the functional properties of these receptor species. In this application we propose to determine a molecular basis for transmembrane signalling by utilizing both in vivo and in vitro approaches to generate various hybrid receptor species. In vitro assembly of various receptor hybrid combinations will be used to systematically dissect the specific binding, kinase and intramolecular subunit interactions responsible for ligand- stimulated transmembrane signalling. In addition, the relationship between intramolecular autophosphorylation with that of cross phosphorylation (intermolecular trans) will be assessed. Controlled expression of hybrid receptors in vivo will be used to directly determine the ligand-mediated receptor signalling via the homologous insulin and IGF-1 receptors as well as by the insulin/IGF-1 hybrid receptor species. Further, transgenic mice expressing the wild type and kinase-defective human insulin receptor will be prepared and characterized. The development of a transgenic mouse model of transdominant insulin and/or IGF-1 resistance will be an extremely valuable system to further define the molecular defects associated with certain types of diabetes in vivo..
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