The overall goal of this project is to understand the mechanisms of peptide hormone-receptor interaction and subsequent signal transduction at the molecular (protein) level. In addition to the insulin receptor that we have been purifying, we have established purification of the insulin-like growth factor (IGF)-I will be studied as model systems in this proposal. The two receptors share similar but complex structure. The subunit structure is disulfide-linked in a beta-alpha-alpha-beta form. Ligand-binding and tyrosine-specific protein kinase (TPK) activities are separately carried by the alpa and beta subunits, respectively. Upon ligand binding, this kinase is activated, which is now thought to be a prerequisite for signal transduction. This proposal covers the following specific aims: 1) Characterization of insulin and IGF-I receptor tyrosine protein kinases: Insulin and IGF-I receptors are generally believed to mediate metabolic and growth effects, respectively. Although 84% identical at the primakry structure level, their kinases should be qualitatively different if the two receptors have different roles and signal pathways. We will screen substrates, inhibitors and activators that are specific for each kinase. We will then use them for biochemical characterization of the kinases to understand differences in their signal transduction pathways. 2) Substructural domain analysis of the insulin receptor by limited proteolysis and protein microanalyses: Although x-ray chrystallography analysis has to be made to further reveal the exact structure of the insulin receptor, protein chemical studies such as domain sanalysis are essential to fill the gap existing between primary and three-dimensional structures. 3) Structural-functional relationships of the IGF-I receptor in comparison with the well-studied insulin receptor: Studies on the IGF-1 receptor have become more feasible since IGF-I and 125I-labeled IGF-I are now commercially available. In addition to the purified placental receptor, we will construct vectors using the IGF-I cDNA and express the intact receptor and the kinase domain in mammalian or insect expression systems. This will allow us to further characterize binding and kinase activities of the IGF-I receptor. The comparison of similarities and differences of the """"""""isoreceptors"""""""" in the proposed studies should lead to better understanding of IGF-I receptor function at the molecular level. These studies should also allow us to project three-dimensional organization of the two receptors from one- dimensional (primary structural) information.
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