STRUCTURAL AND FUNCTIONAL ANALYSIS OF HUMAN IGFBP-4

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DESCRIPTION: This new small grant application will study the molecular mechanisms by which insulin-like growth factor binding protein-4 (IGFBP-4) inhibits the action of insulin-like growth factor (IGF). Previous studies from this laboratory indicate that IGFBP-4 inhibits the proliferation of osteoblasts by specifically preventing the binding of IGF to its membrane receptor. Preliminary data also suggest that the putative sequence essential for IGF binding is located in the region between Thr92 and Gln114. A series of well designed recombinant DNA approaches will be utilized to first localize the IGF binding domain within the IGF binding sequence using mutagenesis. It is proposed that the IGF binding domain of IGFBP-4 is localized within the variable non-conserved region of the gene. The second hypothesis proposes that the potency of IGFBP-4 analogs to inhibit osteoblast proliferation is directly proportional to their affinity for IGFs as well as their ability to compete with IGF receptor for IGF binding. Recombinant DNA techniques including DNA deletion, mutagenesis and polypeptide synthesis will be used to localize the IGF binding domain. Studies described in Aim 2 will then test the ability of resultant IGFBP-4 analogs to prevent IGF binding to its cell surface and a purified receptor. In addition, their ability to inhibit IGF-induced cell proliferation in bone cells will be determined. Completion of these studies will lead to the identification of the IGF binding domain in IGFBP-4 and to the future development of new IGF analogs that are resistant to IGFBP-4. In addition, these studies will contribute to future IGFBP-4 structure-function studies that may lead to the development of more potent IGFBP-4 analogs that can be used to treat certain cancerous cells whose proliferation is dependent on the autocrine/paracrine actions of the IGFs.