CONTROL OF IGF-1 ACTIVITY BY INTERACTION WITH CELL ASSOCIATED BINDING PROTEINS

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The importance of the insulin-like growth factors (IGF-I and IGF-II) on local growth processes and in cell and tissue differentiation is now well-establish. Mechanisms that tightly control the expression and activity of IGF-I must exist because numerous cell types produce and respond to IGF-I. Many actions of both IGFs are mediated by the IGF-I receptor, a heterotetrameric tyrosine-specific protein kinase structurally related to the insulin receptor. The long-term goal of my laboratory has been to understand the functional interactions between IGF-I and its cell surface receptor and binding proteins. Our previous studies have elucidated the interactions between IGF-I receptor subunits that are necessary for high-affinity IGF-I binding and for IGF-I- stimulated autophosphorylation. In this application, two mechanisms that may control IGF-I activity, specifically, the interaction of IGF-I with cell-associated IGF binding proteins and the production of IGF-I as two proproteins which may require activation by limited proteolysis of their E domains, will be investigated. Three specific aims are proposed. First, the interaction of IGF binding proteins with the cell surface and/or extracellular matrix in human fibroblasts will be defined. We have previously characterized fibroblasts from a child with short stature (CSC) that produce abundant cell-associated IGF binding proteins. The interacting cellular molecules in CSC fibroblasts will be localized and purified, and their recognition sites will be characterized. Second, the structures of the oligosaccharide units of IGFBP-3, the major IGF binding protein in serum and in fibroblasts, will be analyzed. The functional importance of glycosylation for the translocation of IGFBP-3 through the cell, for its cellular association, and/or for its stability to proteolytic degradation will be examined. Third, the biological properties and activation of human IGF-IA and IGF-IB will be determined. In collaboration with Dr. Peter Rotwein (Project #1), we have expressed IGF-IA in a baculovirus system. Both IGF-IA and IGF-IB will be expressed and purified in amounts that will allow us to compare their bioactivity with mature 70 residue IGF-I and to elucidate their post-translational processing. It is anticipated that these studies will lead to a better understanding of the mechanisms that control IGF-I activity in normal growth.