The overall goal of these studies is to investigate the mechanisms by which the cellular responses to the insulin-like growth factors (IGFs) are modified by the specific extracellular IGF binding proteins, IGFBP-1 and IGFBP-2. These IGFBPs localize in the extracellular matrix (ECM); the molecular components of the ECM and the structural regions of the IGFBP molecules which determine this localization will be defined. At least one of the IGFBPs (IGFBP-1) exists in a phosphorylated form. The mechanisms and regulation of IGFBP phosphorylation, and the effect of phosphorylation on IGFBP binding to ECM and to cell surfaces will be determined. Finally, the effects of ECM localization and phosphorylation of IGFBP-1 and IGFBP-2 on the cellular responses to IGF will be investigated. A longer range goal of this effort is to investigate the alterations of IGFBP, and to determine the contributions of IGFBP and IGF to the cellular proliferation of these lesions. To determine the structural regions of the IGFBP-1 and IGFBP-2 molecules that are necessary for binding to ECM and to cell surfaces, mutant forms of the molecules will be prepared by site-directed mutagenesis. The mutated cDNAs will be ligated into expression vectors and transfected into Chinese hamster ovary cells. Regions of the IGFBP molecules appropriate for mutagenesis will be selected on the basis of structurally determined regions that are necessary for binding. The mutant proteins will be used to precisely identify the key regions of the IGFBP molecules necessary for binding. To determine the binding site for IGFBP in the ECM, defined components of ECM as well as intact ECM isolated from cultured cells will be assayed for binding affinity to IGFBPs. The proteoglycan component of intact ECM from these cells will be extracted and, if found to bind to IGFBPs, it will be purified. The cell surface receptor for the IGFBPs will be purified, and analyzed for similarities to the integrin family of cell membrane receptors. The capacity of multiple cell types to phosphorylate IGFBP-1 and IGFBP-2 will be evaluated. The location (intracellular vs. cell surface associated) and regulation of the kinases responsible will be determined. The specific amino acids that are phosphorylated in both IGFBP-1 and IGFBP- 2 will be identified. The effects of phosphorylation on binding to ECM, cell surfaces, and to IGF will be determined. The biological effects of the binding of IGFBPs to ECM and to cells will be determined. Cells that express the mutated IGFBPs which have lost the ability to bind either to ECM or to cell surfaces will be used. Cell growth, DNA synthesis, and indicators of differentiation in response to IGF will be determined for these cells and compared to the same parameters in cells transfected with wild-type IGFBP. The biological effects of phosphorylation will be determined in a similar way, using cells expressing IGFBPs that have been mutated at phosphorylation sites and therefore are not phosphorylated.
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