The fibroblast growth factors (FGFs) are a family of heparin- binding polypeptides which have a role in a wide array of biological processes including cell growth, differentiation angiogenesis, tissue repair and transformation FGFs mediate their biological effects by binding to high affinity cell surface receptors. The FGF receptor family contains four members and each of these receptors is capable of binding and responding to more than one type of FGF. Similar to other growth factor receptors, the FGF receptors possess intrinsic tyrosine kinase activity. Thus, FGF binding to the extracellular domain of a receptor leads to receptor autophosphorylation and substrate tyrosine phosphorylation. Although a number of substrates of other tyrosine kinase growth factor receptors have been identified, the substrates of the FGF receptors remain to be characterized. Thus, the mechanisms whereby FGF receptors transduce ligand binding into specific responses in target cells are still poorly understood. Furthermore, it is unclear why different cell types show distinct responses to FGFs. In order to bing to address these questions, the array of FGF receptors present on the surface of a cell which differentiates in response to FGF (PC12 cells) was compared with that present on the surface of a cell which proliferates in response to FGF (Swiss 3T3 fibroblasts). Both cell types have exclusively FGFR-1 on their surfaces, suggesting that there are cell type-specific FGF receptor substrates which connect receptor activation to a varety of distinct pathways. The research proposed in this grant application is designed to characterize two of these cell type-specific FGFR-1 signaling pathways. As a first approach to characterizing these signaling pathways, we have carried out a two hybrid screen utilizing the cytoplasmic domain of FGFR-1 as the bait and a PC12 cDNA library as the prey. A number of positive clones were obtained and we propose to use a combination of immunological, molecular and biological techniques to study the role of these putative receptor substrates in FGF receptor signaling in both PC12 cells and Swiss 3T3 fibroblasts. In addition, we plan to use a variety of different enzyme inhibitors to further characterize the proliferation and differentiation pathways initiated by activation of FGFR-1. In this way, we should be able to identify key factors involved in the regulation of cell type-specific signaling by FGFs. In addition, each of the substrates identified will provide a possible site at which the activities of FGFs on cells could be modulated, perhaps allowing for the development of new therapeutic strategies for the treatment of FGF-dependent disease.
