This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Fibroblast growth factors are involved in a multitude of key cellular process such as angiogenesis, morphogenesis, differentiation and wound healing. The biological effects of FGFs are mediated by binding to their cell surface receptors (FGFRs). The constituents of the FGF signaling complex include the ligand (FGF), heparin and the extracellular ligand binding domain (ECD). We propose to characterize the structure of the FGF signaling complex using a variety of biophysical techniques including NMR spectroscopy. The three-dimensional solution structures of the three Ig-like extracellular domains (D1, D2 and D3) and the entire ECD domain will be determined using multidimensional NMR techniques. The affinity of the D1, D2 and D3 domains and the entire ECD to heparin and ligand (FGF) would be investigated using isothermal titration calorimetric studies. The conformational changes that possibly accompany ligand (FGF)-receptor interactions would be monitored by fluorescence and circular dichroism spectroscopy. Heparin and ligand binding sites on the individual extracellular domains (D1, D2 and D3) and the entire ECD domain would be mapped using a variety of NMR techniques such as, 15N/13C-filtered HSQC NOESY, transverse cross saturation, 1H-15N chemical shift perturbation and amide proton exchange monitored by 1H-15N HSQC spectra. Finally, we plan to determine the three-dimensional structures of the various gain-of-function mutants of FGFR to understand the structural basis for the various craniosynostosis syndromes. Successful achievement of the objectives of the proposal will provide valuable information for the rational design of therapeutic principles against FGF-induced disorders
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