The fibroblast growth factor (FGF) family is a ubiquitous intrinsic mediator of development and adult homeostasis whose dysfunction potentially underlies tissue-specific pathologies. The FGF signal transduction complex signals changes in the local environment through assembly of its three participants into an oligomeric complex that undergoes trans-activation. The FGF polypeptides have grown to seventeen genetically distinct homologues. The transmembrane tyrosine kinase receptor (FGFR) consists of the products of at least 16 single site splice variations in four genes, which form combinatorial variants in the extracellular, intracellular juxtamembrane and C-terminal intracellular domains. The complex is an integral part of the peri-cellular matrix through its heparan sulfate subunit. Using combined immunochemical, biochemical, molecular and cellular approaches and collaborations for direct structural analysis, this continuation project will apply a modular approach to attempt to directly determine structure of the FGFR ectodomain by x-ray crystallography. It will determine whether isolated heparin-Ig module II from the four FGFR exhibits preference for FGF ligands and will determine the role of individual residues and subdomains in the 16-residue homeo-interaction sequence of FGFR in direct interaction, signaling by activation of Fire-Luc, acquisition of mitogenesis in epithelial cells and stoichiometry of the FGFR complex. The Fire-Luc system has been validated as a useful transcriptional reporter for FGFR activity. The project will determine the preference and interaction characteristics of defined oligosaccharides of heparin with FGF-1, FGF-7, FGF-10 and isolated Ig module II from FGFR1-4, and use the optimal oligosaccharides for co-crystallization with the respective molecules. The novel Fire-Luc transcriptional reporter for FGFR1 kinase activity and the acquisition of an FGFR1 mitogenic response in epithelial cells to characterize the role and specificity of the intracellular juxtamembrane sequence, the kinase core, the variant C-terminus and three specific phosphotyrosines and their combinations will be employed. The project will try to complete the resolution of the FGF-7 structure without and with bound heparin mimics and heparin oligosaccharides, as well as FGF-10 and a structural/functional chimeric FGF-7/FGF-1 construct. The work will perform and refine site-directed mutagenesis studies on FGF-7, FGF-10 and chimeric derivatives. These results are essential to understand the molecular basis of the multiple functions of FGF action in health and disease and the rational design of both ligand and receptor antagonists.
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