16 research outputs found
The Alternatively Spliced Acid Box Region Plays a Key Role in FGF Receptor Autoinhibition
SummaryUncontrolled fibroblast growth factor (FGF) signaling can lead to human malignancies necessitating multiple layers of self-regulatory control mechanisms. Fibroblast growth factor receptor (FGFR) autoinhibition mediated by the alternatively spliced immunoglobulin (Ig) domain 1 (D1) and the acid box (AB)-containing linker between D1 and Ig domain 2 (D2) serves as the first line of defense to minimize inadvertent FGF signaling. In this report, nuclear magnetic resonance and surface plasmon resonance spectroscopy are used to demonstrate that the AB subregion of FGFR electrostatically engages the heparan sulfate (HS)-binding site on the D2 domain in cis to directly suppress HS-binding affinity of FGFR. Furthermore, the cis electrostatic interaction sterically autoinhibits ligand-binding affinity of FGFR because of the close proximity of HS-binding and primary ligand-binding sites on the D2 domain. These data, together with the strong amino acid sequence conservation of the AB subregion among FGFR orthologs, highlight the universal role of the AB subregion in FGFR autoinhibition
Compositional Analysis of Heparin/Heparan Sulfate Interacting with Fibroblast Growth Factor·Fibroblast Growth Factor Receptor Complexes
Loss-of-function fibroblast growth factor receptor-2 mutations in melanoma
We report that 10% of melanoma tumors and cell lines harbor mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. These novel mutations include three truncating mutations and 20 missense mutations occurring at evolutionary conserved residues in FGFR2 as well as among all four FGFRs. The mutation spectrum is characteristic of those induced by UV radiation. Mapping of these mutations onto the known crystal structures of FGFR2 followed by in vitro and in vivo studies show that these mutations result in receptor loss of function through several distinct mechanisms, including loss of ligand binding affinity, impaired receptor dimerization, destabilization of the extracellular domains, and reduced kinase activity. To our knowledge, this is the first demonstration of loss-of-function mutations in a class IV receptor tyrosine kinase in cancer. Taken into account with our recent discovery of activating FGFR2 mutations in endometrial cancer, we suggest that FGFR2 may join the list of genes that play context-dependent opposing roles in cancer
Differential interactions of FGFs with heparan sulfate control gradient formation and branching morphogenesis
Analysis of Mutations in Fibroblast Growth Factor (FGF) and a Pathogenic Mutation in FGF Receptor (FGFR) Provides Direct Evidence for the Symmetric Two-End Model for FGFR Dimerization
Two competing models for fibroblast growth factor (FGF) receptor (FGFR) dimerization have recently emerged based on ternary FGF-FGFR-heparin crystal structures. In the symmetric two-end model, heparin promotes dimerization of two FGF-FGFR complexes by stabilizing bivalent interactions of the ligand and receptor through primary and secondary sites and by stabilizing direct receptor-receptor contacts. In the asymmetric model, there are no protein-protein contacts between the two FGF-FGFR complexes, which are bridged solely by heparin. To identify the correct mode of FGFR dimerization, we abolished interactions at the secondary ligand-receptor interaction site, which are observed only in the symmetric two-end model, using site-directed mutagenesis. Cellular studies and real-time binding assays, as well as matrix-assisted laser desorption ionization-time of flight analysis, demonstrate that loss of secondary ligand-receptor interactions results in diminished FGFR activation due to decreased dimerization without affecting FGF-FGFR binding. Additionally, structural and biochemical analysis of an activating FGFR2 mutation resulting in Pfeiffer syndrome confirms the physiological significance of receptor-receptor contacts in the symmetric two-end model and provides a novel mechanism for FGFR gain of function in human skeletal disorders. Taken together, the data validate the symmetric two-end model of FGFR dimerization and argue against the asymmetric model of FGFR dimerization
Structural basis by which alternative splicing modulates the organizer activity of FGF8 in the brain
Two of the four human FGF8 splice isoforms, FGF8a and FGF8b, are expressed in the mid-hindbrain region during development. Although the only difference between these isoforms is the presence of an additional 11 amino acids at the N terminus of FGF8b, these isoforms possess remarkably different abilities to pattern the midbrain and anterior hindbrain. To reveal the structural basis by which alternative splicing modulates the organizing activity of FGF8, we solved the crystal structure of FGF8b in complex with the “c” splice isoform of FGF receptor 2 (FGFR2c). Using surface plasmon resonance (SPR), we also characterized the receptor-binding specificity of FGF8a and FGF8b, the “b” isoform of FGF17 (FGF17b), and FGF18. The FGF8b-FGFR2c structure shows that alternative splicing permits a single additional contact between phenylalanine 32 (F32) of FGF8b and a hydrophobic groove within Ig domain 3 of the receptor that is also present in FGFR1c, FGFR3c, and FGFR4. Consistent with the structure, mutation of F32 to alanine reduces the affinity of FGF8b toward all these receptors to levels characteristic of FGF8a. More importantly, analysis of the mid-hindbrain patterning ability of the FGF8b(F32A) mutant in chick embryos and murine midbrain explants shows that this mutation functionally converts FGF8b to FGF8a. Moreover, our data suggest that the intermediate receptor-binding affinities of FGF17b and FGF18, relative to FGF8a and FGF8b, also account for the distinct patterning abilities of these two ligands. We also show that the mode of FGF8 receptor-binding specificity is distinct from that of other FGFs and provide the first biochemical evidence for a physiological FGF8b-FGFR1c interaction during mid-hindbrain development. Consistent with the indispensable role of FGF8 in embryonic development, we show that the FGF8 mode of receptor binding appeared as early as in nematodes and has been preserved throughout evolution