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Fibroblast Growth Factor Receptor (FGFR) is connected to numerous downstream signalling cascades regulating cellular behavior. Any dysregulation leads to a plethora of illnesses, including cancer. Therapeutics are available, but drug resistance driven by gatekeeper mutation impedes the treatment. Ponatinib is an FDA-approved drug against BCR-ABL kinase and has shown effective results against FGFR-mediated carcinogenesis. Herein, we undertake molecular dynamics simulation-based analysis on ponatinib against all the FGFR isoforms having Val to Met gatekeeper mutations. The results suggest that ponatinib is a potent and selective inhibitor for FGFR1, FGFR2, and FGFR4 gatekeeper mutations. The extensive electrostatic and van der Waals interaction network accounts for its high potency. The FGFR3_VM mutation has shown resistance towards ponatinib, which is supported by their lesser binding affinity than wild-type complexes. The disengaged molecular brake and engaged hydrophobic spine were believed to be the driving factors for weak protein-ligand interaction. Taken together, the inhibitory and structural characteristics exhibited by ponatinib may aid in thwarting resistance based on Val-to-Met gatekeeper mutations at an earlier stage of treatment and advance the design and development of other inhibitors targeted at FGFRs harboring gatekeeper mutations., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The cartilages of marine fish, such as sharks and sturgeon, are important resources of the bioactive chondroitin sulfate (CS). To explore glycosaminoglycans from marine fish, polysaccharides from the cartilage of the sturgeon, Acipenser schrenckii, were extracted. Using enzyme-assisted extraction and anion-exchange chromatography, an uronic acid-containing polysaccharide, YG-1, was isolated. YG-1 is composed of GlcN, GlcUA, GalN, and Gal, in the ratio of 1.4: 3.4: 3.7: 1.0, and its molecular weight was determined to be 3.0 × 10 5  Da. YG-1 was confirmed to be chondroitin 4-sulfate (CS) composed of →4GlcAβ1→3GalNAc4Sβ1→ and minor →4GlcAβ1→3GalNAcβ1→, which was confirmed using IR spectroscopy, disaccharide composition analysis, and NMR. Bioactivity studies, including MTT assay and scratch-wound assays revealed that CS from Acipenser schrenckii had significant proliferation activity. The proliferation activity of the polysaccharide, YG-1, was related to Fibroblast growth factor 2 (FGF2). GalNAc 4S of YG-1 could be the binding sites of FGF2 and FGFR., Competing Interests: Declaration of competing interest None., (Copyright © 2020. Published by Elsevier B.V.)

Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated., Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN., Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fibroblast growth factor receptors (FGFRs) regulate diverse cellular behaviors that should be exquisitely controlled in space and time. We engineered an optically controlled FGFR (optoFGFR1) by exploiting cryptochrome 2, which homointeracts upon blue light irradiation. OptoFGFR1 can rapidly and reversibly control intracellular FGFR1 signaling within seconds by illumination with blue light. At the subcellular level, localized activation of optoFGFR1 induced cytoskeletal reorganization. Utilizing the high spatiotemporal precision of optoFGFR1, we efficiently controlled cell polarity and induced directed cell migration. OptoFGFR1 provides an effective means to precisely control FGFR signaling and is an important optogenetic tool that can be used to study diverse biological processes both in vitro and in vivo., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Kallmann syndrome (KS) is a developmental disease that expresses in patients as hypogonadotropic hypogonadism and anosmia. KS is commonly associated with mutations in the extracellular D2 domain of the fibroblast growth factor receptor (FGFR). In this study, for the first time, the molecular basis for the FGFR associated KS mutation (A168S) is elucidated using a variety of biophysical experiments, including multidimensional NMR spectroscopy. Secondary and tertiary structural analysis using far UV circular dichroism, fluorescence and limited trypsin digestion assays suggest that the KS mutation induces subtle tertiary structure change in the D2 domain of FGFR. Results of isothermal titration calorimetry experiments show the KS mutation causes a 10-fold decrease in heparin binding affinity and also a complete loss in ligand (FGF-1) binding. (1)H-(15)N chemical perturbation data suggest that complete loss in the ligand (FGF) binding affinity is triggered by a subtle conformational change that disrupts crucial structural interactions in both the heparin and the FGF binding sites in the D2 domain of FGFR. The novel findings reported in this study are expected to provide valuable clues toward a complete understanding of the other genetic diseases linked to mutations in the FGFR., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Heparin binding (HB) proteins mediate a wide range of important cellular processes, which makes this class of proteins biopharmaceutically important. Engineering HB proteins may bring many advantages, but it necessitates cost effective and efficient purification methodologies compared to currently available methods. One of the most important classes of HB proteins are fibroblast growth factors (FGFs) and their receptors (FGFRs). In this study, we report an efficient off-column purification of FGF-1 from soluble fractions and purification of the D2 domain of FGFR from insoluble inclusion bodies, using a weak Amberlite cation (IRC) exchanger. FGF-1 and the D2 domain have been expressed in Escherichia coli and purified to homogeneity using IRC resin. This approach is an alternative to conventional affinity column chromatography, which exhibits several disadvantages, including time-consuming experimental procedures for purification and regeneration and results in the expensive production of recombinant proteins. Results of the heparin binding chromatography and steady state fluorescence experiments show that the FGF-1 and the D2 are in a native conformation. The findings of this study will not only aid an in-depth investigation of this class of proteins but will also provide avenues for inexpensive and efficient purification of other important biological macromolecules., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The fibroblast growth factor receptor tyrosine kinases (FGFR1, 2, 3, and 4) represent promising therapeutic targets in a number of cancers. We have developed the first potent and selective irreversible inhibitor of FGFR1, 2, 3, and 4, which we named FIIN-1 that forms a covalent bond with cysteine 486 located in the P loop of the FGFR1 ATP binding site. We demonstrated that the inhibitor potently inhibits Tel-FGFR1-transformed Ba/F3 cells (EC(50) = 14 nM) as well as numerous FGFR-dependent cancer cell lines. A biotin-derivatized version of the inhibitor, FIIN-1-biotin, was shown to covalently label FGFR1 at Cys486. FIIN-1 is a useful probe of FGFR-dependent cellular phenomena and may provide a starting point of the development of therapeutically relevant irreversible inhibitors of wild-type and drug-resistant forms of FGFR kinases., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCgamma binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCgamma as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR.

The 22 members of the fibroblast growth factor (FGF) family have been implicated in cell proliferation, differentiation, survival, and migration. They are required for both development and maintenance of vertebrates, demonstrating an exquisite pattern of affinities for both protein and proteoglycan receptors. Recent crystal structures have suggested two models for the complex between FGFs, FGF receptors (FGFRs) and the proteoglycan heparan sulphate that mediates signalling, and have provided insight into how FGFs show differing affinities for the range of FGFRs. However, the physiological relevance of the two different models has not been made clear. Here, we demonstrate that the two complexes can be prepared from the same protein components, confirming that neither complex is the product of misfolded protein samples. Analyses of the complexes with mass spectrometry and analytical ultracentrifugation show that the species observed are consistent with the crystal structures formed using the two preparation protocols. This analysis supports the contention that both of the crystal structures reflect the state of the molecules in solution. Mass spectrometry of the complexes suggests that the stoichiometry of the complexes is 2 FGF1:2 FGFR2:1 heparin, regardless of the method used to prepare the complexes. These observations suggest that the two proposed complex architectures may both have relevance to the formation of an in vivo signalling complex, with a combination of the two interactions contributing to the formation of a larger focal complex.

Some protein kinases are known to acquire resistance to selective small molecule inhibitors upon mutation of a conserved threonine at the ATP binding site to a larger residue. Here, we performed a comprehensive mutational analysis of this structural element and determined the cellular sensitivities of several disease-relevant tyrosine kinases against various inhibitors. Mutant kinases possessing a larger side chain at the critical site showed resistance to most compounds tested, such as ZD1839, PP1, AG1296, STI571, and a pyrido[2,3-d]pyrimidine inhibitor. In contrast, indolinones affected both wild-type and mutant kinases with similar potencies. Resistant mutants were established for pharmacological analysis of betaPDGF receptor-mediated signaling and allowed the generation of a drug-inducible system of cellular Src kinase activity. Our data establish a conserved structural determinant of protein kinase sensitivity relevant for both signal transduction research and drug development.

Fibroblast growth factors (FGFs) regulate a wide range of important cellular processes. The biological activities of FGFs are mediated by cell surface receptors (FGFRs). In the present study for the first time we report the cloning, expression, and characterization of the ligand (FGF)-binding D2 domain of human FGFR2. D2 domain is expressed in Escherichia coli in high yields (10 mg/L) as inclusion bodies. The protein is recovered by dissolving the inclusion bodies in 8 M urea and subsequently refolding on nickel affinity column. The protein is purified (to approximately 97% purity) to homogeneity using heparin-Sepharose affinity column. Far-UV circular dichroism data and chemical shift index plot based on 1H-alpha, 13C-alpha, 13C-beta, and 13carbonyl group chemical shifts suggest that D2 domain is an all beta-sheet protein consisting of 9 beta-strands. Isothermal titration calorimetry and equilibrium urea unfolding experiments show that recombinant D2 domain is in a biologically active conformation and binds strongly to its ligand (FGF) and to the heparin analog, sucrose octasulfate (SOS). Using a variety of triple resonance NMR experiments, complete assignment of 1H, 15N, and 13C resonances in D2 domain has been accomplished. The findings of the present study not only pave way for an in-depth investigation of the molecular mechanism(s) underlying the activation of FGF signaling but also provide avenues for the rational design of potent inhibitors against FGF-mediated pathogenesis.

GSK-3beta is a regulatory serine/threonine kinase with a plethora of cellular targets. Consequently, selective small molecule inhibitors of GSK-3beta may have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize the active site of GSK-3beta, we determined crystal structures of unphosphorylated GSK-3beta in complex with selective and non-selective ATP-mimetic inhibitors. Analysis of the inhibitors' interactions with GSK-3beta in the structures reveals how the enzyme can accommodate a number of diverse molecular scaffolds. In addition, a conserved water molecule near Thr138 is identified that can serve a functional role in inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the design of second-generation GSK-3beta inhibitors.

The use of a novel inducible FGF signalling system in the frog Xenopus laevis is reported. We show that the lipophilic, synthetic, dimerizing agent AP20187 is able to rapidly activate signalling through an ectopically expressed mutant form of FGFR1 (iFGFR1) in Xenopus embryos. iFGFR1 lacks an extracellular ligand binding domain and contains an AP20187 binding domain fused to the intracellular domain of mouse FGFR1. Induction of signalling by AP20187 is possible until at least early neurula stages, and we demonstrate that ectopically expressed iFGFR1 protein persists until late neurula stages. We show that activation of signalling through iFGFR1 can mimic a number of previously reported FGF activities, including mesoderm induction, repression of anterior development, and neural posteriorization. We show that competence to morphological posteriorization of the anteroposterior axis by FGF signalling only extends until about stage 10.5. We demonstrate that the competence of neural tissue to express the posterior markers Hoxa7 and Xcad3, in response to FGF signalling, is lost by the end of gastrula stages. We also show that activation of FGF signalling stimulates morphogenetic movements in neural tissue until at least the end of the gastrula stage.

We have previously cloned the alternatively spliced isoform of fibroblast growth factor receptor 3 (FGFR3DeltaAB) that lacks the acid box in the extracellular region. To understand the biological functions and signal transduction of these FGFR3 isoforms, we analyzed the effect of FGF1 in ATDC5 cells, chondroprogenitor cell lines overexpressing these isoforms. In response to FGF1, FGFR3 induced a marked cell-morphology change to a round shape, while FGFR3DeltaAB did not. Furthermore, FGFR3 induced complete growth arrest, whereas FGFR3DeltaAB induced only moderate growth inhibition. Both receptors induced the expression of the CDK inhibitor p21(CIP1). However, only FGFR3 induced STAT1 phosphorylation that mediates the transcriptional induction of p21(CIP1), although both FGFR3 isoforms could induce a strong activation of mitogen-activated protein (MAP) kinases. Taken together, the different biological responses mediated by FGFR3 and FGFR3DeltaAB appear to be due to a difference in their ability to utilize STAT1 pathway and signals involved in cell rounding., ((c)2002 Elsevier Science.)

This report describes 2 patients with a clinical and hematologic diagnosis of chronic myeloid leukemia (CML) in chronic phase who had an acquired t(8;22)(p11;q11). Analysis by fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR) indicated that both patients were negative for the BCR-ABL fusion, but suggested that the BCR gene was disrupted. Further FISH indicated a breakpoint within fibroblast growth factor receptor 1 (FGFR1), the receptor tyrosine kinase that is known to be disrupted in a distinctive myeloproliferative disorder, most commonly by fusion to ZNF198. RT-PCR confirmed the presence in both cases of an in-frame messenger RNA fusion between BCR exon 4 and FGFR1 exon 9. Expression of BCR-FGFR1 in the factor-dependent cell line Ba/F3 resulted in interleukin 3-independent clones that grew at a comparable rate to cells transformed with ZNF198-FGFR1. The growth of transformed cells was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002, the farnesyltransferase inhibitors L744832 and manumycin A, the p38 inhibitors SB202190 and SB203580 but not by the MEK inhibitor PD98059. The growth of BaF3/BCR-FGFR1 and BaF3/ZNF198-FGFR1 was not significantly inhibited by treatment with STI571, but was inhibited by SU5402, a compound with inhibitory activity against FGFR1. Inhibition with this compound was associated with decreased phosphorylation of ERK1/2 and BCR-FGFR1 or ZNF198-FGFR1, and was dose dependent with an inhibitory concentration of 50% of approximately 5 microM. As expected, growth of BaF3/BCR-ABL was inhibited by STI571 but not by SU5402. The study demonstrates that the BCR-FGFR1 fusion may occur in patients with apparently typical CML. Patients with constitutively active FGFR1 fusion genes may be amenable to treatment with specific FGFR1 inhibitors.

Models of the oligomeric FGF signaling complex, including those derived from crystal structures, vary in stoichiometry and arrangement of the three subunits comprised of heparin/heparan sulfate chains, FGFR tyrosine kinase and activating FGF. Here, using covalent affinity crosslinking of radiolabeled FGF7 to binary complexes of FGFR2IIIb and heparin, we show that two molecules of FGF7 contact each FGFR2IIIb. This supports models that propose a dimeric complex of two units with stoichiometry 1 FGF:1 FGFR in which each FGF contacts both FGFR. The bivalent FGF7 contact was dependent on the full-length amino terminus of FGF7alpha and the intracellular domain of FGFR2IIIb extending through the juxtamembrane domain and the beta1 and beta2 strands of the kinase which is required for ATP binding. We propose that the differences in crosslinking report differences in relationships among subunits in the ectodomain of the complex that are affected by the amino terminus of FGF and the FGFR intracellular domain. From this, we suggest the corollary that conformational relationships among subunits in the ectodomain are transmitted to the intracellular and ATP binding domains during activation of the complex., (Copyright 2001 Academic Press.)

An acquired chromosomal translocation, t(8;13)(p11;q11-12), observed in a distinctive type of stem cell leukemia/lymphoma syndrome, leads to the fusion of the 5' portion of ZNF198 and the 3' portion of FGFR1. ZNF198-FGFR1 fusion transcripts encode 4 to 10 zinc fingers, a proline-rich region, and the intracellular portion of the FGFR1 (fibroblast growth factor receptor 1) receptor tyrosine kinase. We demonstrate that the ZNF198 proline-rich region constitutes a novel self-association domain. When fused to the intracellular domain of FGFR1, the ZNF198 proline-rich region is sufficient to cause oligomerization, FGFR1 tyrosine kinase activation, and transformation of Ba/F3 cells to IL-3 independent growth. (Blood. 2000;96:699-704)

Epithelial cells, which express FGFR2IIIb, bind and respond to FGF-1, FGF-7 and FGF-10, but not FGF-2. Stromal cells, which bind and respond to FGF-1 and FGF-2, but not FGF-7 and FGF-10, express FGFR2IIIc or FGFR1IIIc. Here we show that when both isolated FGFR2betaIIIb and FGFR2betaIIIc or their common Ig module II are allowed to affinity select heparin from a mixture, the resultant binary complexes bound FGF-1, FGF-2, and FGF-7 with nearly equal affinity. In addition, FGF-2 and FGF-7 bound to both heparin-Ig module IIIb and IIIc complexes, but FGF-1 bound to neither Ig module III. The results show that in isolation both Ig modules II and III of FGFR2 can interact with heparin and that each exhibits a binding site for FGF. We suggest that the specificity of FGFR2IIIb and FGFR2IIIc is dependent on the cell membrane environment and heparin/heparan sulfate. Ig modules II and III cooperate both within monomers and across dimers with cellular heparan sulfates to confer cell type-dependent specificity of the FGFR complex for FGF., (Copyright 2000 Academic Press.)

A general procedure for the reliable preparation of large quantities of insoluble transmembrane peptides has been developed. Optimal couplings were obtained during synthesis by using high-temperature couplings in conjunction with O-(7-azabenzotriazol-1-yl)-1,1,3, 3-tetramethyluronium hexafluorophosphate (HATU) activation. Improved purification schemes were developed that use reverse- phase HPLC on a C1 column and elution with a 2:1 mixture of 1-propanol:1-butanol. Using these methods three very different transmembrane peptides all longer than 25 amino acids have been prepared: glycophorin-A, prion (110-137), and fibroblast growth factor receptor (368-397)., (Copyright 1999 Academic Press.)

Syndecans are a family of heparan sulfate proteoglycans implicated in cell-cell and cell-matrix interactions. To investigate the roles of syndecans in early development, we identified three syndecan family members in Xenopus laevis: Xsyn-1, Xsyn-2, and Xsyn-3. Xsyn-1 and Xsyn-2 are maternal mRNAs localized to the animal pole in blastulae, and are expressed in the ectoderm of gastrulae. In neurulae, Xsyn-1 is restricted to non-neural ectoderm and Xsyn-2 is restricted to neural ectoderm. In tailbud embryos, the three syndecans are expressed in adjacent, non-overlapping patterns. Xsyn-2 is expressed in the heart while Xsyn-1 is expressed in the underlying anterior endoderm. Xsyn-3 is expressed in the hindbrain, midbrain, and forebrain, while Xsyn-2 is expressed in the intervening regions. These results suggest that different members of the syndecan family have distinct developmental roles, perhaps acting as barriers to define tissue boundaries.

K-SAM/FGFR2 gene encodes a receptor tyrosine kinase which belongs to the fibroblast growth factor receptor family and is amplified and overexpressed in KATO-III gastric cancer cells. To characterize K-sam proteins in cancer cells, anti-K-sam rabbit polyclonal antibody PK1-2 was raised and used for the immunoprecipitation analysis. 135, 125, and 110-kDa transmembrane proteins were detected in KATO-III cell lysate, while a soluble truncated 85-kDa K-sam protein was found in the conditioned medium. The molecular size of the soluble K-sam protein does not match with those predicted from the secreted forms of the K-sam cDNA which have been cloned so far. The soluble K-sam protein was highly N-glycosylated like the transmembrane versions, and N-glycosylation appeared to be necessary for its release.

Interactions between the two classes of fibroblast growth factor receptors 1) the high affinity receptors (HAR) a membrane glycoprotein containing an intrinsic tyrosine kinase activity, 2) low affinity receptors (LAR) cell surface proteoglycans containing heparan sulfate side chains (HSPG), and aFGF (MW: 15.5 kDa) were studied in bovine lens epithelial (BEL) cells. By Scatchard analysis of the aFGF binding to the BEL cell surface, we show that heparin at 10 micrograms/ml abolishes completely aFGF binding to LAR and reduces by half the number of aFGF HAR. By using cross-linking experiments, aFGF-HAR complexes are present in two forms (150 kDa and 135 kDa). Addition of heparin at 10 micrograms/ml abolishes the formation of the 150 kDa complex and does not affect the 135 kDa complex. Furthermore, binding of aFGF to LAR induces the spontaneous formation of a 31 kDa aFGF dimer. The dimerization process of aFGF on LAR is abolished by addition of heparin. During aFGF internalization at 37 degrees C, we have shown that aFGF-dimer is internalized, accumulated and degraded in the cell as is the 15.5 kDa native form. Heparin at 10 micrograms/ml suppresses specifically aFGF dimer internalization and reduces by half the total amount of internalized aFGF native form. Moreover, after aFGF binding and internalization, the affinity of HAR for aFGF increases concomitantly with its downregulation. Heparin does not seem to affect this phenomenon. All these results strongly suggest that an heteroreceptor dimer-aFGF complex (150 kDa) is formed by one molecule of HAR associated to one molecule of LAR through their respective interaction with a very stable homodimer of aFGF. Such a three component receptor complex induced by FGF dimerization may be a general process of FGF receptor activation which could explain the diversity of the biological response to FGF of different cell type expressing different HAR and LAR or HSPG.