Biology subjects

Takada, Y. K.

Publications and source records attributed to Takada, Y. K..

7 recordsLinked to original sources

bioRxiv2024

FGF2 binds to the allosteric site (site 2) and activates αvβ3 integrin and FGF1 binds to site 2 but suppresses integrin activation by FGF2: a potential mechanism of anti-inflammatory action of FGF1

FGF1 is known as an anti-inflammatory and has suppresses insulin resistance. Its homologue FGF2 is pro-inflammatory. Mechanism of FGF1s anti-inflammatory action and FGF2s pro-inflammatory action are unknown. Several inflammatory cytokines (e.g., CX3CL1, CCL5, and CXCL12, and CD40L) bind to the classical ligand (RGD)-binding site (site 1) of integrin v{beta}3. In addition, they bind to the allosteric site (site 2) of v{beta}3, which is distinct from site 1, and allosterically activate v{beta}3. Site 2 is involved in inflammatory signals since inflammatory lipid mediator 5-hydroxycholesterol binds to site 2 and induces integrin activation and inflammatory signals (e.g., TNF and IL-6 secretion). We thus hypothesized that FGF1 and FGF2 bind to site 2 and affect activation status of integrins. Here we describe that FGF2 bound to site 2 and allosterically activated v{beta}3 integrin. Point mutations in the site 2-binding interface of FGF2 suppressed this activation, indicating that FGF2 binding to site 2 is required for inducing integrin activation. In contrast, FGF1 bound to site 2 but did not activate v{beta}3, and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2. These findings suggest that FGF1s anti-inflammatory action is mediated by blocking site 2. FGF1 has potential as an anti-inflammatory agent, but is not appropriate for long-term use since it is potent mitogen. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of v{beta}3, suppressed v{beta}3 activation by FGF2 as effectively as WT FGF1. We propose that FGF1 R50E has therapeutic potential for inflammatory diseases.

biochemistry↗

bioRxiv2024

FGF2 binds to the allosteric site (site 2) and activates integrin αIIbβ3 and FGF1 binds to site 2 but suppresses integrin activation by FGF2: A potential mechanism of anti-thrombotic action of FGF1.

It has been believed that platelet integrin IIb{beta}3 recognizes fibrinogen and several ECM proteins, and we recently showed that IIb{beta}3 binds to several inflammatory cytokines (e.g., CCL5, and CXCL12), which are stored in platelet granules. These ligands bind to the classical ligand (RGD)-binding site (site 1) of integrin IIb{beta}3. Also, they bind to the allosteric site (site 2) of IIb{beta}3, which is distinct from site 1, and allosterically activate IIb{beta}3. Site 2 is known to be involved in allosteric integrin activation and inflammatory signaling. FGF2 is also stored in platelet granules and known to be pro-thrombotic, but it is unclear if FGF2 binds to IIb{beta}3. We studied if FGF2 and its homologue FGF1 bind to IIb{beta}3 and induce allosteric activation. FGF1 (not stored in platelet granules) is known to be anti-thrombotic. Mechanism of FGF1s anti-thrombotic action is unknown. Here we describe that FGF1 and FGF2 bound to site 1 of IIb{beta}3, indicating that IIb{beta}3 is a new receptor for FGF1/2. Notably, FGF2 bound to site 2 and allosterically activated IIb{beta}3. Point mutations in the site 2-binding interface of FGF2 suppressed this activation, indicating that FGF2 binding to site 2 is required for activation (FGF2 is an agonist to site 2). In contrast, FGF1 bound to site 2 but did not activate IIb{beta}3, and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of v{beta}3, suppressed IIb{beta}3 activation by FGF2 as effectively as WT FGF1. We propose that FGF1 R50E has therapeutic potential for anti-thrombosis.

biochemistry↗

bioRxiv2023

FGF9, a potent mitogen, is a new ligand for integrin αvβ3, and the FGF9 mutant defective in integrin binding acts as an antagonist.

FGF9 is a potent mitogen and survival factor, but FGF9 protein level is generally low and restricted to a few adult organs. Aberrant expression of FGF9 usually results in cancer. However, the mechanism of FGF9 action has not been fully established. Previous studies showed that FGF1 and FGF2 directly bind to integrin v{beta}3 and this interaction is critical for signaling functions (FGF-integrin crosstalk). FGF1 and FGF2 mutants defective in integrin binding were defective in signaling, whereas the mutants still bound to FGFR, and suppressed angiogenesis and tumor growth, indicating that they act as antagonists. We hypothesize that FGF9 requires direct integrin binding for signaling. Here we show that docking simulation of interaction between FGF9 and v{beta}3 predicted that FGF9 binds to the classical ligand-binding site of v{beta}3. We showed that FGF9 actually bound to integrin v{beta}3, and generated an FGF9 mutants in the predicted integrin-binding interface. An FGF9 mutant (R108E) was defective in integrin binding, activating FRS2 and ERK1/2, inducing DNA synthesis, cancer cell migration, and invasion in vitro. R108E suppressed DNA synthesis induced by WT FGF9 and suppressed DNA synthesis and activation of FRS2 and ERK1/2 induced by WT FGF9 (dominant-negative effect). These findings indicate that FGF9 requires direct integrin binding for signaling and that R108E has potential as an antagonist to FGF9 signaling.

biochemistry↗

bioRxiv2023

The heparin-binding domain of VEGF165 directly binds to integrin αvβ3 and plays a critical role in signaling.

VEGF-A is a key cytokine in tumor angiogenesis and a major therapeutic target for cancer. VEGF165 is the predominant isoform and is the most potent angiogenesis stimulant. VEGFR2/KDR domains 2 and 3 (D2D3) bind to the N-terminal domain (NTD, residues 1-110) of VEGF165. Since removal of the heparin-binding domain (HBD, residues 111-165) markedly reduced the mitogenic activity of VEGF165, it has been proposed that the HBD plays a critical role in the mitogenicity of VEGF165. Integrin v{beta}3 has been shown to bind to VEGF165, but the role of integrin v{beta}3 in VEGF165 signaling are unclear. Here we describe that v{beta}3 specifically bound to the isolated HBD, but not to the NTD. We identified several critical amino acid residues in HBD for integrin binding (Arg-123, Arg-124, Lys-125, Lys-140, Arg-145, and Arg-149) by docking simulation and mutagenesis, and generated full-length VEGF165 that is defective in integrin binding by including mutations in the HBD. The full-length VEGF165 mutant defective in integrin binding (R123A/R124A/K125A/K140A/R145A/R149A) was defective in ERK1/2 phosphorylation, integrin {beta}3 phosphorylation, and KDR phosphorylation, although the mutation did not affect KDR binding to VEGF165. We propose a model in which VEGF165 induces KDR (through NTD)-VEGF165 (through HBD)-integrin v{beta}3 ternary complex formation on the cell surface and this process is critically involved in potent mitogenicity of VEGF165.

biochemistry↗

bioRxiv2022

Pro-inflammatory chemokines CCL5, CXCL12, and CX3CL1 bind to and activate platelet integrin αIIbβ3 in an allosteric manner.

Previous studies showed that pro-inflammatory chemokines CX3CL1 and CXCL12 bound to the allosteric binding site (site 2) of integrins and allosterically activated integrins, in addition to the classical ligand-binding site (site 1). We showed that CCL5 also bound to site 2, in addition to site 1, and activated soluble integrin v{beta}3. Platelet integrin IIb{beta}3, a fibrinogen receptor, is critical for hemostasis and thrombus formation and activation of IIb{beta}3 is a key event for thrombus formation. Activation of IIb{beta}3 is known to be mediated exclusively by inside-out signaling. We studied if IIb{beta}3 can be allosterically activated. We discovered that CCL5, CXCL12, and CX3CL1 are new ligands for IIb{beta}3. Notably they enhanced the binding of monovalent ligand to soluble IIb{beta}3 in 1 mM Ca2+ by binding to site 2. They activated cell-surface IIb{beta}3 on CHO cells quickly (half maximal response <1 min) and at low concentrations (1-10 ng/ml) compared to soluble IIb{beta}3, probably because chemokines bind to cell surface proteoglycans. Notably, activation of IIb{beta}3 by the chemokines was several times more potent than 1 mM Mn2+. Since CCL5 and CXCL12 are stored in platelet granules and rapidly transported to the surface upon platelet activation, we hypothesized that they are released from the granules and allosterically activate IIb{beta}3 by binding to site 2. Transmembrane CX3CL1 on activated endothelial cells likely mediates platelet-endothelial interaction by binding to and activating IIb{beta}3. Also, over-produced chemokines during inflammation may trigger IIb{beta}3 activation, which is a possible missing link between inflammation and thrombosis.

biochemistry↗

bioRxiv2022

Anti-PF4 (heparin-independent)/PF4 complex induces allosteric activation of integrins αIIbβ3 and αvβ3, a potential mechanism of vaccine-induced thrombotic thrombocytopenia (VITT) and autoimmune diseases

The classical immune-mediated heparin-induced thrombocytopenia (HIT) is induced by autoantibody against platelet-factor 4 (PF4)/heparin complex. Vaccine-induced thrombotic thrombocytopenia (VITT) and autoimmune HIT (aHIT) are induced by anti-PF4 in a heparin-independent manner. Activation of platelet integrin IIb{beta}3 is a key event that leads to IIb{beta}3 binding to fibrinogen and platelet aggregation, but is not involved in current models of HIT or VITT. Anti-PF4 (heparin-independent) is also detected in autoimmune diseases (e.g., SLE). However, the role of anti-PF4 in these diseases is unknown. Previous studies showed that several pro-inflammatory chemokines potently activated integrins by binding to the allosteric site (site 2). PF4 is known to be inhibitory since it inhibits angiogenesis and tumor growth. Here we describe that PF4 was predicted to bind to site 2 of IIb{beta}3 by docking simulation, but did not activate it. However, PF4/anti-PF4 mAb (RTO, heparin-independent) complex potently activated it at biological concentrations of PF4 (<1 g/ml), but anti-PF4/heparin (KKO) did not. This indicates that RTO changed the phenotype of PF4. We generated PF4 mutants defective in site 2 binding to integrin by introducing mutations in the predicted site 2 binding site of PF4. A PF4 mutant/RTO complex was defective in activating integrins. Furthermore, this PF4 mutant acted as an antagonist of PF4/RTO-induced integrin activation. We obtained similar results with vascular integrin v{beta}3. We propose that a potential mechanism, in which PF4/RTO complex binds to site 2 and activates integrins and triggers thrombocytopenia or autoimmune diseases. The inhibitory PF4 mutant may have potential as a therapeutic.

immunology↗

bioRxiv2022

The C-type lectin domain of CD62P (P-selectin) is an integrin ligand.

CD62P (P-selectin) is confined to the inside of platelets and endothelial cells, and is translocated to the surface upon activation of platelets or endothelial cells. In current models, CD62P recognizes sialyl-Lewis X on PSGL-1 and mediates rapid rolling of leukocyte over vascular surfaces during the initial steps in inflammation. Docking simulation using integrin v{beta}3 as a target predicted that the C-type lectin domain of CD62P is a potential integrin ligand. It has not been tested if CD62P binds to integrins. Here we describe that the lectin domain of CD62P specifically bound to soluble integrins v{beta}3, IIb{beta}3, 4{beta}1 and 5{beta}1. Known inhibitors of CD62P-PSGL-1 interaction did not suppress the binding of the lectin domain to integrins. We found that the R16E/K17E mutation in the predicted integrin-binding interface of the lectin domain strongly inhibited CD62P binding to IIb{beta}3 and v{beta}3 in 1 mM Mn2+. R16E/K17E is outside of the glycan binding site. Mutating Glu-88 to Asp (the E88D mutation) in the lectin domain, which is known to strongly disrupt glycan binding, only slightly affected integrin binding, indicating that glycan binding and integrin binding sites are distinct. Also, the lectin domain of CD62P supported cell adhesion in a cation-dependent manner. CD62P-integrin interaction is potentially important since integrins are widely expressed compared to PSGL-1, which is limited to leukocytes. These findings indicate that CD62P-integrin interaction plays potentially important role in a wide variety of cell-cell interaction in addition to CD62P-glycan interaction.

cell biology↗