-
[show abstract]
[hide abstract]
ABSTRACT: Integrin αvβ3 plays a role in insulin-like growth factor-1 (IGF1) signaling (integrin-IGF1 receptor (IGF1R) cross-talk). The specifics of the cross-talk are, however, unclear. In a current model, "ligand occupancy" of αvβ3 (i.e. the binding of extracellular matrix proteins) enhances signaling induced by IGF1 binding to IGF1R. We recently reported that IGF1 directly binds to αvβ3 and induces αvβ3-IGF1-IGF1R ternary complex formation. Consistently, the integrin binding-defective IGF1 mutant (R36E/R37E) is defective in inducing ternary complex formation and IGF signaling, but it still binds to IGF1R. Like αvβ3, integrin α6β4 is overexpressed in many cancers and is implicated in cancer progression. Here, we discovered that α6β4 directly bound to IGF1, but not to R36E/R37E. Grafting the β4 sequence WPNSDP (residues 167-172), which corresponds to the specificity loop of β3, to integrin β1 markedly enhanced IGF1 binding to β1, suggesting that the WPNSDP sequence is involved in IGF1 recognition. WT IGF1 induced α6β4-IGF1-IGF1R ternary complex formation, whereas R36E/R37E did not. When cells were attached to matrix, exogenous IGF1 or α6β4 expression had little or no effect on intracellular signaling. When cell-matrix adhesion was reduced (in poly(2-hydroxyethyl methacrylate-coated plates), IGF1 induced intracellular signaling and enhanced cell survival in an α6β4-dependent manner. Also IGF1 enhanced colony formation in soft agar in an α6β4-dependent manner. These results suggest that IGF binding to α6β4 plays a major role in IGF signaling in anchorage-independent conditions, which mimic the in vivo environment, and is a novel therapeutic target.
Journal of Biological Chemistry 02/2012; 287(15):12491-500. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Crosstalk between integrins and FGF receptors has been implicated in FGF signaling, but the specifics of the crosstalk are unclear. We recently discovered that 1) FGF1 directly binds to integrin alphavbeta3, 2) the integrin-binding site and FGF receptor (FGFR) binding site are distinct, and 3) the integrin-binding-defective FGF1 mutant (R50E) is defective in inducing FGF signaling although R50E still binds to FGFR and heparin and induces transient ERK1/2 activation.
We tested if excess R50E affect DNA synthesis and cell survival induced by WT FGF1 in BaF3 mouse pro-B cells expressing human FGFR1. R50E suppressed DNA synthesis and cell proliferation induced by WT FGF1. We tested if WT FGF1 and R50E generate integrin-FGF1-FGFR ternary complex. WT FGF1 induced ternary complex formation (integrin-FGF-FGFR1) and recruitment of SHP-2 to the complex in NIH 3T3 cells and human umbilical endothelial cells, but R50E was defective in these functions. It has been reported that sustained ERK1/2 activation is integrin-dependent and crucial to cell cycle entry upon FGF stimulation. We thus determined the time-course of ERK1/2 activation induced by WT FGF1 and R50E. We found that WT FGF1 induced sustained activation of ERK1/2, but R50E was defective in this function.
Our results suggest that 1) R50E is a dominant-negative mutant, 2) Ternary complex formation is involved in FGF signaling, 3) The defect of R50E to bind to integrin may be directly related to the antagonistic action of R50E. Taken together, these results suggest that R50E has potential as a therapeutic in cancer.
PLoS ONE 01/2010; 5(4):e10273. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: It has been proposed that ligand occupancy of integrin alphavbeta3 with extracellular matrix ligands (e.g. vitronectin) plays a critical role in insulin-like growth factor-1 (IGF-1) signaling. We found that expression of alphavbeta3 enhanced IGF-1-induced proliferation of Chinese hamster ovary cells in serum-free conditions (in the absence of vitronectin). We hypothesized that the direct integrin binding to IGF-1 may play a role in IGF-1 signaling. We demonstrated that alphavbeta3 specifically and directly bound to IGF-1 in cell adhesion, enzyme-linked immunosorbent assay-type binding, and surface plasmon resonance studies. We localized the amino acid residues of IGF-1 that are critical for integrin binding by docking simulation and mutagenesis. We found that mutating two Arg residues at positions 36 and 37 in the C-domain of IGF-1 to Glu (the R36E/R37E mutation) effectively reduced integrin binding. Interestingly, although the mutant still bound to IGF1R, it was defective in inducing IGF1R phosphorylation, AKT and ERK1/2 activation, and cell proliferation. Furthermore wild type IGF-1 mediated co-precipitation of alphavbeta3 and IGF1R, whereas the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between alphavbeta3 and IGF1R. These results suggest that the direct binding to IGF-1 to integrin alphavbeta3 plays a role in IGF-1 signaling through ternary complex formation (alphavbeta3-IGF-IGF1R), and integrin-IGF-1 interaction is a novel target for drug discovery.
Journal of Biological Chemistry 08/2009; 284(36):24106-14. · 4.77 Impact Factor
-
Seiji Mori,
Chun-Yi Wu, Satoshi Yamaji,
Jun Saegusa,
Biao Shi,
Zi Ma,
Yasuko Kuwabara,
Kit S Lam,
R Rivkah Isseroff,
Yoko K Takada,
Yoshikazu Takada
[show abstract]
[hide abstract]
ABSTRACT: Integrins play a role in fibroblast growth factor (FGF) signaling through cross-talk with FGF receptors (FGFRs), but the mechanism underlying the cross-talk is unknown. We discovered that FGF1 directly bound to soluble and cell-surface integrin alphavbeta3 (K(D) about 1 microm). Antagonists to alphavbeta3 (monoclonal antibody 7E3 and cyclic RGDfV) blocked this interaction. alphavbeta3 was the predominant, if not the only, integrin that bound to FGF1, because FGF1 bound only weakly to several beta1 integrins tested. We presented evidence that the CYDMKTTC sequence (the specificity loop) within the ligand-binding site of beta3 plays a role in FGF1 binding. We found that the integrin-binding site of FGF1 overlaps with the heparin-binding site but is distinct from the FGFR-binding site using docking simulation and mutagenesis. We identified an FGF1 mutant (R50E) that was defective in integrin binding but still bound to heparin and FGFR. R50E was defective in inducing DNA synthesis, cell proliferation, cell migration, and chemotaxis, suggesting that the direct integrin binding to FGF1 is critical for FGF signaling. Nevertheless, R50E induced phosphorylation of FGFR1 and FRS2alpha and activation of AKT and ERK1/2. These results suggest that the defect in R50E in FGF signaling is not in the initial activation of FGF signaling pathway components, but in the later steps in FGF signaling. We propose that R50E is a useful tool to identify the role of integrins in FGF signaling.
Journal of Biological Chemistry 07/2008; 283(26):18066-75. · 4.77 Impact Factor
-
Seiji Mori,
Chun-Yi Wu, Satoshi Yamaji,
Jun Saegusa,
Biao Shi,
Zi Ma,
Yasuko Kuwabara,
Kit S. Lam,
R. Rivkah Isseroff,
Yoko K. Takada,
Yoshikazu Takada
[show abstract]
[hide abstract]
ABSTRACT: Integrins play a role in fibroblast growth factor (FGF) signaling through cross-talk with FGF receptors (FGFRs), but the mechanism
underlying the cross-talk is unknown. We discovered that FGF1 directly bound to soluble and cell-surface integrin αvβ3 (KD about 1 μm). Antagonists to αvβ3 (monoclonal antibody 7E3 and cyclic RGDfV) blocked this interaction. αvβ3 was the predominant, if not
the only, integrin that bound to FGF1, because FGF1 bound only weakly to several β1 integrins tested. We presented evidence
that the CYDMKTTC sequence (the specificity loop) within the ligand-binding site of β3 plays a role in FGF1 binding. We found
that the integrin-binding site of FGF1 overlaps with the heparin-binding site but is distinct from the FGFR-binding site using
docking simulation and mutagenesis. We identified an FGF1 mutant (R50E) that was defective in integrin binding but still bound
to heparin and FGFR. R50E was defective in inducing DNA synthesis, cell proliferation, cell migration, and chemotaxis, suggesting
that the direct integrin binding to FGF1 is critical for FGF signaling. Nevertheless, R50E induced phosphorylation of FGFR1
and FRS2α and activation of AKT and ERK1/2. These results suggest that the defect in R50E in FGF signaling is not in the initial
activation of FGF signaling pathway components, but in the later steps in FGF signaling. We propose that R50E is a useful
tool to identify the role of integrins in FGF signaling.
Journal of Biological Chemistry 06/2008; 283(26):18066-18075. · 4.77 Impact Factor
