Direct Binding of Integrin αvβ3 to FGF1 Plays a Role in FGF1 Signaling

Seiji Mori,Kit S. Lam,Yoshikazu Takada,Yoko K. Takada,Yasuko Kuwabara,Biao Shi,Zi Ma,Satoshi Yamaji,R. Rivkah Isseroff,Chun-Yi Wu,Jun Saegusa

doi:10.1074/jbc.m801213200

Seiji Mori, Kit S. Lam + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m801213200

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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.

Highlights

FGF1 and FGF2 the most extensively studied
We found that recombinant soluble ␣v␤3 bound to immobilized WT FGF1 in a dose-dependent manner but did not bind to heat-denatured FGF1 in ELISA-type integrin binding assays (Fig. 1a)
We found that K562 erythroleukemic cells (␣5␤1ϩ) expressing exogenous ␣v␤3 (␣v␤3-K562, ␣5␤1ϩ/␣v␤3ϩ) cells adhered to FGF1, but mock-transfected K562 cells showed only weak adhesion to FGF1 (Fig. 1c)

Summary

EXPERIMENTAL PROCEDURES

Recombinant soluble ␣v␤3 was synthesized in CHO K1 cells using the soluble ␣v and ␤3 expression constructs provided by Tim Springer (Center for Blood Research, Boston) and purified by nickel-nitrilotriacetic acid affinity chromatography as described [24]. The WT FGF1 and its mutants were expressed in Escherichia coli BL21 (DE3) and purified by glutathione affinity chromatography as described by the manufacturer’s instructions (GE Healthcare). A DNA fragment encoding amino acid residues 140 –365 of the immunoglobulin-like D2 and D3 domains of FGFR1 was amplified by PCR with the full-length human FGFR1 cDNA in the pcDNA3 vector (gift from Ann Hanneken, the Scripps Research Institute, La Jolla, CA) as a template. The HBS-P buffer with 1 mM of Mn2ϩ was injected at 50 ␮l/min for 3 min to allow the bound FGF1s to dissociate from the integrin. WT or mutant FGF1 was 2-fold serially diluted from 800 to 50 nM in HBS-P buffer containing 1 mM of Mn2ϩ and injected at a flow rate 30 ␮l/min for 3 min. The same buffer was injected at the same flow rate for 3 min to measure the dissociation of the bound FGF1 from FGFR1 D2D3

Docking Simulation

Integrin Binding Assays

DNA Synthesis

Western Blot Analysis

In Vitro Wound Healing Assay

RESULTS

We assessed the ability of the

We assessed the ability of these

BrdUrd incorporation than WT

DISCUSSION