It has been proposed that ligand occupancy of integrin αvβ3 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 αvβ3 enhanced IGF-1-induced proliferation of CHO 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 αvβ3 specifically and directly bound to IGF-1 in cell adhesion, ELISA-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, while 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 αvβ3 and IGF1R, while the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between αvβ3 and IGF1R. These results suggest that the direct binding to IGF-1 to integrin αvβ3 plays a role in IGF-1 signaling through ternary complex formation (αvβ3-IGF-IGF1R), and integrin-IGF-1 interaction is a novel target for drug discovery. (Fig. 1)

We discovered that fibroblast growth factor (FGF)-1 directly bound to soluble and cell–surface integrin avb3 (KD about 1 mM). Antagonists to avb3 (monoclonal antibody 7E3 and cyclic RGDfV) blocked this interaction. avb3 was a predominant, if not only, integrin that bound to FGF-1, since FGF1 only weakly bound to several b1 integrins tested. We presented evidence that the CYDMKTTC sequence (the specificity loop) within the ligand-binding site of b3 plays a role in FGF-1 binding. We found that the integrin-binding site of FGF-1 overlaps with the heparin-binding site, but is distinct from the FGFR-binding site using docking simulation and mutagenesis. We identified an FGF-1 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 FGF-1 is critical for FGF signaling. Nevertheless R50E induced phosphorylation of FGFR1 and FRS2a, 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. (Fig. 2)

Fig. 1. Docking simulation of IGF-1- αvβ3 interaction

a). A model of IGF-1-integrin interaction. Docking simulation of the interaction between IGF-1 and integrin αvβ3 (PDB code 1L5G) was performed using AutoDock3. The headpiece of 1L5G was used as a receptor. The pose in the cluster 1 with the lowest docking energy –19.46 Kcal/mol is shown. This pose represents the most stable pose of 1GF1 when IGF-1 interacts with integrin αvβ3.


b). Positions of several amino acid residues at the predicted interface between IGF-1 and αvβ3. Arg36 and Arg37 within the predicted integrin-binding site in IGF-1 were selected for mutagenesis. Amino acid residues in MIDAS (the metal ion dependent adhesive site) (Asp119, Ser121, Ser123, Glu220, and Asp251) in b3 (red) and the specificity loop (light blue) are predicted to be close to IGF-1 (blue) in the IGF-1-integrin complex, but not directly interact with Arg36/Arg37. Arg36 and Arg37 are predicted to be close to Asp150, Tyr178, and Asp218 of av (green).


c). Arg36 and Arg37 are distinct from IGFBP4-binding site in IGF-1. Positions of Arg36 and Arg37 are shown in the IGF-1-IGFBP4 complex (PDB code 1WQJ). Tyr residues at positions 24, 31, and 60 of IGF-1 that are critical for IGF1R (pink) binding are also shown.

Fig. 2. Identification of the integrin-binding site in FGF-1. a. A model of FGF-1-integrin interaction. Docking simulation of the interaction between FGF-1 (PDB code 1AXM) and integrin αvβ3 (PDB code 1L5G) was performed using AutoDock3. Model a of 1AXM was used for docking. The headpiece of 1L5G was used as a receptor. The pose in the cluster 1 with the lowest docking energy –26.3 Kcal/mol is shown. This pose represents the most stable pose of FGF1 when FGF-1 interacts with integrin αvβ3. b. Positions of amino acid residues that are selected for mutagenesis at the predicted interface between FGF-1 and αvβ3. Several amino acid residues within the predicted integrin-binding site in FGF-1 were selected for mutagenesis. c. Positions of the amino acid residues (E102A, Y109A, and Q110A) at the FGFR-binding site selected for mutagenesis. Note that the predicted integrin-binding site is distinct from the FGFR-binding site.

Takada lab continues working on the role of integrins in growth factor signaling including other growth factors. We are trying to generate something useful for wound healing, or treating inflammation and cancer.