Figure 1.
FKN-CD activates αvβ3 integrin in cell-free conditions (through direct integrin binding).
a. Activation of soluble αvβ3 by FKN-CD as a function of γC399tr concentration. Binding of soluble αvβ3 (5 µg/ml) to immobilized γC399tr in the presence or absence of WT FKN-CD (40 µg/ml) was measured by ELISA. Data are shown as means ± SEM of three independent experiments. b. Activation of soluble αvβ3 by FKN-CD as a function of FKN-CD concentration. Binding of soluble αvβ3 (5 µg/ml) to immobilized γC399tr (100 µg/ml) in the presence or absence of WT FKN-CD or R36E/R37E was measured by ELISA. Data are shown as means ± SEM of three independent experiments. c. Activation of soluble αvβ3 by FKN-CD using ADAM-15 as a ligand. Experiments were done as described in (a), except that ADAM-15 and 20 µg/ml FKN-CD were used. Data are shown as means ± SEM of three independent experiments.
Figure 2.
FKN-CD activates αvβ3 integrin on the surface of cells that do not express CX3CR1 (through direct integrin binding).
a. Activation of αvβ3 by WT FKN-CD, but not by K36E/R37E (integrin-binding defective), in αvβ3-K562 cells (CX3CR1-negative). Cells were incubated with FITC-labeled γC399tr in the presence of WT FKN-CD or K36E/R37E. Binding of γC399tr to cells was measured by flow cytometry. Data are shown as means ± SEM of median fluorescent intensity (MFI) of three independent experiments. b. αvβ3 activation by FKN-CD as measured by adhesion to ADAM-15. Adhesion assays were performed as described in the methods. Data are shown as means ± SEM of three independent experiments. c. Activation of αvβ3 by FKN-CD in β3-CHO cells (CX3CR1-negative). Experiments were performed as described in (a) except that β3-CHO cells (CX3CR1-negative) were used instead of αvβ3-K562 cells. Data are shown as means ± SEM of MFI of three independent experiments. d. Activation of αvβ3 by FKN-CD in β3-CHO cells at low FKN-CD concentrations. Experiments were performed as described in (c) except that FKN-CD was used at 0.1 and 1 µg/ml. Data are shown as means ± SEM of MFI of three independent experiments.
Figure 3.
Docking simulation of FKN-CD binding to αvβ3 with an inactive conformation predicts a new ligand-binding site (site 2).
a. A docking model of FKN-CD-integrin αvβ3 (active) interaction [25]. The headpiece of ligand-bound form of integrin αvβ3 (PDB code 1L5G) was used as a target. The model predicts that FKN-CD (PDB code 1F2L, red) binds to the classical RGD-binding site of the integrin αvβ3 headpiece (site 1). b. A docking model of FKN-CD-integrin αvβ3 (inactive) interaction. The headpiece of an inactive form of integrin αvβ3 (PDB code 1JV2) was used as a target. The model predicts the position of the second FKN-CD-binding site (site 2). c. Superposition of two models shows that the positions of two predicted FKN-CD binding sites are distinct. d. Position of the β3 peptide (267–287, blue) in site 2 (S2-β3). Most of amino acid residues in this peptide are predicted to interact with FKN-CD (Table 1).
Figure 4.
A peptide derived from the predicted site 2 of αvβ3 (S2-β3) binds to FKN-CD and suppresses CX3CR1-independent FKN-CD-induced αvβ3 activation.
a. Binding of S2-β3 peptide to immobilized FKN-CD. The binding of the peptide to immobilized FKN-CD was measured by ELISA. Data are shown as means ± SEM of three independent experiments. b. Pull-down assays. FKN-CD (with 6His tag) was incubated with S2-β3 or S2-β1 peptide (GST fusion protein) and the complexes were analyzed by Western blotting. c. Binding of site 2 peptides from different integrin β subunits (S2-β1, β2, β3, and β4) to immobilized FKN-CD. The binding of peptides to immobilized FKN-CD was measured as described in (a). Data are shown as means ± SEM of three independent experiments. d. Binding of S2-β3 peptide to FKN-CD. The binding of the peptide to immobilized FKN-CD, γC399tr, FN-H120, FN-8-11 (5 µM) was measured as described in (a). Data are shown as means ± SEM of three independent experiments. e. Effect of S2-β3 peptide on FKN-CD induced integrin activation in αvβ3-K562 cells. Cells were incubated with FITC-labeled γC399tr in the presence of FKN-CD or the mixture of FKN-CD and S2-β3 peptide. FKN-CD (20 µg/ml) were preincubated with S2-β3 (300 µg/ml) in PBS for 30 min at room temperature. Binding of FITC-labeled γC399tr to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. f. Effect of S2-β3 peptide on FKN-CD induced integrin activation in β3-CHO cells. The binding of γC399tr to cells was measured as described in e). Data are shown as means ± SEM of MFI of three independent experiments.
Table 1.
Amino acid residues involved in the interaction between FKN-CD and integrin αvβ3.
Figure 5.
FKN-CD activates α4β1 integrin in a CX3CR1-independent manner through the binding to site 2.
a. Activation of α4β1 by FKN-CD in α4-K562 cells (CX3CR1-negative). The binding of FITC-labeled H120 (specific ligand to α4β1) to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. b. Adhesion of α4-K562 cell to VCAM-1. Cell adhesion to immobilized VCAM-1 was measured as described in the methods. Data are shown as means ± SEM of three independent experiments. c. Effect of S2-β3 on FKN-CD induced α4β1 activation. α4-K562 cells were incubated with FITC-labeled H120 in the presence of FKN-CD or the mixtures of FKN-CD and S2-β3. FKN-CD (20 µg/ml) was preincubated with S2-β3 (300 µg/ml) in PBS for 30 min at room temperature. Binding of FITC-labeled H120 to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. d. Activation of α4β1 by FKN-CD in α4-CHO cells (CX3CR1-negative) in a CX3CR1-independent manner. The binding of FITC-labeled H120 (specific ligand to α4β1) was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. e. Activation of α4β1 by FKN-CD in α4-CHO cells at low FKN-CD concentrations. Experiments were performed as described in (d) except that FKN-CD and K36E/R37E were used at 0.1 and 1 µg/ml. Data are shown as means ± SEM of MFI of three independent experiments. f. Effect of S2-β3 peptide on FKN-CD induced integrin activation in α4-CHO cells. Experiments were performed as descibed in c) except that α4-CHO cells were used. Data are shown as means ± SEM of MFI of three independent experiments.
Figure 6.
FKN-CD activates α5β1 integrin in a CX3CR1-independent manner through the binding to site 2.
a. Activation of α5β1 by FKN-CD in K562 cells (CX3CR1-negative). The binding of FITC-labeled FN8-11 (specific ligand to α5β1) was measured as described in the methods. Data are shown as means ± SEM of MFI of three independent experiments. b. K562 cells adhesion to FN8-11. Cell adhesion to immobilized FN8-11 was measured as described in the methods. Data are shown as means ± SEM of three independent experiments. c. Effect of S2-β3 on FKN-CD induced integrin activation in K562 cells. Cells were incubated with FITC-labeled FN8-11 in the presence of FKN-CD or the mixtures of FKN-CD and S2-β3. FKN-CD (20 µg/ml) was preincubated with S2-β3 (300 µg/ml) in PBS for 30 min at room temperature. Binding of FITC-labeled FN8-11 to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. d. Activation of α5β1 by FKN-CD in CHO cells (CX3CR1-negative) in a CX3CR1-independent manner. The binding of FITC-labeled FN8-11 (specific ligand to α5β1) was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. e. Activation of α5β1 by FKN-CD in CHO cells at low FKN-CD concentrations. Experiments were performed as described in (d) except that FKN-CD and K36E/R37E were used at 0.1 and 1 µg/ml. Data are shown as means ± SEM of MFI of three independent experiments. f. Effect of S2-β3 peptide on FKN-CD induced integrin activation in CHO cells. Experiments were performed as descibed in c) except that CHO cells were used. Data are shown as means ± SEM of MFI of three independent experiments.