The Chemokine Fractalkine Can Activate Integrins without CX3CR1 through Direct Binding to a Ligand-Binding Site Distinct from the Classical RGD-Binding Site

<div><p>The chemokine domain of fractalkine (FKN-CD) binds to the classical RGD-binding site of αvβ3 and that the resulting ternary complex formation (integrin-FKN-CX3CR1) is critical for CX3CR1 signaling and FKN-induced integrin activation. However, only certain cell types express CX3CR1. Here we studied if FKN-CD can activate integrins in the absence of CX3CR1. We describe that WT FKN-CD activated recombinant soluble αvβ3 in cell-free conditions, but the integrin-binding defective mutant of FKN-CD (K36E/R37E) did not. This suggests that FKN-CD can activate αvβ3 in the absence of CX3CR1 through the direct binding of FKN-CD to αvβ3. WT FKN-CD activated αvβ3 on CX3CR1-negative cells (K562 and CHO) but K36E/R37E did not, suggesting that FKN-CD can activate integrin at the cellular levels in a manner similar to that in cell-free conditions. We hypothesized that FKN-CD enhances ligand binding to the classical RGD-binding site (site 1) through binding to a second binding site (site 2) that is distinct from site 1 in αvβ3. To identify the possible second FKN-CD binding site we performed docking simulation of αvβ3-FKN-CD interaction using αvβ3 with a closed inactive conformation as a target. The simulation predicted a potential FKN-CD-binding site in inactive αvβ3 (site 2), which is located at a crevice between αv and β3 on the opposite side of site 1 in the αvβ3 headpiece. We studied if FKN-CD really binds to site 2 using a peptide that is predicted to interact with FKN-CD in site 2. Notably the peptide specifically bound to FKN-CD and effectively suppressed integrin activation by FKN-CD. This suggests that FKN-CD actually binds to site 2, and this leads to integrin activation. We obtained very similar results in α4β1 and α5β1. The FKN binding to site 2 and resulting integrin activation may be a novel mechanism of integrin activation and of FKN signaling.</p></div

Docking simulation of FKN-CD binding to αvβ3 with an inactive conformation predicts a new ligand-binding site (site 2).

<p>a. A docking model of FKN-CD-integrin αvβ3 (active) interaction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096372#pone.0096372-Fujita1" target="_blank">[25]</a>. 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096372#pone-0096372-t001" target="_blank">Table 1</a>).</p

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.

<p>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.</p

FKN-CD activates αvβ3 integrin in cell-free conditions (through direct integrin binding).

<p>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.</p

Amino acid residues involved in the interaction between FKN-CD and integrin αvβ3.

<p>Amino acid residues within 0.6 nm between FKN-CD and αvβ3 were selected using pdb viewer (version 4.1). Amino acid residues in β3 site 2 peptide (S2-β3) are shown in bold.</p

FKN-CD activates α5β1 integrin in a CX3CR1-independent manner through the binding to site 2.

<p>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.</p

FKN-CD activates αvβ3 integrin on the surface of cells that do not express CX3CR1 (through direct integrin binding).

<p>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.</p

mCRP binds to U937 monocytic cells, and induces robust chemotaxis in an integrin-dependent manner.

<p>a).<u> Adhesion of U937 cells to CRP isoforms</u>. Wells of 96 well microtiter plate were coated with mCRP or pCRP and remaining protein-binding sites were blocked with BSA. Wells were incubated with U937 cells (10⁵ cells per well) for 1 h in RPMI1640 and bound cells were quantified. b) and c). <u>Effect of antagonists to αvβ3 and α4β1 on adhesion of U937 cells to mCRP</u>. In b), 2.5 μM coating concentration of mCRP was used. Antibodies used were mAb 7E3 (to human β3, 25 μg/ml), mAb SG73 (to human α4, 25 μg/ml), and AIIB2 (to human β1, 25 μg/ml). “mIgG” represents purified mouse IgG used as a control. Antagonists used were cyclic RGDfV (to αvβ3, 10 μM) and BIO1211 (to α4β1, 1 μM). DMSO was used as a control. Adhesion assay was performed in RPMI. d) mCRP induces AKT activation in U937 cells, but not ERK1/2 activation. U937 cells were serum-starved and stimulated with pCRP and mCRP (100 μg/ml) and cell lysates were analyzed by western blotting. d) <u>mCRP, and less effectively pCRP, induce chemotaxis of U937 cells in an integrin-dependent manner.</u> Chemotaxis was measured in modified Boyden chambers (Transwells). 50 μg/ml mCRP or pCRP in 600 μl RPMI 1640 medium was placed in the lower chamber, and U937 cells (5×10⁵ cells per well) were placed in the upper chamber. U937 cells were preincubated with antibodies (25 μg/ml) for 30 min at 37°C. After 4 h incubation, migrated cells were counted. e) A PI3K inhibitor, not MEK inhibitor, suppresses mCRP-induced chemotaxis of U937 cells. LY294002 (PI3K inhibitor) or PD98059 (MEK inhibitor) were added at 50 μM in the chemotaxis medium.</p

Docking simulation predicts that mCRP binds to integrin αvβ3 but pCRP does not.

<p>a) The headpiece of integrin αvβ3 (PDB code 1LG5) was used as a target. The docking model predicts that mCRP (red) binds to the RGD-binding site of the integrin αvβ3 headpiece (green and blue). Amino acid residues involved in αvβ3-mCRP interaction are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093738#pone-0093738-t001" target="_blank">Table 1</a>. Cations (Mn) and cyclic RGD peptide and specificity loop of β3 are close to the predicted mCRP-binding site in integrin αvβ3. Cations and cyclic RGD peptide in 1LG5 were removed during docking simulation. The predicted integrin-binding site in mCRP is also close to the phosphocholine-binding site and the RQD motif in mCRP. b) To check if pCRP binds to the integrin, we superposed the pentameric CRP (pCRP, orange and red) to the bound mCRP (red). Interestingly, there are steric clashes between pentameric CRP and αvβ3. This predicts that pentameric CRP can not fully access to mCRP-binding site in integrins due to steric hindrance.</p

Amino acid residues involved in mCRP-αvβ3 interaction.

<p>Amino acid residues in integrin αvβ3 and mCRP within 6 Å to each other in the docking model were identified using Swiss-pdb viewer v. 4.1.</p