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The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.

Fujita M, Takada YK, Takada Y - PLoS ONE (2014)

Bottom Line: Notably the peptide specifically bound to FKN-CD and effectively suppressed integrin activation by FKN-CD.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California, United States of America.

ABSTRACT
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.

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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.
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pone-0096372-g005: 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.

Mentions: We studied if FKN-CD activates integrins other than αvβ3 in a CX3CR1-independent manner. We measured the binding of FITC-labeled H120 to K562 and CHO cells that overexpress recombinant α4β1 (designated α4-K562 and α4-CHO cells, respectively) and the adhesion of α4-K562 cells to H120 (Figure 5). We also studied α5β1 activation by FKN-CD using parent K562 or CHO cells that express α5β1. We measured the binding of FITC-labeled FN8-11 and cell adhesion to FN8-11 (Figure 6). We obtained very similar results in α4β1 and α5β1 to that of αvβ3: WT FKN-CD activated α4β1 and α5β1, but K36E/R37E did not. FKN-CD at 1 µg/ml or less induced detectable α4β1 and α5β1 activation. S2-β3 peptide suppressed FKN-CD-induced α4β1 and α5β1 activation, but control GST or S2-β3scr peptide did not. These results suggest that WT FKN-CD activates integrins α4β1 and α5β1 in a CX3CR1-independent manner through binding to site 2.


The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.

Fujita M, Takada YK, Takada Y - PLoS ONE (2014)

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.
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pone-0096372-g005: 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.
Mentions: We studied if FKN-CD activates integrins other than αvβ3 in a CX3CR1-independent manner. We measured the binding of FITC-labeled H120 to K562 and CHO cells that overexpress recombinant α4β1 (designated α4-K562 and α4-CHO cells, respectively) and the adhesion of α4-K562 cells to H120 (Figure 5). We also studied α5β1 activation by FKN-CD using parent K562 or CHO cells that express α5β1. We measured the binding of FITC-labeled FN8-11 and cell adhesion to FN8-11 (Figure 6). We obtained very similar results in α4β1 and α5β1 to that of αvβ3: WT FKN-CD activated α4β1 and α5β1, but K36E/R37E did not. FKN-CD at 1 µg/ml or less induced detectable α4β1 and α5β1 activation. S2-β3 peptide suppressed FKN-CD-induced α4β1 and α5β1 activation, but control GST or S2-β3scr peptide did not. These results suggest that WT FKN-CD activates integrins α4β1 and α5β1 in a CX3CR1-independent manner through binding to site 2.

Bottom Line: Notably the peptide specifically bound to FKN-CD and effectively suppressed integrin activation by FKN-CD.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus