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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.

ABSTRACT: The chemokine domain of fractalkine (FKN-CD) binds to the classical RGD-binding site of ?v?3 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 ?v?3 in cell-free conditions, but the integrin-binding defective mutant of FKN-CD (K36E/R37E) did not. This suggests that FKN-CD can activate ?v?3 in the absence of CX3CR1 through the direct binding of FKN-CD to ?v?3. WT FKN-CD activated ?v?3 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 ?v?3. To identify the possible second FKN-CD binding site we performed docking simulation of ?v?3-FKN-CD interaction using ?v?3 with a closed inactive conformation as a target. The simulation predicted a potential FKN-CD-binding site in inactive ?v?3 (site 2), which is located at a crevice between ?v and ?3 on the opposite side of site 1 in the ?v?3 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 ?4?1 and ?5?1. The FKN binding to site 2 and resulting integrin activation may be a novel mechanism of integrin activation and of FKN signaling.

SUBMITTER: Fujita M

PROVIDER: S-EPMC4008574 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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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 Masaaki M Takada Yoko K YK Takada Yoshikazu Y

PloS one 20140502 5

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 (K3 ...[more]

PMID: 24789099

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Project description:Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.

Dataset Information

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

Publications

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

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