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