Integrins αvβ3 and α4β1 Act as Coreceptors for Fractalkine, and the Integrin-Binding Defective Mutant of Fractalkine Is an Antagonist of CX3CR1

The membrane-bound chemokine fractalkine (FKN, CX3CL1) on endothelial cells plays a role in leukocyte trafficking. The chemokine domain (FKN-CD) is sufficient for inducing FKN signaling (e.g., integrin activation), and FKN-CD binds to its receptor CX3CR1 on leukocytes. Whereas previous studies suggest that FKN-CD does not directly bind to integrins, our docking simulation studies predicted that FKN-CD directly interacts with integrin alpha(v)beta(3). Consistent with this prediction, we demonstrated that FKN-CD directly bound to alpha(v)beta(3) and alpha(4)beta(1) at a very high affinity (K-D of 3.0 x 10(-10) M to alpha(v)beta(3) in 1 mM Mn2+). Also, membrane-bound FKN bound to integrins alpha(v)beta(3) and alpha(4)beta(1), suggesting that the FKN-CD/integrin interaction is biologically relevant. The binding site for FKN-CD in alpha(v)beta(3) was similar to those for other known alpha(v)beta(3) ligands. Wild-type FKN-CD induced coprecipitation of integrins and CX3CR1 in U937 cells, suggesting that FKN-CD induces ternary complex formation (CX3CR1, FKN-CD, and integrin). Based on the docking model, we generated an integrin-binding defective FKN-CD mutant (the K36E/R37E mutant). K36E/R37E was defective in ternary complex formation and integrin activation, whereas K36E/R37E still bound to CX3CR1. These results suggest that FKN-CD binding to CX3CR1 is not sufficient for FKN signaling, and that FKN-CD binding to integrins as co-receptors and the resulting ternary complex formation are required for FKN signaling. Notably, excess K36E/R37E suppressed integrin activation induced by wild-type FKN-CD and effectively suppressed leukocyte infiltration in thioglycollate-induced peritonitis. These findings suggest that K36E/R37E acts as a dominant-negative CX3CR1 antagonist and that FKN-CD/integrin interaction is a novel therapeutic target in inflammatory diseases. The Journal of Immunology, 2012, 189: 5809-5819.