Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine

Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a che moattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagen esis study of fractalkine's chemokine domain was undertaken. The results re veal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48 ) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that A rg-48 is a signaling trigger. This result also shows that signaling residue s are not confined to chemokine N termini, as generally thought. F50A showe d no detectable binding, underscoring its importance to the stability of th e complex. K15A displayed unique signaling characteristics, eliciting a wil d-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine a lso binds the human cytomegalovirus receptor US28, and analysis of the muta nts indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC ch emokine MCP-1 reveals structural details that may account for their dual re cognition by US28 and their selective recognition by host receptors.