Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal 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 Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.Chemokines are proinflammatory proteins that coordinate the immune response by directing the migration of leukocytes (1-3). For humans alone, the chemokine superfamily comprises over 40 members that cluster into four families (CC, CXC, C, and CX3C) according to the number and spacing of conserved cysteines (4). Chemokines mediate their effects by binding and signaling through seven-transmembrane G-protein-coupled receptors (7TMRs), 1 of which 18 human receptors have been identified thus far (2, 5). The patterns of ligand-receptor recognition are complex since chemokines and chemokine receptors typically interact with multiple partners. Furthermore, a given chemokine can act as an agonist, antagonist, or inverse agonist in the context of different receptors (6 -10). Despite the high level of promiscuity, interactions generally occur between receptors and ligands within a particular subfamily, an important exception being viral proteins that typically show broad spectrum recognition profiles (11)(12)(13)(14). A large number of studies have aimed to dissect the molecular determinants of chemokine receptor binding, signaling, and specificity (15-20). However, these details remain poorly understood because structural information on the receptors is difficult to obtain, and the important features of the ligands are encrypted in remarkably conserved tertiary structures. Fractalkine/CX3CL1 and its receptor CX3CR1 are currently the only known members of the CX3C family (21,22). Unlike other chemokines except CXCL16 (23), fractalkine is a membrane-anchored protein consisting of a chemokine module attached to the cell membrane via a mucin-like stalk, which serves as a presentation vehicle (24). A soluble f...