Abstract

Fractalkine (FKN/CX3CL1) is a cell surface-expressed chemokine involved in many aspects of leukocyte trafficking and activation. The various structural domains of FKN play distinct roles in its ability to bind and activate its receptor, CX3CR1. A human herpesvirus 8-encoded chemokine, termed viral macrophage inflammatory protein (vMIP)-II, is structurally similar to FKN; vMIP-II is a nonselective chemokine receptor antagonist (binding multiple chemokine receptors, including CX3CR1). The goal of this study was to identify FKN determinants of selectivity for its receptor and to further refine domains important in affinity and efficacy at CX3CR1. Chimeric and insertional mutagenesis was used to generate mutants of both vMIP-II and FKN, and the expressed proteins were evaluated for chemokine receptor binding affinities and efficacy at CX3CR1. Modification of the intervening amino acids between the first two conserved cysteine residues of FKN or vMIP-II indicated a role of the X3 bulge of FKN in affinity and selectivity for CX3CR1. Substitution of the vMIP-II N terminus with that of FKN created an agonist that was just as potent and efficacious as FKN for binding and stimulating CX3CR1, whereas replacement of the FKN N terminus with the cognate domain of vMIP-II disrupted the ability of FKN to bind CX3CR1. Furthermore, the entire N terminus of FKN was necessary for the high-affinity and full agonist properties of FKN at CX3CR1. These results refine the pharmacophore for chemokine binding to and activation of CX3CR1 and demonstrate the usefulness of modified virally encoded chemokines as templates for the development of selective chemokine receptor antagonists.