Received for publication June 24, 2005.
Revised November 18, 2005.
Accepted for publication November 22, 2005.

The Amino-Terminus and the Third Extracellular Loop of CX3CR1 Contain Determinants Critical for Distinct Receptor Functions

Abstract

The G protein-coupled receptor CX3CR1 is a specific receptor for the CX3C chemokine fractalkine. The aim of this study was to identify receptor elements that contribute independently to agonist binding and receptor activation. Targeted mutation of selected acidic amino acid residues demonstrated that the binding activity of CX3CR1 was critically dependent on the two negatively charged residues D25 and E254 located on the N-terminal domain and third extracellular loop, respectively. In addition, mutation of the uncharged polar residue Y14 in the amino terminus caused a reduction in the ligand binding affinity. In contrast, the three acidic residues E13, D16 and D266 did not contribute to ligand binding but were crucial for receptor activation. The mutant receptors E13A, D16A and D266A bound fractalkine with high affinity but were unable to induce signaling events necessary to support chemotaxis. These acidic residues may engage in electrostatic interactions with basic residues on fractalkine that are necessary for receptor function but not for binding. Our data are consistent with a model of chemokine receptor activation consisting of a multistep mechanism. Step one mediates the high affinity fractalkine binding involving Y14, D25 and E254. The initial interaction then triggers the engagement of E13, D16 and D266, necessary for CX3CR1 activation.

Key words: Chemotactic peptides, MAP Kinase, Receptor binding studies