title: Site-directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16.
abstract: Chemokine receptor CXCR6 mediates the chemotaxis and adhesion of leukocytes to soluble and membrane-anchored forms of CXCL16, and is an HIV-1 co-receptor. Here, we describe the effects of mutation of acidic extracellular CXCR6 residues on receptor function. Although most CXCR6 mutants examined were expressed at levels similar to wild-type (WT) CXCR6, an N-terminal E3Q mutant was poorly expressed, which may explain previously reported protective effects of a similar single nucleotide polymorphism, with respect to late-stage HIV-1 infection. In contrast to several other chemokine receptors, mutation of the CXCR6 N terminus and inhibition of post-translational modifications of this region were without effect on receptor function. Likewise, N-terminal extension of CXCL16 resulted in a protein with decent potency and efficacy in chemotaxis and not, as anticipated, a CXCR6 antagonist. D176N and E274Q CXCR6 mutants were unable to interact with soluble CXCL16, suggesting a critical role for D176 and E274 in ligand binding. Intriguingly, although unable to interact with soluble CXCL16, the E274Q mutant could promote robust adhesion to membrane-anchored CXCL16, suggesting that soluble and membrane-bound forms of CXCL16 possess distinct conformations. Collectively, our data suggest a novel paradigm for the CXCR6:CXCL16 interaction, a finding which may impact the discovery of small-molecule antagonists of CXCR6.
authors.affiliation: NHLI Division, Leukocyte Biology Section, Faculty of Medicine, Imperial College London, London, UK.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Petit
authors.forename: Naomi E
authors.initials: NE
authors.lastname: Chayen
authors.forename: James E
authors.initials: JE
authors.lastname: Pease
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D050197:Atherosclerosis / Q000209:etiology; D001483:Base Sequence; D016466:CHO Cells; D000075743:Chemokine CXCL16; D054427:Chemokine CXCL6 / Q000737:chemistry / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D054387:Receptors, CXCR / Q000737:chemistry / Q000502:physiology*; D000075744:Receptors, CXCR6
chemical_list: D000075743:Chemokine CXCL16; D054427:Chemokine CXCL6; C498926:Cxcl16 protein, mouse; C482906:Cxcr6 protein, mouse; D054387:Receptors, CXCR; D000075744:Receptors, CXCR6