Site‐directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16

Petit, Sarah J.; Chayen, Naomi E.; Pease, James E.

doi:10.1002/eji.200838269

Cited by 27 publications

(27 citation statements)

References 41 publications

(60 reference statements)

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“…Petit et al demonstrated that a point mutation of the CXCL16 receptor CXCR6 (Glu274Gln) abrogated binding of soluble (chemokine domain) CXCL16 but had no effect on adhesion mediated by full-length membrane-bound CXCL16. 40 The authors suggest that the mucin stalk in the full-length molecule causes the chemokine domain to adopt a different conformation compared with soluble chemokine domain CXCL16. These 2 forms may then stabilize different receptor conformations that induce different functional effects on cells expressing the CXCR6 receptor.…”

Section: Shed Versus Membrane-bound Fractalkinementioning

confidence: 99%

Fractalkine: A Survivor's Guide

White

Greaves

2012

ATVB

138

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Abstract-Chemokines are a family of low-molecular-weight proteins essential to the directed migration of cells under homeostatic and pathological conditions. Fractalkine (CX3CL1) is an unusual chemokine that can act as either a soluble or membrane-bound mediator and signals through the G protein-coupled chemokine receptor CX3CR1, expressed on monocytes, natural killer cells, T cells, and smooth muscle cells. Accumulating evidence suggests that fractalkine, in addition to its role in chemotaxis and adhesion of leukocytes, supports the survival of multiple cell types during homeostasis and inflammation. This review presents the evidence obtained from several disease models implying an antiapoptotic function for fractalkine and shows how this is relevant to the pathology of atherosclerosis and other vascular diseases. We discuss whether the key role of fractalkine, unlike other chemokines, is the promotion of cell survival and whether this has implications for vascular disease. (Arterioscler Thromb Vasc Biol. 2012;32:589-594.)Key Words: apoptosis Ⅲ atherosclerosis Ⅲ macrophages Ⅲ vascular biology Ⅲ chemokines F irst recognized in 1987 with the discovery of interleukin-8, the chemokine family today numbers at least 46 members divided into 4 families on the basis of structure. 1,2 As their name-chemotactic cytokinessuggests, all members of the family share the ability to chemoattract cells expressing their cognate G protein-coupled receptors. The variety of cellular functions ascribed to chemokines is vast and growing, including adhesion, proliferation, survival, angiogenesis, and regulation of proinflammatory gene expression. In addition, chemokine receptor expression has been described for virtually every cell type studied, under both homeostatic and inflammatory conditions. Dysregulation of chemokine and chemokine receptor expression is associated with multiple disease states, including cardiovascular disease, cancer, neurodegenerative disease, and systemic inflammatory disease, such as rheumatoid arthritis and systemic lupus erythematosus (reviewed in 3 ).Fractalkine (CX3CL1) is the only member of the CX3C chemokine family and is expressed as a membrane-bound molecule with the chemokine domain attached via a mucinlike stalk to the cell surface. 4 A recent analysis of fractalkine expression using a Cx3cl1 cherry :Cx3cr1 gfp knock-in mouse identified neurons and epithelial cells in the lung, kidney, and intestine as the major sites of fractalkine expression, confirming previous reports. 5,6 Fractalkine can also be expressed by endothelial and smooth muscle cells under inflammatory conditions. 7,8 Cleavage at the base of the mucin stalk is mediated by at least 2 enzymes, ADAM10 and ADAM17, which function under homeostatic and inflammatory conditions, respectively. 9 -11 The structure of membrane-bound and soluble fractalkine is presented in Figure 1. Fractalkine is the unique ligand for the chemokine receptor CX3CR1, which is expressed on monocytes, natural killer cells, T cells, and smooth muscle cells, 12,13 where...

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Section: Shed Versus Membrane-bound Fractalkinementioning

confidence: 99%

Fractalkine: A Survivor's Guide

White

Greaves

2012

ATVB

138

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“…16 HEK293T cells were transiently transfected by calcium phosphate precipitation. For a 24-well plate format, 9ϫ10 4 cells were seeded in 200 L, 1 day before transfection, and the medium was changed 3 hours before transfection.…”

Section: Cell Culturementioning

confidence: 99%

“…16 Expression of CXCL16 on HEK293T cells was detected using biotinylated anti-human CXCL16 (R&D Systems) and streptavidin fluorescein isothiocyanate (Serotec Ltd, Oxford, United Kingdom) with goat IgG as negative control. Expression of CXCL16 on isolated monocytes was detected using allophycocyanin conjugated monoclonal anti-human CXCL16 (R&D Systems).…”

Section: Analysis Of Cxcl16 and Cxcr6 Cell Surface Expression Using Fmentioning

confidence: 99%

“…14,15 Recently, we have shown by site-directed mutagenesis that the stalk region of CXCL16 influences the conformation of the chemokine domain, with a point mutation in the CXCR6 resulting in a receptor that can bind membrane-bound but not soluble CXCL16, thus dissecting the adhesive and chemotactic properties of the ligand. 16 Within exon 4 of the gene encoding the CXCL16 stalk are 2 common nonsynonymous single-nucleotide polymorphisms (SNPs), which are in total linkage disequilibrium: one encoding an I3 T mutation at codon 123, and another encoding an A3 V mutation at codon 181. A previous study by Lundberg et al reported an associ-ation of the V181 allele with increased coronary artery stenosis in postmyocardial infarct patients, although the effects of the mutation on CXCL16 expression, shedding, and function were not determined.…”

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confidence: 99%

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The CXCL16 A181V Mutation Selectively Inhibits Monocyte Adhesion to CXCR6 but Is Not Associated With Human Coronary Heart Disease

Petit

Wise

Chambers³

et al. 2011

ATVB

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Objective-The chemokine CXCL16 serves as a scavenger receptor for oxidized low-density lipoprotein and as an adhesion molecule and chemoattractant for cells expressing the receptor CXCR6. A commonly occurring CXCL16 allele has been described containing 2 nonsynonymous single-nucleotide polymorphisms in complete linkage disequilibrium, although the effects on CXCL16 function are unknown. Here, we examined the effect of the single-nucleotide polymorphisms on CXCL16 function and assessed the association of the mutant allele with coronary heart disease (CHD). Methods and Results-Both wild-type and mutant T123V181-CXCL16 were readily expressed in vitro and were similarly functional in assays of oxidized low-density lipoprotein scavenging and chemotaxis. However, unlike wild-type CXCL16, T123V181-CXCL16 was unable to promote adhesion of CXCR6 ϩ cells. Findings were confirmed ex vivo, with monocytes from donors homozygous for the T123V181 allele unable to facilitate adhesion of CXCR6 transfectants. In the London Life Sciences Prospective Population cohort (nϭ2797), we found that the T123V181 allele was not associated with protection or susceptibility to CHD (adjusted odds ratio, 1.01; 95% CI, 0.95 to 1.10; Pϭ0.74). Key Words: adhesion molecules Ⅲ coronary heart disease Ⅲ cytokines Ⅲ leukotrienes Ⅲ vascular biology A therosclerosis is an inflammatory disease involving the interaction of several different cell types, notably Th 1 -lymphocytes and monocytes/macrophages of the immune system and smooth muscle and endothelial cells of the arteries. 1 The recruitment of cells to the atherosclerotic plaque is coordinated in part by chemokines, of which several have been shown to be expressed in situ. 2 Among these, CXCL16 is preferentially expressed within atherosclerotic plaques of human and murine aortas 2-4 and is produced by several cell types, including macrophages, 5,6 dendritic cells, 7 smooth muscle cells, 8 and endothelial cells. 9 Unlike the majority of chemokines, which are soluble and secreted, CXCL16 is expressed as a type I membrane protein, its chemokine domain presented on the end of a mucin-like stalk, which is released as a soluble form by the action of metalloproteinases. 9,10 CXCL16 is the sole ligand for the receptor CXCR6, and its soluble form induces the directional migration of CXCR6 ϩ cells, such as CD4 ϩ effector memory T cells and natural killer T-cells. 6,7 In its stalk-bound form, CXCL16 has also been shown to facilitate the adhesion of CXCR6 ϩ cells via interactions with the chemokine domain 11 analogous to the interaction between CX3CL1 and CX3CR1. 12 Intriguingly, the chemokine domain of CXCL16 also recognizes oxidized low-density lipoprotein (OxLDL), 5,13 allowing its scavenging by macrophages. The precise role of the CXCL16:CXCR6 axis in atherogenesis is a source of debate, with CXCR6-deficient mice suggesting a proatherogenic role for the receptor and CXCL16-deficient mice suggesting an atheroprotective role for the ligand. 14,15 Recently, we have shown by site-directed mutagenesis that the s...

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“…This model was based on investigations of chemokine interactions with chimeric receptors of CXCR1:3 (Xanthou et al, 2003) and CCR1:3 (Pease et al, 1998). Also supporting this, CX 3 CL1 depends on residues in the N terminus as well as ECL-3 of CX 3 C chemokine receptor 1 (Chen et al, 2006), and CXCL16 depends on all extracellular regions of CXCR6 (Petit et al, 2008). Likewise, we and others have reported that the chemokineinduced activation is critically dependent upon the receptor N terminus in CCR1, CCR2, and CCR8 (Samson et al, 1997;Jensen et al, 2008Jensen et al, , 2012.…”

Section: Role Of Disulfide Bridges In Homologous Chemokinementioning

confidence: 78%

Extracellular Disulfide Bridges Serve Different Purposes in Two Homologous Chemokine Receptors, CCR1 and CCR5

et al. 2013

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In addition to the 7 transmembrane receptor (7TM)-conserved disulfide bridge between transmembrane (TM) helix 3 and extracellular loop (ECL)-2, chemokine receptors (CCR) contain a disulfide bridge between the N terminus and what previously was believed to be ECL-3. Recent crystal and NMR structures of the CXC chemokine receptors (CXCR) CXCR4 and CXCR1, combined with structural analysis of all endogenous chemokine receptors indicate that this chemokine receptor-conserved bridge in fact connects the N terminus to the top of TM-7. By employing chemokine ligands that mainly target extracellular receptor regions and small-molecule ligands that predominantly interact with residues in the main binding crevice, we show that the 7TM-conserved bridge is essential for all types of ligandmediated activation, whereas the chemokine-conserved bridge is dispensable for small-molecule activation in CCR1. However, in striking contrast to previous studies in other chemokine receptors, high-affinity CCL3 chemokine binding was maintained in the absence of either bridge. In the highly related CCR5, a completely different dependency was observed as neither activation nor binding of the same chemokines was retained in the absence of either bridge. In contrast, both bridges were dispensable for activation by the same small molecules. This indicates that CCR5 activity is independent of extracellular regions, whereas in CCR1 the preserved folding of ECL-2 is necessary for activation. These results indicate that conserved structural features in a receptor subgroup do not necessarily provide specific traits for the whole subgroup but rather provide unique traits to the single receptors.

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Site‐directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16

Cited by 27 publications

References 41 publications

Fractalkine: A Survivor's Guide

Fractalkine: A Survivor's Guide

The CXCL16 A181V Mutation Selectively Inhibits Monocyte Adhesion to CXCR6 but Is Not Associated With Human Coronary Heart Disease

Extracellular Disulfide Bridges Serve Different Purposes in Two Homologous Chemokine Receptors, CCR1 and CCR5

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