The chemokine CXC ligand 8 (CXCL8)͞IL-8 and related agonists recruit and activate polymorphonuclear cells by binding the CXC chemokine receptor 1 (CXCR1) and CXCR2. Here we characterize the unique mode of action of a small-molecule inhibitor (Repertaxin) of CXCR1 and CXCR2. Structural and biochemical data are consistent with a noncompetitive allosteric mode of interaction between CXCR1 and Repertaxin, which, by locking CXCR1 in an inactive conformation, prevents signaling. Repertaxin is an effective inhibitor of polymorphonuclear cell recruitment in vivo and protects organs against reperfusion injury. Targeting the Repertaxin interaction site of CXCR1 represents a general strategy to modulate the activity of chemoattractant receptors. L eukocyte trafficking into tissue sites of inflammation is directed by chemokines. Chemokines are grouped into four families based on a cysteine motif in the amino terminus of the protein (1, 2). Human CXC ligand 8 (CXCL8)͞IL-8 and related molecules are polymorphonuclear cells (PMN) chemoattractants. Two high-affinity human CXCL8 receptors are known, CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2). Only one corresponding receptor has been identified in the mouse, and this is recognized by ligands that act as neutrophil attractant, although a mouse orthologue of CXCL8 has not been identified. By recruiting and activating PMN, CXCL8 and related rodent molecules have been implicated in a wide range of disease states characterized by PMN infiltration in organs, including reperfusion injury (RI) (3).G protein-coupled receptors (GPCR) are a prime target for the development of new strategies to control diverse pathologies (4-6). Antichemokine strategies include antibodies, N-terminal modified chemokines, and small-molecule antagonists (7-9). Here we describe a class of GPCR inhibitors that specifically block the inflammatory CXCL8 chemokine receptors CXCR1 and CXCR2 by means of an allosteric noncompetitive mode of interaction and protection against RI. Materials and MethodsReagents. Repertaxin (R)(Ϫ)-2-(4-isobutylphenyl)propionyl methansulfonamide) salified with L-lysine was dissolved in saline. Chemokines were from PeproTech (London). Chemicals, cell culture reagents, and protease inhibitors were from Sigma.Migration. Cell migration of human PMN and monocytes and rodent peritoneal PMN were evaluated in a 48-well microchemotaxis chamber with or without Repertaxin. Agonists (1 nM CXCL8, 10 nM N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), 10 nM CXCL1, 2.5 nM CCL2, 1 nM C5a, 5 nM rat and mouse CXCL1, and 2.5 nM rat and mouse CXCL2) were seeded in the lower compartment. The chemotaxis chamber was incubated for 45 min (human PMN), 1 h (rodent PMN), or 2 h (monocytes). L1.2 migration was evaluated by using 5-m pore-size Transwell filters (Costar) (10). Mutation Analysis of CXCR1 and Signaling. The human CXCR1 ORF was PCR amplified from a CXCR1͞pCEP4 plasmid (kindly provided by P. M. Murphy, National Institutes of Health, Bethesda). Receptor mutants and chimeric re...

Chemokines are chemotactic cytokines orchestrating leukocyte recruitment in physiological and pathological conditions. This complex system includes 42 molecules and 19 receptors and is subjected to different levels of regulation, including ligand production, post-translational modifications and degradation, as well as receptor expression and signaling activity. Here we analyze the chemokine system, with particular attention to available information on clinical situations in which chemokines or their receptors might assume diagnostic value.

Seven transmembrane receptors mediate diverse physiological responses including hormone action, olfaction, neurotransmission, and chemotaxis. Human D6 is a non-signaling seven-transmembrane receptor expressed on lymphatic endothelium interacting with most inflammatory CC-chemokines resulting in their rapid internalization. Here, we demonstrate that this scavenging activity is mediated by continuous internalization and constant surface expression of the receptor, a process involving the clathrin-coated pit-dependent pathway. D6 constitutively associates with the cytoplasmic adaptor ␤-arrestin, and this interaction is essential for D6 internalization. An acidic region, but not the putative phosphorylation sites in the cytoplasmic tail of D6, is critical for receptor interaction with ␤-arrestin and subsequent internalization. Neither the native D6 nor mutants uncoupled from ␤-arrestin activate any Gprotein-mediated signaling pathways. Therefore, D6 may be considered a decoy receptor structurally adapted to perform chemokine scavenging.Leukocyte recruitment into inflamed tissues is mediated by chemokines acting on a distinct subfamily of G-protein-coupled receptors (GPCR). 1 This includes 18 receptors with the ability to activate G ␣i -protein-dependent signaling events and cell migration (1-4). Two other chemokine binding molecules, with homology to chemokine receptors but defective in signaling function, called Duffy antigen and D6, have been identified (5-7) and recently classified as "silent" chemokine receptors (8). Evidence in in vitro models, gene-targeted mice, and individuals with erythrocyte-restricted deficiency suggests that the Duffy antigen silent receptor may facilitate chemokine transport across endothelial cells as well as acting as a chemokine buffering and scavenging system (5, 9 -12). D6 is highly expressed in endothelial cells lining afferent lymphatic vessels (13) and supports rapid internalization and degradation of inflammatory CC-chemokines, acting as a chemokine scavenger (7). D6 was suggested to act as a gatekeeper on afferent lymphatic endothelium, preventing excessive transfer to lymph nodes of inflammatory chemokines and disruptive leukocyte recruitment (7). The molecular mechanisms underlying this scavenging function are unknown.Upon agonist binding, most GPCRs activate a signaling cascade mediated by G-protein activation leading to receptor phosphorylation by a G-protein-coupled receptor kinase, which results in uncoupling of the receptor from G-proteins (14). This desensitization process is further facilitated by the association of the phosphorylated receptors with the cytoplasmic adaptor, ␤-arrestin (15). The receptor-␤-arrestin complex associates with clathrin and accessory proteins involved in the formation of clathrin-coated pits, ultimately leading to receptor internalization (16,17). While mediating the inhibition of G-protein functions, both G-protein-coupled receptor kinases and ␤-arrestins also activate G-protein-independent signaling events by direct association with a numb...