CDB15:0000410 CXCL2 — CXCR2

ABSTRACT: Interleukin-8 (IL-8), growth-related oncogene (GRO) alpha, GRObeta, GROgamma, neutrophil-activating peptide-2 (NAP-2), epithelial cell-derived neutrophil activating peptide- 78 (ENA-78), and granulocyte chemoattractant protein-2 are potent neutrophil chemoattractants 40-90% identical in amino acid sequence that comprise a subgroup of human CXC chemokines defined by the conserved sequence motif glutamic acid-leucine-arginine (ELR). Two human chemotactic receptor subtypes for IL-8, named IL-8 receptors (IL8R) A and B, have been cloned. They are 78% identical in amino acid sequence, coexpressed in neutrophils, and distinguished by their different selectivities for GROalpha and NAP-2. Their selectivity for other ELR+ CXC chemokines has not been previously reported. By measuring calcium flux in human embryonic kidney 293 cells transfected with plasmids encoding IL8RA or IL8RB, we have now defined receptor selectivity for GRObeta, GROgamma, and ENA-78. The rank order of agonist potency, based on inspection of the mean effective concentration values (EC50), for IL8RB was GROgamma (1 nM) > IL-8 (4 nM) approximately GROalpha (5 nM) approximately GRObeta (4 nM) approximately NAP-2 (7 nM) > ENA-78 (11 nM), and for IL8RA was IL-8 (4 nM) >>> ENA-78 (40 nM) approximately NAP-2 (45 nM) > GROalpha (63 nM) approximately GROgamma (65 nM) >> GRObeta. The maximal response of IL8RA to IL-8 was at least 2-fold greater than the other five chemokines. All six agonists for IL8RB competed for high affinity 125I-IL-8, -GROalpha, -NAP-2, and -ENA-78 binding sites at IL8RB. GROalpha, GRObeta, GROgamma, NAP-2, and ENA-78 competed weakly for the high affinity IL-8 binding site at IL8RA. Thus, IL8RA and IL8RB are both highly selective for IL-8 and have similar sequences but differ dramatically in their selectivity for all other ELR+ CXC chemokines tested. These findings have important implications for developing novel neutrophil-specific anti-inflammatory drugs directed against the CXC chemokine signaling system.

ABSTRACT: Murine macrophage inflammatory protein-2 (MIP-2), a member of the alpha-chemokine family, is one of several proteins secreted by cells in response to lipopolysaccharide. Many of the alpha-chemokines, such as interleukin-8, gro-alpha/MGSA, and neutrophil activating peptide-2 (NAP-2), are associated with neutrophil activation and chemotaxis. We describe the expression, purification, and characterization of murine MIP-2 from Pichia pastoris. Circular dichroism spectroscopy reveals that MIP-2 exhibits a highly ordered secondary structure consistent with the alpha/beta structures of other chemokines. Recombinant MIP-2 is chemotactic for human and murine neutrophils and up-regulates cell surface expression of Mac-1. MIP-2 binds to human and murine neutrophils with dissociation constants of 6.4 nM and 2.9 nM, respectively. We further characterize the binding of MIP-2 to the human types A and B IL-8 receptors and the murine homologue of the IL-8 receptor. MIP-2 displays low-affinity binding to the type A IL-8 receptor (Kd > 120 nM) and high-affinity binding to the type B IL-8 receptor (Kd 5.7 nM) and the murine receptor (Kd 6.8 nM). The three-dimensional structure of IL-8 and sequence analysis of six chemokines (IL-8, gro-alpha, NAP-2, ENA-78, KC, and MIP-2) that display high-affinity binding to the IL-8 type B receptor are used to identify an extended N-terminal surface that interacts with this receptor. Two mutants of MIP-2 establish that this region is also involved in binding and activating the murine homologue of the IL-8 receptor. Differences in the sequence between IL-8 and related chemokines identify a unique hydrophobic/aromatic region surrounded by charged residues that is likely to impart specificity to IL-8 for binding to the type A receptor.