BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 232, 663–668 (1997) ARTICLE NO. RC976352 Mapping of the Extracellular Binding Regions of the Human Interleukin-8 Type B Receptor James A. Katancik, Ashu Sharma, Stephen J. Radel, and Ernesto De Nardin 1 Departments of Oral Biology and Microbiology, State University of New York at Buffalo, Buffalo, New York 14214-3092 Received February 12, 1997 factor (TNF), LPS and other agents (1). Although mono- This study was undertaken to define the regions of the cytes and macrophages are the major source of IL-8, human interleukin-8 type B receptor (IL8RB) which are further studies have shown that IL-8 can be produced critical for binding the ligands interleukin-8, NAP-2 and by fibroblasts (6), keratinocytes (7), endothelial cells GROa. Peptides corresponding to the N-terminus region (8) and gingival fibroblasts (9) in response to IL-1b and and the first extracellular loop of the receptor demon- TNF. Neutrophils themselves have also been shown to strated statistically significant (pÅ0.001) inhibition of IL- produce IL-8 in what appears to be a positive feedback 8 control binding levels (inhibition levels of 73.0{5.1% and mechanism for recruitment (10). 89.9{2.2% respectively). In contrast, NAP-2 binding was Neutrophil-activating peptide 2 (NAP-2) was origi- inhibited only by the peptide representing the first extra- nally isolated from stimulated mononuclear cells (11), cellular loop (63.2{2.3%), while GROa binding was inhib- but platelet granules are the major source of NAP-2 ited by portions of the N-terminus (49.7{14.9% and (12). NAP-2 arises from the N-terminal processing of 41.8{14.9%), but not the first extracellular loop. We sug- two platelet a-granule storage proteins, platelet basic gest that: a.) the chemokine receptor IL8RB, known to protein and connective tissue-activating peptide-III, by bind three related ligands with high affinity, seems to do monocyte derived proteases (12). This peptide’s activity so via distinct contact points and b.) the first extracellular loop is significant in the binding event.  1997 Academic Press is therefore strongest in the vasculature, where in- flammation induces platelet activation. Melanoma growth-stimulatory activity alpha (GRO/ MGSA-a; GROa), was originally shown to be mitogenic for cultured human melanoma cells (13). Further studies Interleukin-8 (IL-8), originally known as neutrophil revealed that GRO is a member of a family of bioactive activating protein 1 (NAP-1) (1) and its structurally molecules which includes platelet factor-4 (PF4), b- related analogues, NAP-2 (2) and GRO/MGSA (GRO) thromboglobulin, and IL-8 (14). GRO is secreted by mono- (3), are members of the CXC family of chemokines cytes, macrophages and various tissue cells after stimula- which can attract and activate neutrophils. Although tion with LPS (15). Amino acid sequence comparisons the three agonists are heterologous in overall amino reveal about 40% identity of GRO with IL-8 (14). acid composition (approximately 45% identity), the ma- IL-8 activation of neutrophils seems to be mediated jor determinant of their shared chemokine properties is by two structurally related membrane receptors. These the conserved amino acid sequence Glu-Leu-Arg (ELR) receptors have been designated IL-8 receptor A preceding the first cysteine in the N-terminal domain. (IL8RA) and IL-8 receptor B (IL8RB) and are members Recent evidence also suggests the importance of a sepa- of the rhodopsin-like, heptahelical, G protein-coupled rate hydrophobic area of the C-X-C chemokines that receptor family (16, 17). may be critical for binding the IL-8 receptors (4,5). IL8RA and IL8RB have different binding properties IL-8 is produced by monocytes and tissue cells upon despite the fact that they share 78% amino acid se- stimulation with interleukin-1 (IL-l), tumor necrosis quence identity. IL8RA binds only IL-8, while IL8RB binds GROa and NAP-2 in addition to IL-8. Binding differences of the receptors have been hypothesized to 1 Corresponding author: 210 Foster Hall, 3435 Main Street. Fax: (716) 829-3942. E-mail: ernesto denardin@sdm.buffalo.edu. be due to differences in the amino-terminal segments Abbreviations: IL-8, interleukin-8; NAP-1 and NAP-2, neutrophil (18-21). More recently, Ahuja, et. al. (22), reported that activating protein 1 and 2; IL-1, interleukin-1; TNF, tumor necrosis IL-8, NAP-2 and GRO contact the IL8RB at broadly factor; IL8RA and IL8RB, interleukin-8 receptors A and B; GRO, growth related protein; FMLP, N-formyl-met-leu -phe. distributed multiple domains. They suggest that the 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 663