Interleukin-8 (IL-8) is a potent inflammatory mediator that belongs to the family of C-X-C chemokines. IL-8 promotes the activation and the extravasation of circulating neutrophils to the site of inflammation. Two IL-8 receptor isotypes (type A and B) are identified in human and rabbit neutrophils. IL-8 receptors belongs to the superfamily of G-protein-coupled receptors. Both receptor subtypes A and B bind with high affinity to human IL-8, but they exhibit distinct binding affinity to two functional and structurally related IL-8 peptides, melanoma growth-stimulating activity peptide (MGSA) and neutrophil-activating peptide-2 (NAP-2). Human IL-8 receptor A binds with low affinity to MGSA or NAP-2. In contrast, human IL-8 receptor B binds MGSA with high affinity, and NAP-2 with lesser affinity. Using receptor subtype chimeras, we determined that the N-terminal domain of the receptor confers ligand binding specificity (LaRosa, G. J., Thomas, K. M., Kaufmann, M. E., Mark, R., White, M., Taylor, L., Gray, G., Witt, D., and Navarro, J. (1992) J. Biol. Chem. 267, 25402-25406). In this work, we characterized by molecular cloning and expression a mouse receptor structurally homologous to the IL-8 receptor. We isolated a clone by screening a mouse genomic library with a rabbit IL-8 receptor A cDNA fragment as a probe. The mouse clone exhibited an open reading frame encoding a 359-amino acid protein. Hydropathy plot analysis of the amino acid sequence reveals seven transmembrane domains characteristic of G-protein-coupled receptors. The N terminus and the second extracellular loop contain one and two putative N-glycosylation sites, respectively. The intracellular C-terminal tail contains Ser and Thr residues as potential phosphorylation sites. Northern blot analysis showed that the mouse receptor gene is expressed in mouse neutrophils. The mouse receptor shows 65, 74, 66, and 70% amino acid identity to the rabbit IL-8 receptor subtypes A and B and human IL-8 receptor subtypes A and B, respectively. However, neither mouse neutrophils nor CHO cells expressing the mouse receptor bind human IL-8 in the nanomolar range. To identify the domain(s) conferring high affinity binding to IL-8, we constructed rabbit/mouse receptor chimeras.(ABSTRACT TRUNCATED AT 400 WORDS)