CXC chemokines bind to unique sets of selectivity determinants that can function independently and are broadly distributed on multiple domains of human interleukin-8 receptor B - Determinants of high affinity binding and receptor activitation are distinct

Human interleukin-8 receptors A (IL-8RA) and B (IL-8RB) are seven-transmembrane domain (TMD) neutrophil chemokine receptors with similar sequences (77% amino acid identity) and similar G protein selectivity, but markedly different selectivity for CXC chemokines, IL-8RB is selective for IL-8, growth-related oncogene alpha (GRO alpha) and neutrophil activating peptide-2 (NAP-2), whereas IL-8RA is selective only for IL-8. To identify selectivity determinants, we made eight chimeric receptors exchanging: 1) the three main regions of sequence divergence between IL-8RA and IL-8RB (the N-terminal segment before TMD1, the region from TMD4 to the end of the second extracellular (e2) loop, and the C-terminal tail), and 2) the N-terminal segment of CC chemokine receptor 1, which does not bind CXC chemokines. Chimeras were tested by direct I-125-IL-8, I-125-GRO alpha, and I-125-NAP-2 binding, heterologous competition binding, and calcium flux assays using human embryonic kidney 293 cells stably transfected with receptor DNAs. The following results were obtained: 1) chimeric receptors had binding sites for IL-8, GRO alpha and NAP-2 distinct from those on IL-8RA and IL-8RB; 2) IL-8, GRO alpha and NAP-2 bound to overlapping but distinct sites that mapped differentially to multiple domains on IL-8RB; 3) high affinity radioligand binding and high agonist potency were separable functions for IL-8, GRO alpha and NAP-2, suggesting that the determinants of high affinity binding may not be critical for receptor activation; and 4) determinants of GRO alpha and NAP-2 selectivity were found in both the N-terminal segment before TMD1 and the region from TMD4 to the end of the e2 loop of IL-8RB, and functioned independently of each other, Stated reciprocally, the N-terminal segment of IL-8RA was not a dominant selectivity determinant. These data suggest that both narrow and broad spectrum chemokine antagonists can be developed to block functions mediated by IL-8RB.