Ahuja S K, Lee J C, Murphy P M
Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1996 Jan 5;271(1):225-32. doi: 10.1074/jbc.271.1.225.
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 125I-IL-8, 125I-GRO alpha, and 125I-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.
人白细胞介素8受体A（IL-8RA）和B（IL-8RB）是具有七个跨膜结构域（TMD）的中性粒细胞趋化因子受体，它们的序列相似（氨基酸同一性为77%），对G蛋白的选择性也相似，但对CXC趋化因子的选择性却明显不同。IL-8RB对白细胞介素8、生长相关癌基因α（GROα）和中性粒细胞激活肽2（NAP-2）具有选择性，而IL-8RA仅对白细胞介素8具有选择性。为了确定选择性决定因素，我们构建了八个嵌合受体，交换了：1）IL-8RA和IL-8RB之间序列差异的三个主要区域（TMD1之前的N端片段、从TMD4到第二个细胞外环（e2）末端的区域以及C端尾巴），以及2）CC趋化因子受体1的N端片段，该片段不结合CXC趋化因子。通过使用稳定转染了受体DNA的人胚肾293细胞，进行直接的125I-IL-8、125I-GROα和125I-NAP-2结合、异源竞争结合以及钙流测定来测试嵌合体。得到了以下结果：1）嵌合受体具有与IL-8RA和IL-8RB上不同的白细胞介素8、GROα和NAP-2结合位点；2）白细胞介素8、GROα和NAP-2结合到重叠但不同的位点，这些位点在IL-8RB上的多个结构域上有不同的定位；3）高亲和力放射性配体结合和高激动剂效力对于白细胞介素8、GROα和NAP-2是可分离的功能差异，这表明高亲和力结合的决定因素可能对受体激活并不关键；4）在TMD1之前的N端片段和IL-8RB的从TMD4到e2环末端的区域中都发现了GROα和NAP-2选择性的决定因素，并且它们彼此独立发挥作用。反之，IL-8RA的N端片段不是主要的选择性决定因素。这些数据表明，可以开发窄谱和广谱趋化因子拮抗剂来阻断由IL-8RB介导的功能。
