Horuk R, Colby T J, Darbonne W C, Schall T J, Neote K
Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080.
Biochemistry. 1993 Jun 8;32(22):5733-8. doi: 10.1021/bi00073a002.
In addition to the two human interleukin-8 (IL-8) receptors that have been cloned, IL-8RA and IL-8RB, we recently described a binding protein in human erythrocytes that binds IL-8 and monocyte chemotactic peptide-1 (MCP-1), which we have termed the chemokine (CK) receptor. This communication describes the biochemical characterization, detergent solubilization, and development of a solubilized receptor binding assay for the erythrocyte CK receptor. Competitive 125I-IL-8 binding studies in cells transfected with IL-8RA and IL-8RB revealed that only IL-8 and MGSA were able to displace the radiolabeled IL-8 from these cells. In contrast, a whole array of chemokines were able to cross-compete with 125I-IL-8 for binding to the CK receptor in erythrocyte ghosts. Scatchard analysis of 125I-IL-8 binding to erythrocyte membranes and to dodecyl beta-maltoside solubilized CK receptors revealed a single class of high affinity binding sites in both cases with KD values of 9.5 nM +/- 3.6 and 15.4 nM +/- 5.0, respectively. Chemical cross-linking studies with erythrocyte membranes and with solubilized CK receptors indicated that the CK receptor has a lower molecular mass than the cloned IL-8 receptors (39 kDa compared to 57-69 kDa). Treatment of the cross-linked 47-kDA protein with N-glycanase reduced its molecular mass to 42 kDa.
除了已克隆的两种人白细胞介素-8（IL-8）受体IL-8RA和IL-8RB外，我们最近在人红细胞中描述了一种结合蛋白，它能结合IL-8和单核细胞趋化肽-1（MCP-1），我们将其称为趋化因子（CK）受体。本通讯描述了红细胞CK受体的生化特性、去污剂增溶以及增溶受体结合测定的开发。在用IL-8RA和IL-8RB转染的细胞中进行的竞争性125I-IL-8结合研究表明，只有IL-8和MGSA能够从这些细胞中取代放射性标记的IL-8。相比之下，一系列趋化因子能够与125I-IL-8交叉竞争，以结合红细胞膜中的CK受体。对125I-IL-8与红细胞膜以及十二烷基β-麦芽糖苷增溶的CK受体结合的Scatchard分析表明，在这两种情况下都存在一类单一的高亲和力结合位点，KD值分别为9.5 nM±3.6和15.4 nM±5.0。对红细胞膜和增溶的CK受体进行化学交联研究表明，CK受体的分子量低于克隆的IL-8受体（分别为39 kDa和57 - 69 kDa）。用N-糖苷酶处理交联的47-kDA蛋白可使其分子量降至42 kDa。
