Horuk R, Wang Z X, Peiper S C, Hesselgesser J
Department of Protein Chemistry, Genentech Inc., South San Francisco, California 94080.
J Biol Chem. 1994 Jul 1;269(26):17730-3.
The erythrocyte chemokine receptor is a cell surface protein that binds a wide array of chemokines including interleukin-8 (IL-8), melanoma growth stimulating activity (MGSA), monocyte chemotactic protein-1 (MCP-1), and RANTES (Regulated on Activation, Normal T Expressed and Secreted). This protein has also been identified as the Duffy blood group antigen, a cell surface receptor for the malarial parasite Plasmodium vivax. In the present study, we have identified a chemokine receptor-like binding protein in a human erythroleukemic cell line (HEL), which, based on its molecular properties, may be related to the erythrocyte chemokine receptor. Saturation binding studies with 125I-IL-8 revealed a single class of IL-8 binding sites in HEL cells with a KD of 7.4 +/- 1.9 nM and a receptor density of 12,818 +/- 965 binding sites/cell. In competition studies unlabeled IL-8 MGSA, MCP-1, and RANTES were fully able to inhibit the binding of 125I-IL-8 to HEL cells. Chemical cross-linking with radiolabeled IL-8 resulted in a cross-linked species of 60 kDa in membranes from HEL cells. The labeling was specific since it was inhibited by pre-incubation with 1 microM unlabeled IL-8 or MGSA. A monoclonal antibody (Fy6) to the human erythrocyte Duffy blood group antigen/chemokine receptor blocked the binding of IL-8 and other chemokines to the HEL cell chemokine receptor-like binding protein. Cell membranes from HEL cells and from erythrocyte ghosts were subjected to SDS-PAGE and analyzed by Western blotting with anti-Fy6. The antibody bound to a molecule with a molecular mass of 50 kDa in HEL cell membranes and 40 kDa in erythrocyte ghosts. Northern blot analysis of mRNA revealed that the HEL chemokine-binding protein hybridized to a cDNA probe to the Duffy antigen/chemokine receptor.
红细胞趋化因子受体是一种细胞表面蛋白，可结合多种趋化因子，包括白细胞介素 - 8（IL - 8）、黑色素瘤生长刺激活性因子（MGSA）、单核细胞趋化蛋白 - 1（MCP - 1）和调节激活正常T细胞表达和分泌因子（RANTES）。该蛋白也被鉴定为达菲血型抗原，是间日疟原虫的细胞表面受体。在本研究中，我们在人红白血病细胞系（HEL）中鉴定出一种趋化因子受体样结合蛋白，基于其分子特性，它可能与红细胞趋化因子受体相关。用125I - IL - 8进行的饱和结合研究显示，HEL细胞中存在一类单一的IL - 8结合位点，解离常数（KD）为7.4±1.9 nM，受体密度为12,818±965个结合位点/细胞。在竞争研究中，未标记的IL - 8、MGSA、MCP - 1和RANTES完全能够抑制125I - IL - 8与HEL细胞的结合。用放射性标记的IL - 8进行化学交联导致HEL细胞膜中出现一种60 kDa的交联产物。这种标记是特异性的，因为用1μM未标记的IL - 8或MGSA预孵育可抑制标记。一种针对人红细胞达菲血型抗原/趋化因子受体的单克隆抗体（Fy6）可阻断IL - 8和其他趋化因子与HEL细胞趋化因子受体样结合蛋白的结合。对HEL细胞和红细胞血影的细胞膜进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE），并用抗Fy6抗体进行蛋白质印迹分析。该抗体与HEL细胞膜中分子量为50 kDa的分子以及红细胞血影中分子量为40 kDa的分子结合。对mRNA的Northern印迹分析表明，HEL趋化因子结合蛋白与达菲抗原/趋化因子受体的cDNA探针杂交。
