Identification and characterization of a promiscuous chemokine-binding protein in a human erythroleukemic cell line.
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.
Horuk
R
R
Department of Protein Chemistry, Genentech Inc., South San Francisco, California 94080.
Wang
Z X
ZX
Peiper
S C
SC
Hesselgesser
J
J
D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D018932:Chemokine CCL2; D018946:Chemokine CCL5; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8 / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute; D008222:Lymphokines / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018121:Receptors, Cytokine / Q000378:metabolism*; D013154:Spleen / Q000378:metabolism; D014407:Tumor Cells, Cultured
C475829:CXCL1 protein, human; D018932:Chemokine CCL2; D018946:Chemokine CCL5; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D016207:Cytokines; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D016209:Interleukin-8; D008222:Lymphokines; D012333:RNA, Messenger; D018121:Receptors, Cytokine