Expression of the Duffy antigen in K562 cells. Evidence that it is the human erythrocyte chemokine receptor.
The human malarial parasite Plasmodium vivax invades erythrocytes by binding to a cell surface protein identified as the Duffy blood group antigen. The molecular properties of the Duffy antigen, which was recently cloned, are very similar to those of a chemokine binding protein known as the human erythrocyte chemokine receptor. This has led to the suggestion that these two molecules are the same protein. To further investigate the suspected double identity of the Duffy antigen we have transfected it into a human erythroleukemic cell line, K562. Cells stably expressing the Duffy antigen were isolated and used to characterize the protein. K562 cells transfected with the Duffy antigen displayed specific 125I-melanoma growth-stimulating activity (MGSA) binding while mock transfected cells did not. Comparison of 125I-MGSA binding to the Duffy antigen and the human erythrocyte chemokine receptor showed that the specific 125I-MGSA binding to both proteins was displaced by excess unlabeled MGSA, interleukin-8, RANTES, monocyte chemotactic peptide-1, and platelet factor 4, but not by macrophage inflammatory protein-1 alpha or -1 beta. Scatchard analysis of competition binding studies with these unlabeled chemokines revealed high affinity binding to the Duffy antigen with KD binding values of 24 +/- 4.9, 20 +/- 4.7, 41.9 +/- 12.8, and 33.9 +/- 7 nM for MGSA, interleukin-8, RANTES, and monocyte chemotactic peptide-1, respectively. A monoclonal antibody, Fy6, to the Duffy antigen inhibited 125I-MGSA binding to K562 cells expressing the Duffy antigen. Cell membranes from K562 cells permanently expressing the Duffy antigen were chemically cross-linked with 125I-MGSA. SDS-polyacrylamide gel electrophoresis analysis of the cross-linked products showed covalent incorporation of radiolabeled MGSA into a protein of molecular mass 47 kDa, and cross-linking was inhibited in the presence of unlabeled MGSA. These studies provide evidence that the Duffy blood group antigen is the same protein as the human erythrocyte chemokine receptor.
Chaudhuri
A
A
New York Blood Center, New York 10021.
Zbrzezna
V
V
Polyakova
J
J
Pogo
A O
AO
Hesselgesser
J
J
Horuk
R
R
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002460:Cell Line; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000378:metabolism*; D004375:Duffy Blood-Group System / Q000096:biosynthesis / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D010957:Plasmids; D018121:Receptors, Cytokine / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D004375:Duffy Blood-Group System; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D018121:Receptors, Cytokine; C078862:melanoma growth stimulating activity receptor