CLOSE ASSOCIATION OF THE FIRST AND 4TH EXTRACELLULAR DOMAINS OF THE DUFFY ANTIGEN RECEPTOR FOR CHEMOKINES BY A DISULFIDE BOND IS REQUIRED FOR LIGAND-BINDING/

It has been demonstrated that the promiscuous chemokine binding profil e of the Huffy antigen/reaeptor for chemokines (DARC) is given by its extracellular NH2-terminal region. However, the relationship among the Fy6, Fy(a)/(b), and Fy3 epitopes, localized in the first and fourth e xtracellular domains of DARC, respectively, and the chemokine binding sites remained a matter of controversy. Here, we performed cross-displ acement and cross-inhibition experiments indicating that all anti-Fy6, anti-Fy(a), and anti-Fy3 monoclonal antibodies and interleukin 8 are antagonists for binding to red cells. Biopanning of phage peptide libr aries with an anti-Fy6 monoclonal antibody led to the identification o f the motif phe(22)-Glu(23), th, mutation of which altered the binding of both anti-Fy6 and chemokines (interleukin 8, MGSA, RANTES (regulat ed on activation normal T cell expressed)) to DARC transfectants. Thes e results characterized the core of the Fy6 epitope and provided defin itive proof of the tight relationship between Fy6 and the chemokine re ceptor site. Analysis of red cells treated by sulfhydryl group-modifyi ng reagents suggested that the chemokine receptor function of DARC req uired the integrity of disulfide bond(s) but not that of free sulfhydr yl group(s). Accordingly, mutation of cysteines 51 and 276 abolished c hemokine binding to DARC transfectants. Altogether, our results sugges ted that the chemokine binding pocket of DARC included sequences locat ed in the first and fourth extracellular domains which are brought int o close vicinity by a disulfide bridge.