DISCRETE STEPS IN BINDING AND SIGNALING OF INTERLEUKIN-8 WITH ITS RECEPTOR

The mechanisms by which chemokines bind and signal through their recep tors are complex and poorly understood. in the present study, we sough t to dissect these processes and to map important functional domains o f the two CXC chemokine (interleukin-8) receptors, CXCR1 (formally IL- 8RA) and CXCR2 (formally IL-8RB), using blocking monoclonal antibodies (mAbs) to the receptors and a series of chimeras between CXCR1 and CX CR2. A panel of specific mAbs against CXCR1 or CXCR2, generated by imm unizing mice with transfectants expressing either receptor, were shown to effectively block IL-8- and/or growth-related oncogene a (GRO alph a) -mediated Ligand binding, chemotaxis, elastase release, and VCAM-1 binding in CXCR1 and CXCR2 transfectants and/or human neutrophils. Of particular interest was an anti-CXCR1 mAb, 7D9, that inhibited chemota xis, elastase release, and VCAM-1 binding but had no detectable effect s on Ligand binding. The epitopes of these blocking mAbs were mapped b y using a series of CXCR1/2 chimera transfectants and synthetic peptid es, Most of the anti-CXCR1 antibodies, except 7D9, mapped to the amino acid sequence WDFDDL (CXCR1 residues 10-15), and all the anti-CXCR2 a ntibodies mapped to the amino acid sequence FEDFW (CXCR1 residues 6-10 ). The epitope of mAb 7D9 mainly involved a region within the first 45 residues of CXCR1, and it appeared to be conformation-sensitive. Thes e results support a model in which the binding and signaling of IL-8 w ith its receptor occur in at least two discrete steps involving distin ct domains of the receptor, This model is consistent with the notion t hat discrete conformational changes of the receptor secondary to ligan d binding are required to trigger various biological responses. Moreov er, the ligand binding and chemotaxis properties of each CXCR1/2 chime ric receptor to IL-8 and GRO alpha were determined, It was found that each is distinct in its ability to confer Ligand binding and chemotact ic response to IL-8 and GRO alpha, and two conclusions could be made. 1) The N-terminal segment of CXCR1 is a dominant determinant of recept or subtype selectivity, consistent with previous studies using rabbit/ human CXCR1/2 chimeras; and 2) the specificity determinant for Oil-On binding in CXCR2 involves sequences in the N terminus, distal to the f irst 15 residues, as well as other parts of the receptor.