Construction and activity analysis of chimeric interleukin-8 receptor for chemokine binding

초록

The chemokines [intercrines or platelet factor 4 (PF4) superfamily of cytokines] are involved in a variety of immune and inflammatory responses, acting primarily as chemoattractants and activators for specific leukocytes. PF4, interleukin-8 (IL-8), human protooncogene Gro/melanocyte growth stimulating activity (MGSA), beta-thromboglobulin (beta-TG), neutrophil activating protein-2 (NAP-2),neutrophil activating protein from epithelial cells (ENA-78), and interferon inducing protein-10 (IP-10) are members of chemokine alpha gene family characterized by CXC motif (cysteines separated by any amino acid). Two distinct but homologous human IL-8 receptor(R)s, alpha and beta, bind to alpha chemokines including IL-8. The N-terminus of the receptor may be involved in binding to the ELR (glutamic acid-leucine-arginine) motif of CXC chemokines. We created IL-8R alpha and beta chimera to define interaction of receptor/chemokine ligand. In studies using transiently expressed IL-8R alpha and beta in COS cells, both IL-8Rs bound to IL-8 with high affinity. The chimeric receptor IL-8R alpha/beta, however, bound more [I-125]IL-8 than did with IL-8R alpha or beta, respectively. On the other hand, the binding capacity of chimeric receptor IL-8R beta/alpha was not so active. PF4 did not bind to neither IL-8Rs, but bound to IL-8R alpha/beta with similar to 80% increase in binding affinity. The relative binding capacity of receptor to 1 mu M NAP-2 was similar to 60% for IL-8R alpha, similar to 87% for IL-8R beta, and similar to 55% for IL-8R alpha/beta compared to that of IL-8. Ligand-induced release of inositol phosphates was detected in COS cells which were coexpressed with IL-8Rs and G alpha 16 (G protein a subunit). Both IL-8R alpha and beta released similar amounts of inositol phosphate, but IL-8R alpha/beta and IL-8R beta/alpha resulted lower efficiency of the release than that by IL-8R alpha or beta. In case of NAP-2 response, IL-8R beta is higher than IL-8R alpha, whereas IL-8R alpha/beta and IL-8R beta/alpha showed about the same overall pattern as IL-8. Based on these results, we concluded that the binding of recombinant (r) IL-8, rNAP-2 and rPF4 to the two IL-8Rs, alpha and beta, were not only depending on its N-terminal domain, but also on the receptor conformations expressed at the cell surface.

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