COMPLETE MUTAGENESIS OF THE EXTRACELLULAR DOMAIN OF INTERLEUKIN-8 (IL-8) TYPE-A RECEPTOR IDENTIFIES CHARGED RESIDUES MEDIATING IL-8 BINDINGAND SIGNAL-TRANSDUCTION

We systematically converted each of the amino acids in the extracellul ar domain of the interleukin-8 (IL-8) type A receptor to alanine for t he purpose of identifying amino acids contributing to a IL-8 binding a nd IL-8-mediated signal transduction. We identified 20 mutations which cause a decrease in receptor affinity from a K, of 2 nM to a K-d grea ter than or equal to 25 nM. We then analyzed these receptor mutants fo r their ability to mobilize intracellular calcium upon stimulation wit h 10 nM IL-8. The majority of the mutants were able to produce calcium fluxes at levels approximating that of wild-type IL-8 receptor A, wit h the exception of six mutants (R199A, R203A C30A, C110A, C187A, and C 277A) which showed no significant response. In addition, we performed calcium mobilization experiments to further characterize a series of p reviously constructed mutants which had only been characterized by the ir binding affinities in our previous report and found that mutant D26 5A showed no response upon stimulation with 10 nM IL-8. Our study show s that, besides the extracellular domain cysteines which may be critic al for the overall folding of the receptor, three residues, Arg-199, A rg-203, and Asp-265, are important for IL-8 binding and IL-8-mediated signal transduction.