PARTIAL FUNCTIONAL MAPPING OF THE HUMAN INTERLEUKIN-8 TYPE-A RECEPTOR- IDENTIFICATION OF A MAJOR LIGAND-BINDING DOMAIN

We have previously demonstrated that a basic amino acid residue of int erleukin (IL)-8, namely Arg-6, is critical for the binding of IL-8 to its receptor. We reasoned that this residue is likely to be poised to directly interact with a counterpart acidic residue on the receptor. T o identify this key residue, we systematically mutated to Ala all acid ic residues present on the ligand accessible surface of IL-8 receptor type A. Using this strategy, we demonstrate that two residues which ar e present in extracellular loop 3 of the receptor, namely Glu-275 and Arg-80, are critical for ligand binding. In addition, we show that alt hough Asp-11 is critical for ligand binding, a conservative mutation o f Asp-11 to Glu or a substitution of Asp-11 with Lys (the residue foun d at position 11 in IL-8 receptor type B) does not affect the K(d) of the receptor/ligand interaction. These data suggest that Lys-11 recrui ts a new and favorable interaction with IL-8 (analogous to that of IL- 8 receptor type B with IL-8) or that the cavity created by mutating As p-11 to Ala is particularly deleterious. Finally, we discuss fluoresce nce-activated cell sorter staining data which support the hypothesis t hat the N-terminal region and the extracellular loop 3 of the receptor may lie in close proximity of one another and constitute a major bind ing domain for IL-8.