RECEPTOR RECOGNITION AND SPECIFICITY OF INTERLEUKIN-8 IS DETERMINED BY RESIDUES THAT CLUSTER NEAR A SURFACE-ACCESSIBLE HYDROPHOBIC POCKET

To determine the regions of interleukin-8 (IL-8) that allow high affin ity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chem okines, we produced chimeric proteins containing structural domains fr om IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 ( IL8R2) with high affinity, and from GRO gamma, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity, Receptor binding activ ity was tested by competition of I-125-IL-8 binding to recombinant IL8 R1 and IL8R2 cell lines, Substitution into IL-8 of the GRO gamma seque nces corresponding to either the amino-terminal loop (amino acids 1-18 ) or the first beta-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2, The third beta-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2, Exchanges of the second b eta-sheet (amino acids 32-46) or the carboxyl-terminal alpha-helix (am ino acids 53-72) had no significant effect, When IL-8 sequences were s ubstituted into GRO gamma, a single domain containing the second beta- sheet of IL-8 (amino acids 18-32) was sufficient to confer high affini ty binding for both IL8R1 and IL8R2, The amino-terminal loop (amino ac ids 1-18) and the third beta-sheet (amino acids 46-53) of IL-8 had lit tle effect when substituted individually but showed increased binding to both receptors when substituted in combination. Individual amino ac id substitutions were made at positions where IL-8 and GRO gamma seque nces differ within the regions of residues 11-21 and 46-53, IL-8 mutat ions L49A or L49F selectively inhibited binding to IL8R1, Mutations Y1 3L and F21N enhanced binding to IL8R1 with little effect on IL8R2, A c ombined mutation Y13L/S14Q selectively decreased binding to IL8R2, Res idues Tyr(13), Ser(14), Phe(21), and Lys(49) are clustered in and arou nd a surface-accessible hydrophobic pocket on IL-8 that is physically distant from the previously identified ELR binding sequence, A homolog y model of GRO gamma, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pock et was effectively abolished in GRO gamma, These studies suggest that the surface hydrophobic pocket and/or adjacent residues participate in IL-8 receptor recognition for both IL8R1 and IL8R2 and that the hydro phobic pocket itself may be essential for IL8R1 binding, Thus this reg ion contains a second site for IL-8 receptor recognition that, in comb ination with the Glu(4)-Leu(5)-Arg(6) region, can modulate receptor bi nding affinity and IL8R1 specificity.