Abstract
In order to identify residues required for the binding of interleukin-8 (IL-8) to its receptor, mutants were constructed in which clusters of charged amino acids were systematically replaced with alanine along the entire IL-8 sequence. The mutants were tested for their ability to induce a receptor-mediated rise in cytosolic free Ca2+, a property of wild-type IL-8 which can readily be detected by flow cytometry using neutrophils loaded with the calcium probe Indo-1. Eleven of the 12 mutants caused neutrophil calcium mobilization at 5 nM; the exception being a triple alanine mutant at positions K3, E4, and R6, which was inactive at all concentrations tested (150 nM maximum). A second set of mutants was generated in which residues 1-15 were individually mutated to alanine. Mutants E4A, L5A, or R6A were all inactive in the Ca2+ assay at 5 nM and competed poorly with 125I-IL-8 for neutrophil receptor binding; I10A, E4A, L5A, and R6A had approximately 30-, 100-, 100-, and 1000-fold reduced affinity, as compared with control IL-8, respectively. The nuclear magnetic resonance structure of IL-8 indicates that, in solution, the side chains of E4, L5, R6, and I10 point away from the core of the protein and do not participate in any intramolecular hydrogen bonds or salt bridges (Clore, G. M., and Gronenborn, A. M. (1991) J. Mol. Biol. 217, 611-620).
Highlights
In orderto identify residues required for the binding Tanaka et al (1988)provided the first reporton the activity of interleukin-8 (IL-8) to its receptor, mutants were of peptides based on the IL-8 sequence
If one excludes the possibility that the p-FINAP preparation was contaminated with ~ 1 I% L-8, this observation suggests that -terminal half of IL-8 is sufficient to cause signal transduction but that presence of the C-terminal half of IL-8 is necessary for potency in the subnanomolar range
A number of groups have used synthetic peptides in an attempt to map the domains of IL-8 that are important for receptor binding and signal transduction, but together, these findings present an unclear picture of IL-8 structure/function relationships
Summary
In orderto identify residues required for the binding Tanaka et al (1988)provided the first reporton the activity of interleukin-8 (IL-8) to its receptor, mutants were of peptides based on the IL-8 sequence. The effects on IL-8 binding of conservative mutations of the which the N-terminal sequence Ala-Val-Leu-Pro-Arg is changed to 2 charged residues are presented.
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