Identification of a receptor binding site in the carboxyl terminus of human interleukin-6.

Abstract

To identify a receptor binding site of human interleukin-6 (IL-6), we created a library of IL-6 variants with single amino acid substitutions in the last 15 residues (171-186) in the COOH terminus of IL-6.Twenty-seven IL-6 variants were tested for biological activity on a human hepatoma and a mouse hybridoma cell line.Most variants were additionally tested in a receptor binding assay using a human myeloma cell line.Several single amino acid substitutions in the COOH terminus of IL-6 were found to decrease biological activity significantly.This is especially seen in variants with amino acid substitutions that alter the postulated amphipathical a-helix structure between residues 178 and 183.The two highly conserved Arg residues at positions 180 and 183 seem to play a very important role in biological activity.The loss of biological activity in all inactive variants is completely par- alleled by a decrease of IL-6 receptor binding, as determined by competition binding experiments.One mutant (Leu171) displayed a higher activity on human cells and a higher binding affinity to the receptor and can be considered an IL-6 agonist.It is concluded that the amphipathical a-helix structure in the COOH terminus of IL-6 is critical for ligand receptor interaction.Furthermore, the region between residues Serl" and ArglS3 (Ser-Leu-Arg-Ala-X-Arg) is identified as a receptor binding site in the COOH terminus of human IL-6.Interleukin-6 (IL-6)' is a multifunctional cytokine that is produced by a variety of cells such as B-cells, T-cells, monocytes, fibroblasts, and endothelial cells (f6r review see Refs. 1, 2, and 3).IL-6 exerts several activities including terminal differentiation of B-cells (4), proliferation and differentiation of T-cells (5), regulation of the acute phase response (6), and growth regulation of epithelial cells (7).It also stimulates the