Identification of a receptor binding site in the carboxyl terminus of human interleukin-6.
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 (171-185) 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 alpha-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 paralleled 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 alpha-helix structure in the COOH terminus of IL-6 is critical for ligand receptor interaction. Furthermore, the region between residues Ser178 and Arg183 (Ser-Leu-Arg-Ala-X-Arg) is identified as a receptor binding site in the COOH terminus of human IL-6.
Leebeek
F W
FW
Department of Pathology, University of North Carolina, Chapel Hill 27599.
Kariya
K
K
Schwabe
M
M
Fowlkes
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics* / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000378:metabolism; D016296:Mutagenesis; D011971:Receptors, Immunologic / Q000378:metabolism*; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015850:Interleukin-6; D011971:Receptors, Immunologic; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins