IDENTIFICATION OF KEY CHARGED RESIDUES OF HUMAN INTERLEUKIN-5 IN RECEPTOR-BINDING AND CELLULAR ACTIVATION

Interleukin-5 (IL-5) is a cytokine that plays a major role in the diff erentiation and activation of eosinophils. In order to identify which charged residues of human IL-5 are important in binding to its recepto r and subsequent cellular activation, we have systematically replaced all of the clusters of charged amino acids with alanine residues. The mutants have been expressed in Escherichia coli, renatured, and purifi ed. They were assayed for ability to cause proliferation of the erythr o-leukaemic cell line TF-1 and the up-regulation of eosinophil adhesio n to ICAM-1. In addition, we studied receptor binding using either imm obilized recombinant IL-5 receptor alpha-chain or the alpha/beta-recep tor complex expressed on TF-2 cells. The key charged residue is Glu-12 . This residue is in an identical position to those previously identif ied in IL-3 and granulocyte-macrophage colony-stimulating factor (GM-C SF) involved in binding to the receptor beta-chain. The alpha-chain bi nding site is shown to involve the side chains Arg-90 and Glu-109, loc ated in the second beta sheet and after the end of the fourth helix, r espectively. It is unique to IL-5 and does not occur in IL-3 or GM-CSF . Understanding the topology of the interaction of IL-5 with its recep tor chains will help in the search for rationally designed antagonists of IL-5 function.