SOLUBLE INTERLEUKIN-5 RECEPTOR ALPHA-CHAIN BINDING ASSAYS - USE FOR SCREENING AND ANALYSIS OF INTERLEUKIN-5 MUTANTS

Interleukin-5 (IL-5) is a key cytokine for the production, differentia tion, and activation of eosinophils. IL-5 is a member of the four heli cal bundle family of cytokines, and in common with many members of the cytokine family it binds to a heterodimeric receptor composed of a li gand binding alpha-chain and a signal-transducing beta-chain. We have established two receptor/ligand binding assays based on the extracellu lar domain of the receptor alpha-chain which we have produced as a fus ion protein. One assay is based on scintillation proximity fluoromicro spheres and radiolabeled ligand and the other on detection of biotinyl ated ligand binding to immobilized receptor using a chemiluminescent s ubstrate in a 96-well microtiter plate format. Both receptor binding a ssays have been optimized for high throughput screening for receptor a ntagonists. These assays were also used for analytical purposes and th e binding of ligand to the receptor alpha-chain was compared directly to receptor binding assays performed on TF-1 cells which express the r eceptor alpha beta-heterodimer. These three assays have been used to s tudy site-directed mutants of IL-5 to determine the important residues for interaction of the cytokine with each chain of the receptor (P. G raber ct al. (1995) J. Biol. Chem. 270, 15762-15769). (C) 1995 Academi c Press, Inc.