A two-step selection approach for the identification of ligand-binding determinants in cytokine receptors

We have developed a novel cell-based method for the isolation and selection of mutant cytokine receptors with defects in ligand binding and applied it to the human interleukin-4 receptor. The experimental procedure is based u pon the functional heterologous expression of receptor mutants in eukaryoti c cells followed by a two-step selection procedure. Positive selection for cells that express receptor variants is achieved by means of an agonistic a ntibody that mediates cell survival through receptor dimerization. An IL-4- coupled toxin is subsequently used to select against cells expressing wild- type receptors. Cells expressing mutant receptors that are unable to bind t he cytotoxic ligand survive and can be amplified. The procedure allows the isolation of rare receptor variants from cell pools containing predominantl y wild-type cells. This method, which should be equally applicable to simil ar receptor systems, was used to demonstrate the importance of a critical c harged amino acid residue in the human IL-4 receptor alpha-subunit for IL-4 -induced receptor activation, (C) 1999 Academic Press.