Elsevier

Analytical Biochemistry

Volume 268, Issue 2, 15 March 1999, Pages 179-186
Analytical Biochemistry

Regular Article
A Two-Step Selection Approach for the Identification of Ligand-Binding Determinants in Cytokine Receptors

https://doi.org/10.1006/abio.1998.3078Get rights and content

Abstract

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 upon the functional heterologous expression of receptor mutants in eukaryotic cells followed by a two-step selection procedure. Positive selection for cells that express receptor variants is achieved by means of an agonistic antibody 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 the cytotoxic ligand survive and can be amplified. The procedure allows the isolation of rare receptor variants from cell pools containing predominantly wild-type cells. This method, which should be equally applicable to similar receptor systems, was used to demonstrate the importance of a critical charged amino acid residue in the human IL-4 receptor α-subunit for IL-4-induced receptor activation.

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