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Mutants of Single Chain Interleukin 5 Show Asymmetric Recruitment of Receptor α and βc Subunits
Journal article   Open access   Peer reviewed

Mutants of Single Chain Interleukin 5 Show Asymmetric Recruitment of Receptor α and βc Subunits

Jun Li, Richard Cook and Irwin Chaiken
The Journal of biological chemistry, v 271(49), pp 31729-31734
06 Dec 1996
DOI: https://doi.org/10.1074/jbc.271.49.31729
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
http://www.jbc.org/content/271/49/31729.full.pdfView
Published, Version of Record (VoR) Open
url
https://doi.org/10.1074/jbc.271.49.31729View
Published, Version of Record (VoR) Open

Abstract

Dual asymmetric mutagenesis of single-chain interleukin 5 (scIL5) was used to obtain evidence that the normally homodimeric IL5 molecule, which contains two 4-helix bundle domains arranged symmetrically about a 2-fold axis, can recruit receptor α and βc subunits asymmetrically. Functionally active scIL5 was constructed using recombinant DNA methods by linking two IL5 monomers with a Gly-Gly linker. Mutants were constructed at residues Arg91, Glu110, and Trp111, previously shown to be involved in IL5 receptor α chain binding, and at residue Glu13, known to be involved in signal transduction presumably through interaction with the receptor βc chain. Mutants were examined for receptor α chain binding by an optical biosensor assay and for bioactivity using a cell proliferation assay. Substitution of the two binding site residues R91 and W111 in the same 4-helix bundle domain caused a 5-fold greater reduction in receptor binding affinity than when the two substitutions were distributed one in each domain. Substitution of E13 and R91 either in the same or in opposite domains gave comparable IL5Rα chain binding kinetics, essentially unchanged from those of scIL5. However, in contrast to the binding affinity pattern observed with R91A/W111A dual mutants, distributing the E13A/R91A mutations between the two 4-helix bundle domains caused a 5-6-fold greater loss of bioactivity than when the two changes were in the same domain, leaving the other domain unaltered. Taken with previous mutagenesis data, these results are consistent with a single shared-site model of IL5-IL5Rα chain recognition in which a single α chain can orientate in either of two modes, each one of which is stabilized preferentially by one of the two 4-helix bundles of IL5. Furthermore, the results suggest that a single βc molecule is activated for each IL5, through the Glu13 residue on the same helix bundle domain that dominates the IL5Rα interaction.

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Web of Science research areas
Biochemistry & Molecular Biology
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