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Monitoring Ligand‐Dependent IL‐5 Receptor Activation on Cells Using Fluorescence Resonance Energy Transfer (FRET)
Journal article   Open access   Peer reviewed

Monitoring Ligand‐Dependent IL‐5 Receptor Activation on Cells Using Fluorescence Resonance Energy Transfer (FRET)

Meirav Zaks‐Zilberman, Adrian Harrington, Tetsuya Ishino and Irwin Chaiken
The FASEB journal, v 21(5), pp A250-A250
Apr 2007
DOI: https://doi.org/10.1096/fasebj.21.5.A250-a
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https://doi.org/10.1096/fasebj.21.5.a250-aView
Published, Version of Record (VoR) Open

Abstract

Interleukin‐5 (IL‐5) is important in a number of pathophysiological states, including allergy, asthma, hypereosinophilia syndromes, myeloid leukemia, and certain infectious diseases. IL‐5 exerts its biological functions through binding to its receptor, which is composed of subunits α and βc. To date, the mechanism of receptor subunit interactions in the cellular context is poorly understood. We used fluorescence resonance energy transfer (FRET) to measure the interactions of IL‐5R subunits in intact cells, including the extent of preassembly and the effects of IL‐5 stimulation. Phoenix cells coexpressing IL‐5Rα and βc fused to cyan or yellow fluorescent proteins (CFP or YFP) were used to evaluate protein‐protein interactions by FRET before and after IL‐5 treatment and in the presence or absence of the IL‐5Rα inhibitor AF17121. We observed FRET between IL‐5R βc chains in the absence of ligand, demonstrating that βc receptor subunits exist in preformed homomeric receptor complexes. In the presence of ligand, we observed dose‐dependent FRET between IL‐5Rα and βc consistent with heteromeric receptor subunit assembly. Ligand‐dependent heteromeric subunit assembly, but not βc self‐assembly, was inhibited by the IL‐5Rα inhibitor AF17121. Taken together, these results are consistent with a model in which βc molecules exist in the cell membrane as dimers prior to ligand binding, and where binding of cytokine ligand by IL‐5Rα leads to recruitment of βc dimers into heteromeric complexes.

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