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J. Biol. Chem., Vol. 283, Issue 19, 13398-13406, May 9, 2008

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Interleukin-5 Receptor Subunit Oligomerization and Rearrangement Revealed by Fluorescence Resonance Energy Transfer Imaging^*

From the Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102

Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, and βc. Specific assembly steps of full-length subunits as they occur in cell membranes, ultimately leading to receptor activation, are not well understood. We tracked the oligomerization of IL-5 receptor subunits using fluorescence resonance energy transfer (FRET) imaging. Full-length IL-5R and βc were expressed in Phoenix cells as chimeric proteins fused to enhanced cyan or yellow fluorescent protein (CFP or YFP, respectively). A time- and dose-dependent increase in FRET signal between IL-5R-CFP and βc-YFP was observed in response to IL-5, indicative of heteromeric receptor -βc subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5R. Substantial FRET signals with βc-CFP and βc-YFP co-expressed in the absence of IL-5R demonstrated that βc subunits exist as preformed homo-oligomers. IL-5 had no effect on this βc-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing βc-CFP, βc-YFP, and nontagged IL-5R led to further increase in FRET efficiency. Observation of preformed βc oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on βc assembly in the presence of nontagged IL-5R provide direct evidence that IL-5 can cause higher order rearrangements of βc homo-oligomers. These results suggest that IL-5 and perhaps other βc cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit ), followed by binding of the ligand-subunit complex to, and consequent rearrangement of, a ground state form of βc oligomers.

Received for publication, December 14, 2007 , and in revised form, February 22, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants R01 GM55648 and AI 40462. This work was also supported by National Science Foundation Grant DMI-0422010. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Drexel University College of Medicine, 11102 New College Bldg., 245 N. 15th St., Philadelphia, PA 19102. Tel.: 215-762-4197; Fax: 215-762-4452; E-mail: ichaiken{at}drexelmed.edu.

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