Interleukin-5 receptor subunit oligomerization and rearrangement revealed by fluorescence resonance energy transfer imaging.
Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, alpha and betac. 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-5Ralpha and betac 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-5Ralpha-CFP and betac-YFP was observed in response to IL-5, indicative of heteromeric receptor alpha-betac subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5Ralpha. Substantial FRET signals with betac-CFP and betac-YFP co-expressed in the absence of IL-5Ralpha demonstrated that betac subunits exist as preformed homo-oligomers. IL-5 had no effect on this betac-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing betac-CFP, betac-YFP, and nontagged IL-5Ralpha led to further increase in FRET efficiency. Observation of preformed betac oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on betac assembly in the presence of nontagged IL-5Ralpha provide direct evidence that IL-5 can cause higher order rearrangements of betac homo-oligomers. These results suggest that IL-5 and perhaps other betac cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit alpha), followed by binding of the ligand-subunit alpha complex to, and consequent rearrangement of, a ground state form of betac oligomers.
Zaks-Zilberman
Meirav
M
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.
Harrington
Adrian E
AE
Ishino
Tetsuya
T
Chaiken
Irwin M
IM
D002460:Cell Line; D018360:Crystallography, X-Ray; D053648:Cytokine Receptor Common beta Subunit / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D006801:Humans; D015848:Interleukin-5 / Q000378:metabolism; D053666:Interleukin-5 Receptor alpha Subunit / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D019076:Transgenes
D053648:Cytokine Receptor Common beta Subunit; D015848:Interleukin-5; D053666:Interleukin-5 Receptor alpha Subunit