title: Single cell analysis of ligand binding and complex formation of interleukin-4 receptor subunits.
abstract: Interleukin-4 (IL-4) is an important class I cytokine involved in adaptive immunity. IL-4 binds with high affinity to the single-pass transmembrane receptor IL-4Rα. Subsequently, IL-4Rα/IL-4 is believed to engage a second receptor chain, either IL-2Rγ or IL-13Rα1, to form type I or II receptor complexes, respectively. This ternary complex formation then triggers downstream signaling via intracellular Janus kinases bound to the cytoplasmic receptor tails. Here, we study the successive steps of complex formation at the single cell level with confocal fluorescence imaging and correlation spectroscopy. We characterize binding and signaling of fluorescently labeled IL-4 by flow cytometry of IL-4-dependent BaF3 cells. The affinity to ectopically expressed IL-4Rα was then measured by single-color fluorescence correlation spectroscopy in adherent HEK293T cells that express the components of the type II IL-4R but not type I. Finally, IL-4-induced complex formation was tested by dual-color fluorescence cross-correlation spectroscopy. The data provide evidence for codiffusion of IL-4-A647 bound IL-4Rα and the type II subunit IL-13Rα1 fused to enhanced green fluorescent protein, whereas type I complexes containing IL-2Rγ and JAK3 were not detected at the cell surface. This behavior may reflect hitherto undefined differences in the mode of receptor activation between type I (lymphoid) and type II (epithelial) receptor expressing cells.
authors.affiliation: Biophysics Research Group, Technische Universität Dresden, Dresden, Germany. thomas.weidemann@biotec.tu-dresden.de
authors.forename: Thomas
authors.initials: T
authors.lastname: Weidemann
authors.forename: Remigiusz
authors.initials: R
authors.lastname: Worch
authors.forename: Kristina
authors.initials: K
authors.lastname: Kurgonaite
authors.forename: Martin
authors.initials: M
authors.lastname: Hintersteiner
authors.forename: Christian
authors.initials: C
authors.lastname: Bökel
authors.forename: Petra
authors.initials: P
authors.lastname: Schwille
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D051379:Mice; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D053660:Receptors, Interleukin-13 / Q000378:metabolism*; D019948:Receptors, Interleukin-4 / Q000378:metabolism*; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: C569686:Alexa Fluor 647; D002232:Carbocyanines; D008024:Ligands; D021122:Protein Subunits; D053660:Receptors, Interleukin-13; D019948:Receptors, Interleukin-4
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