title: Dynamics and interaction of interleukin-4 receptor subunits in living cells.
abstract: It has long been established that dimerization of Interleukin-4 receptor (IL-4R) subunits is a pivotal step for JAK/STAT signal transduction. However, ligand-induced complex formation at the surface of living cells has been challenging to observe. Here we report an experimental assay employing trisNTA dyes for orthogonal, external labeling of eGFP-tagged receptor constructs that allows the quantification of receptor heterodimerization by dual-color fluorescence cross-correlation spectroscopy. Fluorescence cross-correlation spectroscopy analysis at the plasma membrane shows that IL-4R subunit dimerization is indeed a strictly ligand-induced process. Under conditions of saturating cytokine occupancy, we determined intramembrane dissociation constants (K(d,2D)) of 180 and 480 receptors per μm(2) for the type-2 complexes IL-4:IL-4Rα/IL-13Rα1 and IL-13:IL-13Rα1/IL-4Rα, respectively. For the lower affinity type-1 complex IL-4:IL-4Rα/IL-2Rγ, we estimated a K(d,2D) of ∼1000 receptors per μm(2). The receptor densities required for effective dimerization thus exceed the typical, average expression levels by several orders of magnitude. In addition, we find that all three receptor subunits accumulate rapidly within a subpopulation of early sorting and recycling endosomes stably anchored just beneath the plasma membrane (cortical endosomes, CEs). The receptors, as well as labeled IL-4 and trisNTA ligands are specifically trafficked into CEs by a constitutive internalization mechanism. This may compensate for the inherent weak affinities that govern ligand-induced receptor dimerization at the plasma membrane. Consistently, activated receptors are also concentrated at the CEs. Our observations thus suggest that receptor trafficking may play an important role for the regulation of IL-4R-mediated JAK/STAT signaling.
authors.affiliation: BIOTEC/Biophysics, Technische Universität Dresden, Dresden, Germany.
authors.forename: Hetvi
authors.initials: H
authors.lastname: Gandhi
authors.affiliation: BIOTEC/Biophysics, Technische Universität Dresden, Dresden, Germany; Institute of Physics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Remigiusz
authors.initials: R
authors.lastname: Worch
authors.affiliation: Center for Regenerative Therapies Dresden, Technische Universität Dresden, Dresden, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Kurgonaite
authors.affiliation: Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Hintersteiner
authors.affiliation: BIOTEC/Biophysics, Technische Universität Dresden, Dresden, Germany; Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Schwille
authors.affiliation: Center for Regenerative Therapies Dresden, Technische Universität Dresden, Dresden, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Bökel
authors.affiliation: BIOTEC/Biophysics, Technische Universität Dresden, Dresden, Germany; Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: weidemann@biochem.mpg.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Weidemann
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D053612:Janus Kinases / Q000378:metabolism; D008024:Ligands; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D019948:Receptors, Interleukin-4 / Q000378:metabolism*; D050791:STAT Transcription Factors / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D021122:Protein Subunits; D019948:Receptors, Interleukin-4; D050791:STAT Transcription Factors; D053612:Janus Kinases