Cells must Accumulate Interleukin-4 Receptor Subunits within Cortical Signaling Endosomes to Drive Complex Formation and Signal Transduction

Abstract

The cytokines Interleukin-4 (IL-4) and IL-13 provide cues for many important immune functions and their upregulation is associated with disorders like allergy and asthma. Signaling requires ligand mediated dimerization of single-pass transmembrane receptors. We recently established epithelial HEK293T cells as a model to characterize ectopically expressed IL-4R subunits with single- and dual-color fluorescence correlation spectroscopy [1]. Here we report an improved experimental setup employing hexahistidine specific dyes and demonstrate for the first time ligand-induced IL-4R complex formation in a native plasma membrane. Furthermore, we quantified the two-dimensional affinity constants for all three combinations of receptor dimers with 120 (IL-4:IL-4Rα/IL-13Rα1), 510 (IL-13:IL-13Rα1/IL-4Rα), and 825 (IL-4:IL-4Rα/IL-2Rγ) receptor molecules per μm2. However, considering physiological surface expression levels of several 100-1000 receptor molecules per cell, such low affinities challenge the traditional view that signaling productive complexes self-assemble in significant numbers in the plasma membrane.Instead, we mount several lines of evidence that signal transduction requires the accumulation of the receptor subunits within a novel class of early endosomes. These cortical signaling endosomes are stably anchored within the actin cortex just beneath the plasma membrane and carry markers of both the early sorting (EEA1, Rab5) and recycling compartments (Rab11). The IL-4R subunits show Rac1/Pak-dependent trafficking from the surface into these endosomes with a time constant of 6-9 min. Using fluorescence lifetime imaging / Forster resonance energy transfer (FLIM/FRET) microscopy, we could demonstrate ligand-dependent complex formation within the cortical signaling endosomes. Importantly, specific inhibition of the endocytosis machinery with drugs abrogates both receptor trafficking and phosphorylation of the downstream signal transducer STAT6. In summary, our findings suggest a unique thermodynamic function for endocytosis upstream of JAK/STAT pathway activation.[1] Weidemann, T., et al. (2011) Biophys. J.101: 2360-69.