| The four members of Janus kinase family, JAK1-3 and TYK2, provide the interconnecting link for a complex matrix of extracellular and cytoplasmic receptor-ligand pairs in cytokine signaling. Here we show first results of our attempt to reconstitute a functional fluorescently tagged Interleukin-4 receptor (IL4R) signaling complex at the plasma membrane. The IL4R type I consists of the IL4R alpha chain (IL4Ra) bound by JAK1 and the co-receptor IL2R gamma chain (IL2Rg, common gamma chain) bound by JAK3. In agreement with literature, we see that cytoplasmic fractions of JAK3 do not exchange with receptor bound fractions as shown by fluorescence recovery after photobleaching (FRAP). In addition, co-expression of JAK3 and IL2Rg leads to aggregation phenomena at the plasma membrane as indicated by the occurrence of fluorescent speckles. We further characterized the aggregation behavior of JAK3-EGFP, and mutants thereof, with fluorescence correlation spectroscopy (FCS) in the cytoplasm. The biophysical data are discussed with respect to downstream signaling. For this we studied IL4-induced and JAK3-mediated target gene activation with a Signal Transducer and Activator of Transcription 6 (STAT6) -responsive luciferase assay. |
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