Biochemical Monitoring of the Early Endocytic Traffic of the Type I Interferon Receptor

The type I interferon (IFN) receptor consists of two transmembrane chains IFNAR1 and IFNAR2, associated with the tyrosine kinases Tyk2 and Jak1, respectively. Binding of IFN to this receptor complex induces activation of Jak/Stat and non-Stat signaling pathways. Ligand binding also drives receptor internalization and sorting toward degradation or recycling. To gain insights into receptor trafficking and its relation to signaling, we performed subcellular organelle fractionation from IFN-stimulated Daudi cells and defined biochemically an early endosomal antigen-1 (EEA1)-positive compartment bearing the activated IFN receptor. Endosomes were thus purified by immunoaffinity isolation on anti-EEA1 antibodies-coated beads. The content of these purified endosomal fractions was analyzed by Western blot and proteomics. Shortly after IFN stimulation, robustly ubiquitinated IFNAR1 and a small amount of IFNAR2 were found in this endosomal compartment, which also contained tyrosine-phosphorylated Tyk2 and Jak1. Thes...