Activation of Endosome-Associated Inert EGF Receptor Following Internalization

Our current understanding of endosomal signaling is incomplete given our inability to generate endosomal signals in isolation from plasma membrane signals. We present a novel method to specifically activate epidermal growth factor (EGF) receptor (EGFR) following its internalization into endosomes. This method will allow us to address questions such as whether endosomal signaling is sufficient to activate signal transduction pathways and produce biological responses. In this method, the cells are treated with EGF in the presence of AG1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks recycling of EGFR. The treatment results in the internalization of nonactivated EGF-EGFR complex into endosomes. Next, the removal of AG1478 and monensin causes endosome-associated EGFR activation. Phosphorylated surface EGFR levels are undetectable during the treatment. This is a unique and novel approach to study endosomal signaling and its physiological relevance. The protocol is transferable to study endosomal signaling of other receptor tyrosine kinases.