Abstract

Despite intensive efforts to understand cell signaling from endosomes, there is no direct evidence demonstrating that endosomal signaling is sufficient to activate signal transduction pathways or that endosomal signaling can produce biological responses. The lack of breakthrough is due in part to the inability to generate endosomal signals in isolation from plasma membrane signals. In this Protocol, we describe a system in which epidermal growth factor (EGF) receptor (EGFR) is specifically activated when it is endocytosed into endosomes. We treated cells with EGF in the presence of AG1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks recycling of EGFR. This treatment led to the internalization of nonactivated EGF-EGFR complex into endosomes. The endosome-associated EGFR was then activated by removing AG1478 and monensin. During this procedure, we did not observe any detectable surface EGFR phosphorylation. We also achieved specific activation of endosome-associated EGFR without using monensin. Specific activation of endosome-associated EGFR provides a unique tool to study endosomal signaling of EGFR. This method may also be applied to other receptor tyrosine kinases to study whether they, too, can signal from endosomes.