title: Internalization of inactive EGF receptor into endosomes and the subsequent activation of endosome-associated EGF receptors. Epidermal growth factor.
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.
authors.affiliation: Department of Cell Biology and Signal Transduction Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Steven
authors.initials: S
authors.lastname: Pennock
authors.forename: Xinmei
authors.initials: X
authors.lastname: Chen
authors.forename: Zhixiang
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D004285:Dogs; D011992:Endosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D066246:ErbB Receptors / Q000276:immunology / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000276:immunology / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011799:Quinazolines; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014407:Tumor Cells, Cultured; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D008565:Membrane Proteins; D010750:Phosphoproteins; D011799:Quinazolines; D020032:Tyrphostins; C101044:RTKI cpd; D019000:Phosphotyrosine; D008985:Monensin; D066246:ErbB Receptors; D017027:Protein Tyrosine Phosphatases