EGF signaling in bowel carcinoma cells utilizes higher order architectures of EGFR and HER2

Adam Wollman, Isabel Llorente-Garcia, Charlotte Fournier, Oliver L Harriman, Alex Hargreaves, Sviatlana Shashkova, Djamila Ouaret, Peng Zhou, Ta-Chun Liu, Jenny Wilding, Akihiro Kusumi, Walter Bodmer, Mark Christian Leake

Research output: Working paperPreprint


Epidermal growth factor (EGF) signaling regulates normal cell development, however EGF receptor (EGFR) overexpression is reported in several carcinomas. Despite structural and biochemical evidence that EGF-EGFR ligation activates signaling through monomer-dimer transitions, live cell mechanistic details remain contentious. We report single-molecule multispectral TIRF of human epithelial carcinoma cells transfected with fluorescent EGFR, and of CHO-K1 cells containing fluorescent EGFR and HER2, enabling super-resolved localization to quantify receptor architectures and spatiotemporal dynamics upon EGF ligation. Using inhibitors that block binding to EGFR, and time-dependent kinetics modelling, we find that pre-activated EGFR consist predominantly of preformed clusters that contain a mixture of EGFR and HER2, whose stoichiometry increases following EGF activation. Although complicated by EGFR internalization and recycling, our observation of an EGFR:EGF stoichiometry >1 for plasma membrane colocalized EGFR/EGF foci soon after activation may indicate preferential binding of EGF ligand to EGFR monomers, negative cooperativity and preferential ligated-unligated dimerization of monomers.
Original languageEnglish
PublisherbioRxiv, at Cold Spring Harbor Laboratory
Publication statusPublished - 12 Jul 2020

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