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

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DatePublished - 12 Jul 2020
PublisherbioRxiv, at Cold Spring Harbor Laboratory
Original languageEnglish

Abstract

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.

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