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

doi:10.1101/2020.07.11.198572

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 paper › Preprint

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.

Original language	English
Publisher	bioRxiv, at Cold Spring Harbor Laboratory
DOIs	https://doi.org/10.1101/2020.07.11.198572
Publication status	Published - 12 Jul 2020

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Cite this

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title = "EGF signaling in bowel carcinoma cells utilizes higher order architectures of EGFR and HER2",

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.",

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

year = "2020",

month = jul,

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doi = "10.1101/2020.07.11.198572",

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T1 - EGF signaling in bowel carcinoma cells utilizes higher order architectures of EGFR and HER2

AU - Wollman, Adam

AU - Llorente-Garcia, Isabel

AU - Fournier, Charlotte

AU - Harriman, Oliver L

AU - Hargreaves, Alex

AU - Shashkova, Sviatlana

AU - Ouaret, Djamila

AU - Zhou, Peng

AU - Liu, Ta-Chun

AU - Wilding, Jenny

AU - Kusumi, Akihiro

AU - Bodmer, Walter

AU - Leake, Mark Christian

PY - 2020/7/12

Y1 - 2020/7/12

N2 - 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.

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

PB - bioRxiv, at Cold Spring Harbor Laboratory

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