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Title: Oligomerization-function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy.
Authors: Yamashita, Hirotaka
Yano, Yoshiaki  kyouindb  KAKEN_id  orcid (unconfirmed)
Kawano, Kenichi  kyouindb  KAKEN_id  orcid (unconfirmed)
Matsuzaki, Katsumi  kyouindb  KAKEN_id  orcid (unconfirmed)
Author's alias: 松崎, 勝巳
Keywords: Dimerization
Live-cell imaging
Issue Date: 11-Mar-2015
Publisher: Elsevier BV
Journal title: Biochimica et biophysica acta
Volume: 1848
Issue: 6
Start page: 1359
End page: 1366
Abstract: The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor-ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag-probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~10%), whereas ~70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~1nM) was lower than that for half-maximum autophosphorylation (~8nM), which suggested the presence of an inactive dimer binding a single EGF molecule.
Rights: © 2015 Elsevier B.V. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International NOTICE: this is the author's version of a work that was accepted for publication in [Biochimica et Biophysica Acta (BBA) - Biomembranes]. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in [Biochimica et Biophysica Acta (BBA) - Biomembranes Volume 1848, Issue 6, Pages 1359–1366] DOI:10.1016/j.bbamem.2015.03.004
This is not the published version. Please cite only the published version.
DOI(Published Version): 10.1016/j.bbamem.2015.03.004
PubMed ID: 25771448
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