title: Effect of phosphorylation on EGFR dimer stability probed by single-molecule dynamics and FRET/FLIM.
abstract: Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (koff). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the koff of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell's proliferation potential.
authors.affiliation: Richard Dimbleby Department of Cancer Research, King's College London, London, UK; Randall Division of Cellular and Molecular Biophysics, King's College London, London, UK. Electronic address: oana.coban@kcl.ac.uk.
authors.forename: Oana
authors.initials: O
authors.lastname: Coban
authors.affiliation: Science and Technology Facilities Council, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK.
authors.forename: Laura C
authors.initials: LC
authors.lastname: Zanetti-Dominguez
authors.affiliation: Richard Dimbleby Department of Cancer Research, King's College London, London, UK; Randall Division of Cellular and Molecular Biophysics, King's College London, London, UK.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Matthews
authors.affiliation: Science and Technology Facilities Council, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rolfe
authors.affiliation: Richard Dimbleby Department of Cancer Research, King's College London, London, UK; Randall Division of Cellular and Molecular Biophysics, King's College London, London, UK.
authors.forename: Gregory
authors.initials: G
authors.lastname: Weitsman
authors.affiliation: Gray Institute for Radiation Oncology & Biology, Department of Oncology, University of Oxford, Oxford, UK.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Barber
authors.affiliation: Richard Dimbleby Department of Cancer Research, King's College London, London, UK; Randall Division of Cellular and Molecular Biophysics, King's College London, London, UK.
authors.forename: Jody
authors.initials: J
authors.lastname: Barbeau
authors.affiliation: Richard Dimbleby Department of Cancer Research, King's College London, London, UK; Randall Division of Cellular and Molecular Biophysics, King's College London, London, UK.
authors.forename: Viviane
authors.initials: V
authors.lastname: Devauges
authors.affiliation: Division of Imaging Sciences, King's College London, The Rayne Institute, St. Thomas Hospital, London, UK.
authors.forename: Florian
authors.initials: F
authors.lastname: Kampmeier
authors.affiliation: Computational Science and Engineering Department, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, UK.
authors.forename: Martyn
authors.initials: M
authors.lastname: Winn
authors.affiliation: Randall Division of Cellular and Molecular Biophysics, King's College London, London, UK; Gray Institute for Radiation Oncology & Biology, Department of Oncology, University of Oxford, Oxford, UK.
authors.forename: Borivoj
authors.initials: B
authors.lastname: Vojnovic
authors.affiliation: Division of Cancer Studies, King's College London, London, UK; Cancer Research UK, London Research Institute, London, UK.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Parker
authors.affiliation: Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Lidke
authors.affiliation: Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico; Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque, New Mexico.
authors.forename: Diane S
authors.initials: DS
authors.lastname: Lidke
authors.affiliation: Richard Dimbleby Department of Cancer Research, King's College London, London, UK; Randall Division of Cellular and Molecular Biophysics, King's College London, London, UK; Division of Cancer Studies, King's College London, London, UK.
authors.forename: Simon M
authors.initials: SM
authors.lastname: Ameer-Beg
authors.affiliation: Science and Technology Facilities Council, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK.
authors.forename: Marisa L
authors.initials: ML
authors.lastname: Martin-Fernandez
authors.affiliation: Richard Dimbleby Department of Cancer Research, King's College London, London, UK; Randall Division of Cellular and Molecular Biophysics, King's College London, London, UK; Division of Cancer Studies, King's College London, London, UK.
authors.forename: Tony
authors.initials: T
authors.lastname: Ng
mesh_terms: D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D010766:Phosphorylation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D054631:Receptor-Like Protein Tyrosine Phosphatases, Class 3 / Q000378:metabolism
chemical_list: D066246:ErbB Receptors; D054631:Receptor-Like Protein Tyrosine Phosphatases, Class 3