title: Dynamic conformational transitions of the EGF receptor in living mammalian cells determined by FRET and fluorescence lifetime imaging microscopy.
abstract: We have revealed a reorientation of ectodomain I of the epidermal growth factor receptor (EGFR; ErbB1; Her1) in living CHO cells expressing the receptor, upon binding of the native ligand EGF. The state of the unliganded, nonactivated EGFR was compared to that exhibited after ligand addition in the presence of a kinase inhibitor that prevents endocytosis but does not interfere with binding or the ensuing conformational rearrangements. To perform these experiments, we constructed a transgene EGFR with an acyl carrier protein sequence between the signal peptide and the EGFR mature protein sequence. This protein, which behaves similarly to wild-type EGFR with respect to EGF binding, activation, and internalization, can be labeled at a specific serine in the acyl carrier tag with a fluorophore incorporated into a 4'-phosphopantetheine (P-pant) conjugate transferred enzymatically from the corresponding CoA derivative. By measuring Förster resonance energy transfer between a molecule of Atto390 covalently attached to EGFR in this manner and a novel lipid probe NR12S distributed exclusively in the outer leaflet of the plasma membrane, we determined the apparent relative separation of ectodomain I from the membrane under nonactivating and activating conditions. The data indicate that the unliganded domain I of the EGFR receptor is situated much closer to the membrane before EGF addition, supporting the model of a self-inhibited configuration of the inactive receptor in quiescent cells.
authors.affiliation: Laboratory of Cellular Dynamics, Max Planck Institute for Biophysical Chemistry, 37077, Göttingen, Germany.
authors.forename: Iwona
authors.initials: I
authors.lastname: Ziomkiewicz
authors.forename: Anastasia
authors.initials: A
authors.lastname: Loman
authors.forename: Reinhard
authors.initials: R
authors.lastname: Klement
authors.forename: Cornelia
authors.initials: C
authors.lastname: Fritsch
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Klymchenko
authors.forename: Gertrude
authors.initials: G
authors.lastname: Bunt
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Jovin
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Arndt-Jovin
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D003412:Cricetulus; D004705:Endocytosis / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011799:Quinazolines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis
chemical_list: D005456:Fluorescent Dyes; D011799:Quinazolines; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors; C088860:4-((3-bromophenyl)amino)-6,7-dimethoxyquinazoline
keywords: EGF; FRET; epidermal growth factor receptor; lifetime imaging; time-correlated single-photon counting; tethered configuration