title: Evidence for extended YFP-EGFR dimers in the absence of ligand on the surface of living cells.
abstract: The epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family of receptors. Structural studies have revealed two distinct conformations of the ectodomain of the EGFR: a compact, tethered, conformation and an untethered extended conformation. In the context of a monomer-dimer transition model, ligand binding is thought to untether the monomeric receptor leading to exposure of a dimerization arm which then facilitates receptor dimerization, kinase activation and signaling. For receptors directed orthogonal to the local plane of the membrane surface, this would lead to a large change in the distance of the receptor N-terminus from the membrane surface. To investigate this experimentally, we produced stable BaF/3 cell lines expressing a biochemically functional yellow fluorescent protein (YFP)-EGFR chimera and determined the vertical separation of the N-terminal YFP tag from the membrane using fluorescence resonance energy transfer (FRET) techniques. Homo-FRET/rFLIM was employed to determine the presence of unliganded dimers and to measure the average distance between the N-terminal tags in those dimers. The results suggest that EGF-induced activation occurs within or between pre-formed and extended dimers with very little change in the extension of the N-terminii from the membrane surface. These results provide constraints on possible models for EGFR activation.
authors.affiliation: Centre for Microphotonics, Swinburne University of Technology, Victoria, Australia.
authors.forename: Noga
authors.initials: N
authors.lastname: Kozer
authors.forename: Christine
authors.initials: C
authors.lastname: Henderson
authors.forename: Jacob T
authors.initials: JT
authors.lastname: Jackson
authors.forename: Edouard C
authors.initials: EC
authors.lastname: Nice
authors.forename: Antony W
authors.initials: AW
authors.lastname: Burgess
authors.forename: Andrew H A
authors.initials: AH
authors.lastname: Clayton
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D008024:Ligands; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D066246:ErbB Receptors