Ligand-induced Dimer-Tetramer Transition during the Activation of the Cell Surface Epidermal Growth Factor Receptor-A Multidimensional Microscopy Analysis
| Autor: | Anthony W Burgess, Francesca Walker, Suzanne G Orchard, Andrew H. A. Clayton, Dominik Fuchs, Julie Rothacker, Christine Henderson, Edouard C. Nice |
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| Rok vydání: | 2005 |
| Předmět: |
Transcriptional Activation
Conformational change Recombinant Fusion Proteins Green Fluorescent Proteins Ligands Models Biological Biochemistry Culture Media Serum-Free Cell Line Mice ErbB Fluorescence Resonance Energy Transfer Animals ERBB3 Epidermal growth factor receptor Phosphorylation Receptor Molecular Biology Chromatography High Pressure Liquid Microscopy Microscopy Confocal Dose-Response Relationship Drug biology Chemistry Kinase Cell Membrane Cell Biology Protein Structure Tertiary ErbB Receptors Kinetics Crystallography Microscopy Fluorescence Biophysics biology.protein Tyrosine Dimerization Tyrosine kinase Fluorescein-5-isothiocyanate |
| Zdroj: | Journal of Biological Chemistry. 280:30392-30399 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m504770200 |
| Popis: | The epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family of receptors. For many years it has been believed that receptor activation occurs via a monomer-dimer transition that is associated with a conformational change to activate the kinase. However, little is known about the quaternary state of the receptor at normal levels of expression (10(5) receptors/cell). We employed multidimensional microscopy techniques to gain insight into the state of association of the human EGFR, in the absence and presence of ligand, on the surface of intact BaF/3 cells (50,000 receptors/cell). Image correlation microscopy of an EGFR-enhanced green fluorescent protein chimera was used to establish an average degree of aggregation on the submicron scale of 2.2 receptors/cluster in the absence of ligand increasing to 3.7 receptors/cluster in the presence of ligand. Energy transfer measurements between mixtures of fluorescein isothiocyanate-EGF and Alexa 555-EGF were performed using fluorescence lifetime imaging microscopy as a function of the donor: acceptor labeling ratio to gain insight into the spatial disposition of EGFR ligand binding sites on the nanometer scale. In the context of a two-state Förster resonance energy transfer (FRET)/non-FRET model, the data are consistent with a minimum transfer efficiency of 75% in the FRET population. The microscopy data are related to biophysical data on the EGFR in the A431 cell line and the three-dimensional structure of the ligated EGFR extracellular domain. In the context of a monomer-dimer-oligomer model, the biophysical data are consistent with a significant fraction of ligated EGFR tetramers comprising two dimers juxtaposed in a side-by-side (or slightly staggered) arrangement. Our data are consistent with a specific higher order association of the ligand-bound EGFR on the nanometer scale and indicate the existence of distinct signaling entities beyond the level of the EGFR dimer which could play an important role in receptor transactivation. |
| Databáze: | OpenAIRE |
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