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Home List of Titles Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor: a multidimensional microscopy analysis
Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/94143
- Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor: a multidimensional microscopy analysis
- Clayton, Andrew H. A.; Walker, Francesca; Orchard, Suzanne G.; Henderson, Christine; Fuchs, Dominik; Rothacker, Julie; Nice, Edouard C.; Burgess, Antony W.
- 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.
- Publication type
- Journal article
- Journal of Biological Chemistry, Vol. 280, no. 34 (2005), pp. 30392-30399
- Publication year
- FOR Code(s)
- 0601 Biochemistry and Cell Biology; 0904 Chemical Engineering
- Cell culture; Conformations; Correlation methods; Dimer-tetramer transitions; Dimers; Energy transfer; Enzymes; Epidermal growth factor receptor; Kinase; Ligands; Spectroscopic analysis
- American Society for Biochemistry and Molecular Biology
- Publisher URL
- Copyright © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
- Peer reviewed