Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor-A multidimensional microscopy analysis.

Authors:
Francesca Walker
Francesca Walker
Ludwig Institute for Cancer Research
Australia
Dr Suzanne G Orchard, BSc (Hons), PhD
Dr Suzanne G Orchard, BSc (Hons), PhD
Monash University
Senior Study Manager
Clinical Trials, Cancer Research
Mlebourne, Vic | Australia
Dominik Fuchs
Dominik Fuchs
University of Heidelberg
Germany
Julie Rothacker
Julie Rothacker
Ludwig Institute for Cancer Research
Australia
Edouard C Nice
Edouard C Nice
Ludwig Institute for Cancer Research
Australia
Antony W Burgess
Antony W Burgess
Ludwig Institute for Cancer Research
Australia

J Biol Chem 2005 Aug 30;280(34):30392-9. Epub 2005 Jun 30.

Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Victoria 3050, Australia.

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.

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M504770200DOI Listing
August 2005
4 Reads
76 PubMed Central Citations(source)
4.57 Impact Factor

Publication Analysis

Top Keywords

epidermal growth
8
growth factor
8
monomer-dimer transition
4
receptor egfr
4
occurs monomer-dimer
4
factor receptor
4
transition associated
4
microscopy analysis
4
analysis epidermal
4
egfr member
4
activation occurs
4
years believed
4
kinase family
4
receptors years
4
tyrosine kinase
4
believed receptor
4
member erbb
4
receptor activation
4
erbb tyrosine
4
multidimensional microscopy
4

Altmetric Statistics

Similar Publications

Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation.

J Cell Biol 1995 Jun;129(6):1543-58

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Goettingen, Federal Republic of Germany.

The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. Read More

View Article
June 1995

Unligated epidermal growth factor receptor forms higher order oligomers within microclusters on A431 cells that are sensitive to tyrosine kinase inhibitor binding.

Biochemistry 2007 Apr 24;46(15):4589-97. Epub 2007 Mar 24.

The Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, P.O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia.

Characterization of the association states of the unligated epidermal growth factor receptor (EGFR) is important in understanding the mechanism of EGFR tyrosine kinase activation in a tumor cell environment. We analyzed, in detail, the association states of unligated, immunotagged EGFR on the surface of intact epidermoid carcinoma A431 cells, using AlexaFluor488 and AlexaFluor546 anti-EGFR antibody, mAb528, as probes. Image correlation microscopy revealed the presence of unligated EGFR in submicron scale clusters containing an average of 10-30 receptors (mean cluster density = 32 +/- 9 clusters per square micron). Read More

View Article
April 2007

Predominance of activated EGFR higher-order oligomers on the cell surface.

Growth Factors 2008 Dec;26(6):316-24

Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Parkville, Victoria, Australia.

The epidermal growth factor receptor (EGFR) kinase is generally considered to be activated by either ligand-induced dimerisation or a ligand-induced conformational change within pre-formed dimers. We report the relationship between ligand-induced higher-order EGFR oligomerization and EGFR phosphorylation on the surface of intact cells. We have combined lifetime-detected Forster resonance energy transfer, as a probe of the receptor phosphorylation state and image correlation spectroscopy, to extract the relative association state of activated versus unactivated EGFR, to determine the ratio of the average number of receptors for active (phosphorylated) and inactive clusters. Read More

View Article
December 2008

Differential and synergistic effects of epidermal growth factor receptor antibodies on unliganded ErbB dimers and oligomers.

Biochemistry 2011 May 15;50(18):3581-90. Epub 2011 Apr 15.

Ludwig Institute for Cancer Research, Melbourne-Parkville Branch, Royal Melbourne Hospital, Victoria 3050, Australia.

Antibodies directed against the epidermal growth factor receptor (EGFR) offer a potentially powerful therapeutic approach against cancers driven by the EGFR pathway. EGFR antibodies are believed to halt cell surface activation by blocking ligand-induced receptor tyrosine kinase activation, i.e. Read More

View Article
May 2011