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.
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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
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
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
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