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Home List of Titles Unligated epidermal growth factor receptor forms higher order oligomers within microclusters on A431 cells that are sensitive to tyrosine kinase inhibitor binding
Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/94154
- Unligated epidermal growth factor receptor forms higher order oligomers within microclusters on A431 cells that are sensitive to tyrosine kinase inhibitor binding
- Clayton, Andrew H. A.; Tavarnesi, Maria L.; Johns, Terrance G.
- 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). Lifetime-based Förster resonance energy transfer (FRET) techniques as a function of acceptor:donor labeling ratio disclosed a clustering of the unligated EGFR in clusters containing an average of four receptors on the nanometer (<10 nm) scale. The relationship between the nanoscale and submicron scale associations was determined using a new analysis that combines nanoscale information from lifetime-detected FRET imaging with submicron scale information obtained with image correlation microscopy. This analysis revealed the presence of monomers (or small oligomers) and larger clusters containing 15-30 receptors that were partially associated on the sub-10 nm scale. Pretreatment of the cells with the tyrosine kinase inhibitor AG1478 caused a partial dispersal of the submicron clusters (mean cluster density = 85 ± 15 clusters per square micron; mean degree of association = 4-10 receptors per cluster) and reduced the level of FRET down to our limit of detection. These results are consistent with a higher order nanoscale receptor organization of the unligated receptor population that is partially controlled by the kinase domains. The ramifications of the results to mechanisms of EGFR activation in a tumor cell environment are discussed.
- Publication type
- Journal article
- Biochemistry, Vol. 46, no. 15 (Apr 2007), pp. 4589-4597
- Publication year
- FOR Code(s)
- 0601 Biochemistry and Cell Biology; 1101 Medical Biochemistry and Metabolomics
- Cells; Energy transfer; Epidermal growth factor receptor; Growth kinetics; Image correlation microscopy; Microclusters; Oligomers; Resonance; Submicron clusters; Tumors
- American Chemical Society
- Publisher URL
- Copyright © 2007 American Chemical Society. The American Chemical Society does not allow institutions to archive either the accepted manuscript or the published version of the article.
- Peer reviewed