Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/94154

Title

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

Author(s)

Clayton, Andrew H. A.;  Tavarnesi, Maria L.;  Johns, Terrance G.

Abstract

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

Source

Biochemistry, Vol. 46, no. 15 (Apr 2007), pp. 4589-4597

Publication year

2007

FOR Code(s)

0601 Biochemistry and Cell Biology;  1101 Medical Biochemistry and Metabolomics

Keyword(s)

Cells;  Energy transfer;  Epidermal growth factor receptor;  Growth kinetics;  Image correlation microscopy;  Microclusters;  Oligomers;  Resonance;  Submicron clusters;  Tumors

Publisher

American Chemical Society

ISSN

0006-2960

Publisher URL

http://dx.doi.org/10.1021/bi700002b

Copyright

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