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J. Cell Sci. 115 (22): 4251-4262

Research Article

Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2

Peter Nagy1,2,3, György Vereb1, Zsolt Sebestyén1, Gábor Horváth1, Stephen J. Lockett4,*, Sándor Damjanovich1,2, John W. Park5, Thomas M. Jovin3, and János Szöllosi1,{ddagger}

1 Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, POB 39, Debrecen H-4012, Hungary
2 Cell-biophysical Workgroup of the Hungarian Academy of Sciences, University of Debrecen, POB 39, Debrecen H-4012, Hungary
3 Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen D-37077, Germany
4 Bioimaging Group, Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA
5 Department of Medicine, Division of Hematology/Oncology, University of California at San Francisco, San Francisco, CA 94143, USA
* Present address: NCI-FCRDC, Boyle Street, Frederick, MD 21702, USA

{ddagger} Author for correspondence (e-mail: szollo{at}jaguar.dote.hu)

Accepted for publication 6 August 2002.

Abstract: The ErbB family of transmembrane receptor tyrosine kinases plays an important role in the pathogenesis of many cancers. The four members of the family, ErbB1-4, form various homo- and heterodimers during the course of signal transduction. A second hierarchical level of molecular associations involving 102-103 molecules, termed large-scale clustering, has also been identified, but the regulatory factors and biological consequences of such structures have not been systematically evaluated. In this report, we describe the states of association of ErbB2 and their relationship to local ErbB3 density and lipid rafts based on quantitative fluorescence microscopy of SKBR-3 breast cancer cells. Clusters of ErbB2 colocalized with lipid rafts identified by the GM1-binding B subunit of cholera toxin. Pixel-by-pixel analysis of fluorescence resonance energy transfer between labeled antibodies indicated that the homoassociation (homodimerization) of ErbB2 was proportional to the local density of ErbB2 and inversely proportional to that of ErbB3 and of the raft-specific lipid GM1. Crosslinking lipid rafts with the B subunit of cholera toxin caused dissociation of the rafts and ErbB2 clusters, an effect that was independent of the cytoskeletal anchoring of ErbB2. Crosslinking also decreased ErbB2-ErbB3 heteroassociation and the EGF- and heregulin-induced tyrosine phosphorylation of Shc. When cells were treated with the anti-ErbB2 monoclonal antibody 4D5 (parent murine version of Trastuzumab used in the immunotherapy of breast cancer), internalization of the antibody was inhibited by crosslinking of lipid rafts, but the antiproliferative activity of 4D5 was retained and even enhanced. We conclude that local densities of ErbB2 and ErbB3, as well as the lipid environment profoundly influence the association properties and biological function of ErbB2.

Key Words: ErbB proteins • Lipid rafts • Breast cancer • Fluorescence resonance energy transfer

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