Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2.
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 10(2)-10(3) 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.
Nagy
Peter
P
Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, POB 39, Debrecen H-4012, Hungary.
Vereb
György
G
Sebestyén
Zsolt
Z
Horváth
Gábor
G
Lockett
Stephen J
SJ
Damjanovich
Sándor
S
Park
John W
JW
Jovin
Thomas M
TM
Szöllosi
János
J
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism*; D002772:Cholera Toxin / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000187:drug effects / Q000378:metabolism; D019281:Dimerization; D005057:Eukaryotic Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D046911:Macromolecular Substances; D021962:Membrane Microdomains / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018739:Oncogene Proteins v-erbB / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011506:Proteins / Q000187:drug effects / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D020893:Receptor, ErbB-3 / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000235:genetics; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D002772:Cholera Toxin; D003598:Cytoskeletal Proteins; D046911:Macromolecular Substances; D018739:Oncogene Proteins v-erbB; D011506:Proteins; D020794:Receptor Protein-Tyrosine Kinases; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D011956:Receptors, Cell Surface; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; C528024:SHC1 protein, human