GM3 IN THE REGULATION OF THE EXPRESSION AND ACTIVATION OF ErbB-2/EGFR HETERODIMERS

Gangliosides are a large and heterogeneous family of sialic acid containing glycosphingolipids, ubiquitous components of all eukaryotic cell membranes. They can partially segregate, together with cholesterol and specific signaling transduction proteins, such as receptor tyrosine-kinases, into unique more or less stable clusters or microdomains, indicated as “glycolipid-enriched domains”, “lipid rafts”, “caveolae”, contributing to the membrane structure, organization and function (1). Gangliosides are well-characterized modulators of receptor tyrosine-kinase (RTKs) phophorylation and dimerization (2). Our interest is particularly directed to study the relationship between gangliosides and two members of the tyrosine kinase ErbB receptor family: the epidermal growth factor receptor, EGFR or ErbB-1, and the structurally related protein ErbB-2. Unlike EGFR, ErbB-2 is a ligandless receptor: it can be activated by heterodimerization and cross-phosphorylation by other ligand-activated ErbB receptors (3,4). Our previous experimental evidence supports the functional relationship between ErbB-2 and gangliosides, in particular GM3 (5,6). In the present study, using the HC11 mouse mammary epithelial cell line, we investigated the ErbB-2 activation state and its tendency to form stable molecular complexes with EGFR and with ganglioside GM3, before and after EGF stimulation, by co-immunoprecipitation experiments with anti-ErbB-2 antibody and Western blot analyses. As HC11 cells express different ganglioside species, the exclusive association of GM3 with ErbB-2 and EGFR was ascertained by high performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of gangliosides extracted from the immunoprecipitates. Results display that in EGF-stimulated HC11 cells stable and tyrosine-phosphorylated ErbB2/EGFR dimers are formed and that GM3 is the unique ganglioside tightly associated to ErbB-2/EGFR dimers and to EGFR monomers, but not to ErbB2 monomers. In cells not stimulated with EGF a spontaneous but unproductive dimerization of ErbB2 and EGFR takes place and no ganglioside is found associated to the receptors. These observations indicate that the modulation of ErbB2 activation by GM3 may be mediated by EGFR, but that EGF stimulation is indispensable. After ganglioside depletion by [D]-PDMP, phosphorylated EGFR monomers are observed both before and after EGF stimulation, whereas ErbB-2 is present in the monomeric and unphosphorylated state even after EGF stimulation, suggesting that GM3 might have a bivalent key role in modulating the activation of ErbB-2 and EGFR. References 1. Fivaz, M., Abrami, L., Van Der Goot. F.G. Trends Cell Biol. 9(6), 212-213 (1999); 2. Bremer, E.G., Current topics in membranes 40, 387-411(1999); 3. Qian, X., LeVea, C.M., Freeman, J.K., Dougall, W.C., Greene, M.I., Proc. Natl. Acad. Sci. USA 91, 1500-1504 (1994); 4. Gulliford, T.J., Huang, G.C., Ouyang, X., Epstein, R.J., Oncogene 15, 2219-2223 (1997); 5. Sottocornola E., Berra B., and Colombo I., Biochim. Biophys. Acta-Molecolar and cell Biology of Lipids, 1635, 55-66 (2003); 6. Sottocornola E., Misasi R., Mattei V., Ciarlo L., Gradini R., Garofalo T., Berra B., Colombo I., and Sorice M., FEBS J. 273, 1821-1830 (2006).