Gangliosides influence EGFR/ErbB2 heterodimer stability but they do not modify EGF-dependent ErbB2 phosphorylation

Gangliosides are well-known regulators of cell differentiation through specific interactions with growth factor receptors. Previously, our group provided the first evidence about stable association of ganglioside GM₃ to EGFR/ErbB2 heterodimers in mammary epithelial cells. Goals of the present study were to better define the role of gangliosides in EGFR/ErbB2 heterodimerization and receptor phosphorylation events and to analyze their involvement in mammary cell differentiation. Experiments have been conducted using the ceramide analogue (+/−)-treo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride (D]-PDMP), which inhibits ceramide glucosyltransferase resulting in the endogenous ganglioside depletion, and the lactogenic hormone mix DIP (dexamethasone, insulin, prolactin), which induces cell differentiation and β-casein mRNA synthesis. In addition, treatments of ganglioside-depleted cells with exogenous GM₃ have been carried out to ascertain the specific involvement of this ganglioside. Results from co-immunoprecipitation and Western blot experiments have shown that the endogenous ganglioside depletion resulted in the disappearance of SDS-stable EGFR/ErbB2 heterodimers and in the appearance of tyrosine-phosphorylated EGFR also in the absence of EGF stimulation; exogenous GM₃ added in combination with D]-PDMP reversed both these effects. In contrast, the tyrosine phosphorylation of ErbB2 in ganglioside-depleted cells occurred only after EGF stimulation. Moreover, when ganglioside-depleted cells were treated with DIP in absence of EGF, β-casein gene expression appeared strongly down-regulated, and β-casein mRNA levels were partially restored by exogenous GM₃ treatment. Altogether, although the involvement of other ganglioside species cannot be excluded, these findings sustain the ganglioside GM₃ as an essential molecule for EGFR/ErbB2 heterodimer stability and important regulator of EGFR tyrosine phosphorylation, but it is not crucial for tyrosine phosphorylation of the heterodimerization partner ErbB2. Moreover, modulation of EGFR phosphorylation may explain how gangliosides contribute to regulate the lactogenic hormone-induced mammary cell differentiation.