Ganglioside GM(3) is stably associated to tyrosine-phosphorylated ErbB2/EGFR receptor complexes and EGFR monomers, but not to ErbB2.
Gangliosides are known to modulate the activation of receptor tyrosine-kinases (RTKs). Recently, we demonstrated the functional relationship between ErbB2 and ganglioside GM(3) in HC11 epithelial cell line. In the present study we investigated, in the same cells, the ErbB2 activation state and its tendency to form stable molecular complexes with the epidermal growth factor receptor (EGFR) and with ganglioside GM(3) upon EGF stimulation. Results from co-immunoprecipitation experiments and western blot analyses indicate that tyrosine-phosphorylated ErbB2 and EGFR monomers and stable ErbB2/EGFR high molecular complexes (heterodimers) are formed following EGF stimulation, even if the receptors co-immunoprecipitates also in the absence of the ligand; these data suggest the existence of pre-dimerization inactive receptor clusters on the cell surface. High performance-thin layer chromatography (HP-TLC) and TLC-immunostaining analyses of the ganglioside fractions extracted from the immunoprecipitates demonstrate that GM(3), but not other gangliosides, is tightly associated to the tyrosine-phosphorylated receptors. Furthermore, we show that GM(3) is preferentially and in a SDS-resistant manner associated to the activated ErbB2/EGFR complexes and EGFR monomer, but not to ErbB2. Altogether our data support the hypothesis that the modulating effects produced by GM(3) on ErbB2 activation are mediated by EGFR.
Milani
Simona
S
Institute of General Physiology and Biological Chemistry, University of Milan, Via Trentacoste 2-20134, Milan, Italy.
Sottocornola
Elena
E
Zava
Stefania
S
Berselli
Patrizia
P
Berra
Bruno
B
Colombo
Irma
I
D000818:Animals; D015153:Blotting, Western; D019281:Dimerization; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005679:G(M3) Ganglioside / Q000378:metabolism*; D047468:Immunoprecipitation; D051379:Mice; D019000:Phosphotyrosine / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*
D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D005679:G(M3) Ganglioside; D019000:Phosphotyrosine; D018719:Erb-b2 Receptor Tyrosine Kinases