GM 3 content modulates the EGF-activated p185 c-neu levels, but not those of the constitutively activated oncoprotein p185 neu

Abstract

The functional relationship between ganglioside GM 3 and two tyrosine-kinase receptors, the normal protein p185 c-neu and the mutant oncogenic protein p185 neu, was examined in HC11 cells and in MG1361 cells, respectively. In the former, p185 c-neu expression and activation are controlled by EGF addition to the culture medium and by epidermal growth factor receptor (EGFR) activity, whereas the latter express unchangingly high levels of constitutively activated p185 neu. Studies were carried out using (±)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ganglioside biosynthesis resulting in ganglioside depletion, and addition of exogenous GM 3 to the culture medium. In HC11 cells treated with only [D]-PDMP, p185 c-neu levels remain similar to control cells, whereas levels of tyrosine-phosphorylated p185 c-neu increase after treatment with [D]-PDMP in combination with EGF. When exogenous GM 3 is added in combination with [D]-PDMP and EGF, the enhanced phosphorylated-p185 c-neu returns to control levels. Interestingly, EGFR levels also vary and, analogously to phosphorylated-p185 c-neu, the increase of EGFR content consequent to the [D]-PDMP and EGF addition is reversed by exogenous GM 3. In contrast, the addition of neither [D]-PDMP nor exogenous GM 3 modifies expression and tyrosine-phosphorylation levels of p185 neu in MG1361 cells. These findings indicate that changes in GM 3 content modulate the tyrosine-phosphorylated p185 c-neu levels in a reversible manner, but this is not specific for p185 c-neu because EGFR levels are also modified. Furthermore, these data suggest that GM 3 may play a functional role by affecting the internalisation pathway of p185 c-neu/EGFR heterodimers, but not of p185 neu homodimers.