Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor.

Glycosphingolipids added exogenously to 3T3 cells in culture were shown to inhibit cell growth, alter the membrane affinity to platelet-derived growth factor binding, and reduce platelet-derived growth factor-stimulated membrane phosphorylation (Bremer, E., Hakomori, S., Bowen-Pope, D. F., Raines, E., and Ross, R. (1984) J. Biol. Chem. 259, 6818-6825). This approach has been extended to the epidermal growth factor (EGF) receptor of human epidermoid carcinoma cell lines KB and A431. GM3 and GM1 gangliosides inhibited both KB cell and A431 cell growth, although GM3 was a much stronger inhibitor of both KB and A431 cell growth. Neither GM3 nor GM1 had any affect on the binding of 125I-EGF to its cell surface receptor. However, GM3 and, to a much lower extent, GM1 were capable of inhibiting EGF-stimulated phosphorylation of the EGF receptor in membrane preparations of both KB and A431 cells. Further characterization of GM3-sensitive receptor phosphorylation was performed in A431 cells, which had a higher content of the EGF receptor. The following results were of particular interest. (i) EGF-dependent tyrosine phosphorylation of the EGF receptor and its inhibition by GM3 were also demonstrated on isolated EGF receptor after adsorption on the anti-receptor antibody-Sepharose complex, and the receptor phosphorylation was enhanced on addition of phosphatidylethanolamine. (ii) Phosphoamino acid analysis of the EGF receptor indicated that the reduction of phosphorylation induced by GM3 was entirely in the phosphotyrosine and not in the phosphoserine nor phosphothreonine content. (iii) The inhibitory effect of GM3 on EGF-dependent receptor phosphorylation could be reproduced in membranes isolated from A431 cells that had been cultured in medium containing 50 nmol/ml GM3 to effect cell growth inhibition. The membrane fraction isolated from such growth-arrested cells was found to be less responsive to EGF-stimulated receptor phosphorylation. These results suggest that membrane lipids, especially GM3, can modulate EGF receptor phosphorylation in vitro as well as in situ.