Abstract
The addition of gangliosides to tissue culture cells causes a decrease in the tyrosine protein kinase activity of the epidermal growth factor (EGF) receptor and an inhibition of EGF-stimulated growth. Based on these data, the hypothesis that the EGF receptor is physiologically regulated by gangliosides has been proposed by E.G. Bremer, J. Schlessinger, and S. Hakomori (J. Biol. Chem. (1986) 261, 2434-2440). To test this hypothesis, a mutant Chinese hamster ovary cell line (clone Idl D) that has a reversible defect in the biosynthesis of gangliosides (Kingsley, D.M., Kozarsky, K. F., Hobbie, L., and Krieger, M. (1986) Cell 44, 749-759) was investigated. The human EGF receptor cDNA was expressed in the mutant cells, and the properties of the EGF receptor were examined using cells grown under permissive and nonpermissive conditions. Changes in ganglioside expression were not observed to cause any significant alterations in the affinity or number of EGF receptors detected at the cell surface. However, decreased levels of ganglioside expression were associated with 1) increased EGF receptor autophosphorylation on tyrosine residues, and 2) increased EGF-stimulated cellular proliferation. The inverse correlation observed between the level of ganglioside expression and signal transduction by the EGF receptor is consistent with the hypothesis that the function of the EGF receptor is physiologically regulated by gangliosides.
Highlights
The addition of gangliosides to tissue culture cells causes a decrease in the tyrosine protein kinase activity of the epidermal growth factor (EGF) receptor and an inhibition of EGF-stimulated growth
The inverse correlation observed between the level of ganglioside expression and signal transduction by the EGF receptor is consistent with the hypothesis that the function of the EGF receptor is physiologically regulated by gangliosides
The alterations in ganglioside metabolism observed during oncogenic transformation, the cell cycle, and density-dependent growth inhibition indicate that sphingolipid metabolism may play a role in the regulation of cell growth [1, 2]
Summary
Muteri&-EGF was isolated from mouse submaxillary glands [16, 17] and iodinated as described [18]. Colonies obtained were isolated using cloning rings and screened for expression of EGF receptors by measuring the cell surface binding of lz51-EGF at 0 OC. Autophasphonktion of the EGF Receptor in Intact CeLs-CHO cells were grown in 35-mm dishes and washed with serum-free medium. The EGF receptors were isolated by immunoprecipitation and polyacrylamide gel electrophoresis as described [24]. 100 PM Na:,VOs. EGF receptors were isolated by immunoprecipitation and polyacrylamide gel electrophoresis as described [24]. Incorporation-CHO cells were grown in 16.mm wells and transferred to medium supplemented with 0.1% calf serum for 24 h. Phosphorylation of S$hetic Peptide 2’669-CHO cells were grown in 35mm wells and incubated in 1 ml of 120 mM NaCl, 6 mM KCl, 1.2 mM CaCl?, 1 mM MgCl?, 25 mM Hepes (pH 7.4), and 30 PM bovine serum albumin for 30 min at 37 “C. The phosphorylated peptide was identified by autoradiography, and the incorporation of radioactivity into the peptide was quantitated by liquid scintillation counting [19]
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