Biological activities of EGF‐receptor mutants with individually altered autophosphorylation sites

• Published in: The EMBO journal 1988-10, Vol.7 (10), p.3045-3052
• Main Authors: Honegger, A., Dull, T. J., Bellot, F., Van Obberghen, E., Szapary, D., Schmidt, A., Ullrich, A., Schlessinger, J.
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Cell receptors; Cell structures and functions; DNA Mutational Analysis; Endocytosis; Epidermal Growth Factor - metabolism; ErbB Receptors - genetics; Fundamental and applied biological sciences. Psychology; Mice; Mitogens; Molecular and cellular biology; Phosphoproteins - genetics; Phosphorylation; Protein-Tyrosine Kinases - genetics; Structure-Activity Relationship; Tetradecanoylphorbol Acetate - pharmacology; Transfection
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1988.tb03169.x

In vitro site‐directed mutagenesis was used to replace individually the three known autophosphorylation sites of the epidermal growth factor (EGF)‐receptor (i.e. Tyr1173, Tyr1148 and Tyr1068) by phenylalanine, a residue which cannot serve as a phosphate acceptor site. In another mutant, Tyr1173 was substituted by a serine residue. The cDNA constructs encoding either mutant or wild‐type EGF‐receptors were transfected into NIH‐3T3 cells devoid of endogenous EGF‐receptors. The mutant receptors were expressed on the cell surface and displayed typical high‐ and low‐affinity binding sites for [125I]EGF. Phorbol ester (PMA) modulated the binding affinity of wild‐type and mutant receptors in a similar manner. Mutant EGF‐receptors exhibited EGF‐dependent tyrosine kinase activity leading to self‐phosphorylation and phosphorylation of exogenous substrates both in vitro and in living cells. The internalization and degradation of EGF‐receptors were not affected by the mutations. Cells expressing mutant EGF‐receptors became mitogenically responsive to EGF, indicating that none of the vital functions of the EGF‐receptor were critically impaired by the loss of individual autophosphorylation sites. Maximal mitogenic stimulation correlated with the number of wild‐type or mutant receptors per cell, highly expressing cells showing higher maximal stimulation. However, the dose‐response curves of cells expressing mutant receptors were slightly shifted to lower concentrations of EGF, rendering the cells mitogenically responsive to lower doses of EGF than cells expressing normal EGF‐receptor at similar expression levels. Basal [3H]thymidine incorporation in the presence of 0.5% calf serum was consistently higher for cells expressing mutant receptors, while the response to stimulation with 10% calf serum was not affected.