Biological activities of EGF-receptor mutants with individually altered autophosphorylation sites.
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.
Honegger
A
A
Rorer Biotechnology, Inc., King of Prussia, PA 19406.
Dull
T J
TJ
Bellot
F
F
Van Obberghen
E
E
Szapary
D
D
Schmidt
A
A
Ullrich
A
A
Schlessinger
J
J
D000818:Animals; D004252:DNA Mutational Analysis; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics*; D051379:Mice; D008934:Mitogens; D010750:Phosphoproteins / Q000235:genetics*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D008934:Mitogens; D010750:Phosphoproteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D013755:Tetradecanoylphorbol Acetate