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

EMBO J. 1988 Oct;7(10):3045-52.

Abstract

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.

MeSH terms

  • Animals
  • DNA Mutational Analysis
  • Endocytosis
  • Epidermal Growth Factor / metabolism
  • ErbB Receptors / genetics*
  • Mice
  • Mitogens
  • Phosphoproteins / genetics*
  • Phosphorylation
  • Protein-Tyrosine Kinases / genetics*
  • Structure-Activity Relationship
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transfection

Substances

  • Mitogens
  • Phosphoproteins
  • Epidermal Growth Factor
  • ErbB Receptors
  • Protein-Tyrosine Kinases
  • Tetradecanoylphorbol Acetate