SHC AND GRB-2 ARE CONSTITUTIVELY ACTIVATED BY AN EPIDERMAL GROWTH-FACTOR RECEPTOR WITH A POINT MUTATION IN THE TRANSMEMBRANE DOMAIN

A single point mutation, Glu(627) --> Val, equivalent to the activatin g mutation in the Neu oncogene, was inserted in the transmembrane doma in of the human epidermal growth factor (EGF) receptor. Unlike the wil d type, Glu(627)-EGF receptor, transfected in NIH3T3 cells, gave rise to focal transformation and growth in agar even in the absence of EGF. Constitutive activity of mutant EGF receptor amounted to 20% of that of wild type receptor stimulated by EGF. In addition, the mutant recep tor was more sensitive to EGF, reaching maximum transforming activity at 5 ng/ml EGF, NIH3T3 cells expressing Glu(627)-EGF receptor showed a transformed phenotype and were not arrested in G(0) upon serum depriv ation, The mutant receptor was constitutively autophosphorylated. and several other cellular proteins were phosphorylated on tyrosine in abs ence of the ligand. Among these, the SHC adaptor protein was phosphory lated in absence of EGF, the other adaptor, GRB-2, was constitutively associated with the Glu(627)-EGF receptor in vivo and in vitro, and mi togen-activated protein kinase was constitutively phosphorylated. In c ontrast, other EGF receptor substrates, like phospholipase C gamma, we re not phosphorylated in absence of EGF. The mutant receptor showed a higher sensitivity to cleavage by calpain both in absence and presence of EGF, appeared as a 170- and 150-kDa doublet in cell extracts, and a specific calpain inhibitor blocked the appearance of the 150-kDa for m. Since the calpain cleavage site is located in the receptor cytoplas mic tail, this finding suggests that the Glu(627) mutation induces a s lightly different conformation in the EGF receptor intracellular domai n. In conclusion, our data show that a point mutation in the EGF recep tor transmembrane domain was able to constitutively activate the recep tor and to induce transformation via constitutive activation of the Ra s pathway.