SHC and GRB-2 are constitutively by an epidermal growth factor receptor with a point mutation in the transmembrane domain.
A single point mutation, Glu627--> Val, equivalent to the activating mutation in the Neu oncogene, was inserted in the transmembrane domain of the human epidermal growth factor (EGF) receptor. Unlike the wild type, Glu627-EGF receptor, transfected in NIH3T3 cells, gave rise to focal transformation and growth in agar even in the absence EGF. Constitutive activity of mutant EGF receptor amounted to 20% of that of wild type receptor stimulated by EGF. In addition, the mutant receptor was more sensitive to EGF, reaching maximum transforming activity at 5 ng/ml EGF. NIH3T3 cells expressing Glu627-EGF receptor showed a transformed phenotype and were not arrested in G0 upon serum deprivation. The mutant receptor was constitutively autophosphorylated, and several other cellular proteins were phosphorylated on tyrosine in absence of the ligand. Among these, the SHC adaptor protein was phosphorylated in absence of EGF, the other adaptor, GRB-2 was constitutively associated with the Glu627-EGF receptor in vivo and in vitro, and mitogen-activated protein kinase was constitutively phosphorylated. In contrast, other EGF receptor substrates, like phospholipase C gamma, were 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 form. Since the calpain cleavage site is located in the receptor cytoplasmic tail, this finding suggests that the Glu627 mutation induces a slightly different conformation in the EGF receptor intracellular domain. In conclusion, our data show that a point mutation in the EGF receptor transmembrane domain was able to constitutively activate the receptor and to induce transformation via constitutive activation of the Ras pathway.
Miloso
M
M
Laboratorio di Oncologia Molecolare, DIBIT, HS Raffaele, Milano, Italy.
Mazzotti
M
M
Vass
W C
WC
Beguinot
L
L
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D051966:Phospholipase C gamma; D010766:Phosphorylation; D017354:Point Mutation; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011494:Protein Kinases; D066246:ErbB Receptors; D010738:Type C Phospholipases; D051966:Phospholipase C gamma