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SHC and GRB-2 are constitutively by an epidermal growth factor receptor with a point mutation in the transmembrane
M Miloso1, M Mazzotti, W C Vass
1Laboratorio di Oncologia Molecolare, DIBIT, HS Raffaele, Milano, Italy.
Abstract:
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.
Insights
A specific mutation in the epidermal growth factor (EGF) receptor transmembrane domain causes constitutive activation, leading to cell transformation and Ras pathway activation without EGF signaling.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The epidermal growth factor (EGF) receptor is a key regulator of cell growth and differentiation.
- Activating mutations in receptor tyrosine kinases are implicated in various cancers.
- Understanding the structural and functional consequences of EGF receptor mutations is crucial for targeted therapies.
Purpose of the Study:
- To investigate the functional consequences of a specific point mutation (Glu627Val) in the transmembrane domain of the human EGF receptor.
- To determine if this mutation leads to constitutive receptor activation and cellular transformation.
- To elucidate the downstream signaling pathways affected by the mutant EGF receptor.
Main Methods:
- Site-directed mutagenesis was used to introduce the Glu627Val mutation into the human EGF receptor.
- NIH3T3 cells were transfected with wild-type or mutant EGF receptor constructs.
- Cellular transformation was assessed by focus formation and soft agar growth assays.
- Receptor autophosphorylation and downstream signaling events (e.g., SHC and GRB-2 association, MAPK phosphorylation) were analyzed in the absence and presence of EGF.
- Calpain cleavage of the EGF receptor was investigated using specific inhibitors.
Main Results:
- The Glu627Val mutation in the EGF receptor transmembrane domain induced focal transformation and anchorage-independent growth in NIH3T3 cells, even without EGF.
- The mutant EGF receptor exhibited constitutive autophosphorylation and activated downstream signaling pathways, including Ras.
- The mutant receptor showed increased sensitivity to EGF and was more susceptible to calpain cleavage, suggesting altered intracellular conformation.
- SHC adaptor protein phosphorylation and GRB-2 association were constitutive, and mitogen-activated protein kinase was constitutively phosphorylated.
Conclusions:
- A single point mutation in the EGF receptor transmembrane domain can lead to its constitutive activation.
- This constitutive activation drives cellular transformation through the Ras signaling pathway.
- The findings highlight the importance of the transmembrane domain in regulating EGF receptor activity and suggest potential therapeutic targets.