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Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin
M X Sliwkowski1, G Schaefer, R W Akita
1Genentech, Inc., South San Francisco, California 94080.
Abstract:
The heregulin/neu differentiation factor gene products were purified and cloned based on their ability to stimulate the phosphorylation of a 185-kDa protein in human breast carcinoma cell lines known to express erbB2. However, not all cells that express erbB2 respond to heregulin, indicating that other components besides erbB2 may be required for heregulin binding. Cells that are transfected with the closely related receptor, erbB3, display a single class of lower affinity heregulin binding sites than has been previously observed on breast carcinoma cell lines. Little or no stimulation of tyrosine phosphorylation in response to heregulin occurs in cells that are transfected with erbB3 alone. Transfection of cells with erbB3 and erbB2 reconstitutes a higher affinity binding receptor, which is also capable of generating a tyrosine phosphorylation signal in response to heregulin. A monoclonal antibody to erbB2 will inhibit heregulin activation of tyrosine phosphorylation and binding in cells transfected with both receptors but not with erbB3 alone. In cells expressing erbB2 and erbB3, both proteins become tyrosine-phosphorylated upon interaction with heregulin. Direct interaction between heregulin and the two proteins was demonstrated by chemical cross-linking experiments using 125I-heregulin followed by immunoprecipitation with antibodies specific for erbB2 or erbB3.
Insights
Heregulin binding and signaling require both erbB2 and erbB3 receptors. Co-expression of these receptors reconstitutes high-affinity heregulin binding and tyrosine phosphorylation, crucial for breast cancer cell response.
Area of Science:
- Cell signaling
- Molecular biology
- Cancer research
Background:
- Heregulin/neu differentiation factor stimulates phosphorylation of a 185-kDa protein in erbB2-expressing breast cancer cells.
- Not all erbB2-expressing cells respond to heregulin, suggesting additional binding components are necessary.
Purpose of the Study:
- To investigate the role of erbB3 in heregulin binding and signaling.
- To determine if co-expression of erbB2 and erbB3 reconstitutes heregulin responsiveness.
Main Methods:
- Transfection of cell lines with erbB2 and/or erbB3 receptors.
- Heregulin binding assays.
- Tyrosine phosphorylation stimulation assays.
- Chemical cross-linking and immunoprecipitation experiments.
Main Results:
- Cells transfected with erbB3 alone showed low-affinity heregulin binding and minimal tyrosine phosphorylation.
- Co-transfection with erbB2 and erbB3 reconstituted high-affinity binding and significant heregulin-induced tyrosine phosphorylation.
- Monoclonal antibody against erbB2 inhibited heregulin signaling in cells expressing both receptors.
- Both erbB2 and erbB3 proteins were tyrosine-phosphorylated upon heregulin interaction.
Conclusions:
- Heregulin signaling and binding necessitate the co-expression of both erbB2 and erbB3 receptors.
- The erbB2/erbB3 heterodimer forms a functional heregulin receptor complex.
- This complex is critical for mediating heregulin-driven cellular responses in breast cancer.