c-erbB-3: A nuclear protein in human mammary epithelial cells?

Research project P 13622 c-erB-3: A Nuclear Protein? Thomas W. GRUNT 28.06.1999 C-erbB-3 is - like the other c-erbB receptors - generally considered as a transmembrane receptor tyrosine kinase, that can be activated through extracellular binding of heregulin (HRG), an EGF-like growth factor. Our preliminary data demonstrate high levels of c-erbB-3 expression occurring within the nuclei of an immortalized non-malignant human mammary epithelial cell line (MTSV1-7) and of primary human mammary epithelial cells, whereas in malignant cell lines such as BT-474 and SK-BR-3 c-erbB-3 is found in the cytoplasm. In accordance, flow cytometry reveals a complete absence of c-erbB-3 at the cell membrane of normal, but not of malignant cells. More importantly, however, in nonmalignant cells, we were able to show that the c-erbB-3 ligand HRG causes a shift of c-erbB-3 from the nucleus into cytoplasmic vesicles. This effect was only seen in cell culture systems that preserve epithelial cell polarity and differentiation such as permeable filter supports and 3D collagen gels. These findings on the subcellular localization of c-erbB-3 in. dependence of exogenous stimuli and of the stage of cell differentiation challenge the specific function of c-erbB-3 as a transmembrane receptor protein and argue for some additional, yet unidentified roles of c-erbB-3 within the cells. A combination of biochemical, cell biological and molecular biological approaches will be used in order to draw a clear picture of the significance of the observed nuclear/cytoplasmic distribution of c-erbB-3. Coimmunoprecipitation and immunoblotting of nuclear and cytoplasmic extracts will be performed to identify possible interaction partners of c-erbB-3 and to analyze the state of phosphorylation of c-erbB-3 and its associated proteins. Furthermore, transfected MTSV1-7 cells expressing various parts of c-erbB-3 fused to enhanced green fluorescent protein (EGFP) will be used to analyze the subcellular distribution of c-erbB-3 in more detail and to map possible nuclear localization signals (NLSs) within the c-erbB-3 protein. Once identified, the NLSs will be altered by site-directed mutagenesis and the cell biological consequences of these molecular changes will be analyzed. In addition, inhibition experiments using leptornycin B - a nuclear export inhibitor - will demonstrate, if HRG causes inhibition of nuclear import or activation of nuclear export of c-erbB-3. Soft agar growth of MTSV1-7 transfectants stably expressing mutant (cytoplasmic) or wildtype c-erbB-3 will be used to elucidate whether cytoplasmic c-erbB-3 is associated with malignant transformation of breast epithelial cells. Flow cytometry will demonstrate, if the HRGinduced cytoplasmic accumulation of c-erbB-3, is accompanied by a similar increase at the cell membrane. The proposed experiments will contribute to a better understanding of the function of c-erbB-3 in human mammary epithelial cell growth, differentiation and transformation.

The family of erbB growth factor receptor proteins comprises four members: erbB-1 to -4. To avoid confusion with terminology, erbB-1 is better known as epidermal growth factor receptor, erbB-2 is also referred to as HER-2/neu, erbB-3 as HER-3, and erbB-4 as HER-4. These proteins reside in the cell membranes and act as docking sites for specific extracellular growth factors. Binding of the growth factor to the receptor activates it and triggers a series of enzyme reactions within the cytoplasm. Thereby, the `signal` delivered by the growth factor to the cell gets transformed into enzymatic processes that alter and activate specific cytoplasmic substrate proteins. The last protein in the row is typically a transcription factor, which then enters the cell nucleus and modulates the expression of genes controlling cell growth and differentiation. This process, termed `signal transduction`, causes rapid, but transient biochemical alterations enabling the cell to quickly respond to external stimuli. However, this classical model cannot explain all effects of growth factors such as constantly elevated cell growth. Thus we hypothesized that erbB receptors exert additional, as yet unidentified functions in the cell nucleus. Here we focused on erbB-3, which together with erbB-2 is present at high levels in many cancers incl. breast cancer causing a more severe course of the disease. In normal breast cells and breast cancer cells grown in cell culture we found erbB-3 also within the cell nuclei. The subcellular localization is controlled by the stage of differentiation and by the culture conditions. A growth factor that binds erbB-3 (heregulin) shifts erbB-3 from the nucleus into the cytoplasm and to the cell membrane. Under heregulin-free conditions erbB-3 was found in the nucleoli, which are substructures within the nuclei that control the synthesis of components required for protein production. In summary, we were the first to demonstrate that erbB-3 not only is a membrane-anchored receptor triggering classical signal transduction, but as a nuclear protein exerts additional functions within the cell nuclei. This induces a paradigm shift in the view of erbB-3 functions, which will have impacts on molecular medicine and may provide new targets for anticancer drug development.