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Functional nuclear epidermal growth factor receptors in human choriocarcinoma JEG-3 cells and normal human placenta
1Department of Obstetrics and Gynecology, University of Louisville School of Medicine, Kentucky 40292, USA.
Abstract:
Immunocytochemistry with a monoclonal antiepidermal growth factor (anti-EGF) receptor antibody directed against the extracellular domain which can inhibit ligand binding to the receptors showed that nuclei of choriocarcinoma JEG-3 cells and normal placental trophoblasts were distinctly immunostained for EGF receptors. This finding led us to investigate the structure and function of nuclear EGF receptors. Western immunoblotting revealed that cell membranes, isolated intact pure nuclei, and nuclear membranes contain a 170-kilodalton EGF receptor protein. Covalent receptor cross-linking demonstrated that the 170-kilodalton receptor protein in nuclei and nuclear membranes can bind [125I]EGF just as in cell membranes, and that this binding is inhibited by excess unlabeled EGF. As in cell membranes, the addition of EGF resulted in an increased receptor autophosphorylation in the nuclei and nuclear membranes. In addition, the activated receptor kinase stimulated, and in some cases inhibited, tyrosine phosphorylation of a number of lower molecular size proteins, especially in nuclei and nuclear membranes. Although the identity of these proteins is not known, none of them could bind [125I]EGF. The addition of EGF to isolated nuclei resulted in a time-dependent specific transcriptional inhibition of hCG/LH receptor gene. In summary, our data demonstrating the presence of functional nuclear EGF receptors are novel, potentially important, and go against the traditional concepts of growth factors action. The nuclear receptors have the capacity to transduce signals from EGF and may mediate intracrine and paracrine actions of EGF in the regulation of trophoblast functions.
Insights
Nuclear epidermal growth factor (EGF) receptors were identified in choriocarcinoma cells and placental trophoblasts. These functional nuclear EGF receptors regulate gene transcription, challenging traditional growth factor action concepts.
Area of Science:
- Cell Biology
- Molecular Endocrinology
- Cancer Research
Background:
- Epidermal growth factor (EGF) receptors are typically located on the cell membrane.
- The role of EGF receptors within the cell nucleus is not well understood.
- Trophoblast cells play a crucial role in placental development and function.
Purpose of the Study:
- To investigate the presence and function of EGF receptors in the nucleus.
- To determine if nuclear EGF receptors are functional and can bind EGF.
- To explore the downstream signaling and transcriptional effects of nuclear EGF receptor activation.
Main Methods:
- Immunocytochemistry using anti-EGF receptor antibody.
- Western immunoblotting to detect EGF receptor protein.
- Covalent cross-linking assays to assess EGF binding.
- EGF stimulation assays to measure receptor autophosphorylation and downstream protein phosphorylation.
- Analysis of hCG/LH receptor gene transcription upon EGF addition.
Main Results:
- EGF receptors were detected in the nuclei and nuclear membranes of choriocarcinoma cells and normal placental trophoblasts.
- A 170-kilodalton EGF receptor protein was identified in cell membranes, isolated nuclei, and nuclear membranes.
- Nuclear EGF receptors bound [125I]EGF, with binding inhibited by unlabeled EGF.
- EGF stimulation led to increased autophosphorylation of nuclear EGF receptors and altered tyrosine phosphorylation of other nuclear proteins.
- EGF addition inhibited hCG/LH receptor gene transcription in isolated nuclei.
Conclusions:
- Functional EGF receptors are present in the nucleus of trophoblast cells.
- Nuclear EGF receptors can bind EGF and transduce signals, impacting gene transcription.
- These findings suggest novel intracrine and paracrine roles for EGF in regulating trophoblast functions.
- The presence of functional nuclear EGF receptors challenges conventional understanding of growth factor signaling pathways.