Sorting of the FGF receptor 1 in a human glioma cell line
Regina Irschick1, Tobias Trost, Georg Karp
1Division of Neuroanatomy, Medical University Innsbruck, Muellerstrasse 59, 6020 Innsbruck, Austria.
Abstract:
Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase promoting tumor growth in a variety of cancers, including glioblastoma. Binding of FGFs triggers the intracellular Ras/Raf/ERK signaling pathway leading to cell proliferation. Down-regulation of FGFR1 and, consequently, inactivation of its signaling pathways represent novel treatment strategies for glioblastoma. In this study, we investigated the internalization and endocytic trafficking of FGFR1 in the human glioma cell line U373. Stimulation with FGF-2 induced cell rounding accompanied by increased BrdU and pERK labeling. The overexpression of FGFR1 (without FGF treatment) resulted in enhanced phosphorylated FGFR1 suggesting receptor autoactivation. Labeled ligand (FGF-2-Cy5.5) was endocytosed in a clathrin- and caveolin-dependent manner. About 25 % of vesicles carrying fluorescently tagged FGFR1 represented early endosomes, 15 % transferrin-positive recycling endosomes and 40 % Lamp1-positive late endosomal/lysosomal vesicles. Stimulation with FGF-2 increased the colocalization rate in each of these vesicle populations. The treatment with the lysosomal inhibitor leupeptin resulted in FGFR1 accumulation in lysosomes, but did not enhance receptor recycling as observed in neurons. Analysis of vesicle distributions revealed an accumulation of recycling endosomes in the perinuclear region. In conclusion, the shuttling of receptor tyrosine kinases can be directly visualized by overexpression of fluorescently tagged receptors which respond to ligand stimulation and follow the recycling and degradation pathways similarly to their endogenous counterparts.
Insights
Fibroblast growth factor receptor 1 (FGFR1) internalization was studied in glioma cells. Labeled FGFR1 followed endocytic pathways, with FGF-2 stimulation altering its trafficking and localization.
Area of Science:
- Oncology
- Cell Biology
- Molecular Biology
Background:
- Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase implicated in glioblastoma growth.
- Targeting FGFR1 signaling pathways offers a potential therapeutic strategy for glioblastoma.
Purpose of the Study:
- To investigate the internalization and endocytic trafficking of FGFR1 in human glioma cells.
- To visualize the dynamic movement of FGFR1 in response to ligand stimulation.
Main Methods:
- Overexpression of fluorescently tagged FGFR1 in U373 glioma cells.
- Stimulation with FGF-2 and analysis of receptor internalization via clathrin- and caveolin-dependent pathways.
- Tracking of fluorescently labeled FGFR1 through early endosomes, recycling endosomes, and late endosomal/lysosomal vesicles using microscopy.
Main Results:
- FGF-2 stimulation induced cell rounding and increased proliferation markers (BrdU) and signaling (pERK).
- Overexpressed FGFR1 showed autoactivation, and labeled FGF-2 was internalized via clathrin and caveolin pathways.
- FGFR1 trafficked through various endosomal compartments, with FGF-2 increasing colocalization rates. Lysosomal inhibition led to FGFR1 accumulation, but not enhanced recycling.
Conclusions:
- Overexpression of fluorescently tagged receptors allows visualization of receptor tyrosine kinase shuttling.
- FGFR1 follows specific endocytic and trafficking pathways in glioma cells, similar to endogenous receptors.
- Understanding FGFR1 trafficking provides insights into potential therapeutic targets for glioblastoma.
