Histochemistry and Cell Biology

, Volume 139, Issue 1, pp 135–148 | Cite as

Sorting of the FGF receptor 1 in a human glioma cell line

  • Regina Irschick
  • Tobias Trost
  • Georg Karp
  • Barbara Hausott
  • Maria Auer
  • Peter Claus
  • Lars KlimaschewskiEmail author
Original Paper


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.


Trafficking Recycling Degradation Endosomes Lysosomes Leupeptin U373 cells 



This study was supported by the Austrian Cancer Society (Tyrol) and by the Medical Research Fund (MFF). Special thanks go to Markus Hutterer for providing U373 cells, to Markus Offterdinger for valuable help with microscopy and image analysis, to Roland Irschick for performing data analysis in Fig. S1 and to Ellen M. Haugsten for corrections on the manuscript.

Supplementary material

418_2012_1009_MOESM1_ESM.pdf (390 kb)
Suppl. Fig. 1 (S1) Frequency distribution of vesicles positive for FGFR1 and Lamp1 (A) or FGFR1 and transferrin (B) in three-dimensional datasets. The x-coordinate represents the distance of the cultured cell in z-direction (normalized, 0 is level of coverslip). The y-coordinate represents the frequency distribution of FGFR1- (green line) and Lamp1- or transferrin-positive vesicles (red line). The green and red areas indicate standard deviations of each curve, the yellow area is the overlap of both standard deviations. Quantification reveals significant differences only in the distribution of transferrin (D), not of Lamp1 (C) or FGFR1 (C, D). Mean ± SEM with ≥ 19 cells in each experimental group (one-way-ANOVA followed by Tukey's multiple comparison test, **p < 0.01, ***p < 0.001). (PDF 390 kb)
418_2012_1009_MOESM2_ESM.pdf (552 kb)
Suppl. Fig. 2 (S2) Analysis of number, size and intensity of fluorescence in all EEA1-, Clathrin (rLCA)-, Caveolin1 (Cav1)-, Lamp1- or LysoTracker (LysoTr)-positive vesicles (objects) in U373 cells two hours after treatment with FGF-2 regardless of colocalized FGFR1. Mean ± SEM of at least three independent experiments with ≥ 100 cells in each experimental group (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001). (PDF 522 kb)


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Regina Irschick
    • 1
  • Tobias Trost
    • 1
  • Georg Karp
    • 1
  • Barbara Hausott
    • 1
  • Maria Auer
    • 1
  • Peter Claus
    • 2
  • Lars Klimaschewski
    • 1
    Email author
  1. 1.Division of NeuroanatomyMedical University InnsbruckInnsbruckAustria
  2. 2.Hannover Medical SchoolInstitute of Neuroanatomy, OE 4140HannoverGermany

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