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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

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.

Keywords

Trafficking Recycling Degradation Endosomes Lysosomes Leupeptin U373 cells 

Notes

Acknowledgments

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)

References

  1. Bache KG, Slagsvold T, Stenmark H (2004) Defective downregulation of receptor tyrosine kinases in cancer. EMBO J 23:2707–2712PubMedCrossRefGoogle Scholar
  2. Barysch SV, Aggarwal S, Jahn R, Rizzoli SO (2009) Sorting in early endosomes reveals connections to docking- and fusion-associated factors. Proc Natl Acad Sci USA 106:9697–9702PubMedCrossRefGoogle Scholar
  3. Citores L, Wesche J, Kolpakova E, Olsnes S (1999) Uptake and intracellular transport of acidic fibroblast growth factor: evidence for free and cytoskeleton-anchored fibroblast growth factor receptors. Mol Biol Cell 10:3835–3848PubMedGoogle Scholar
  4. Couet J, Sargiacomo M, Lisanti MP (1997) Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinase activities. J Biol Chem 272:30429–30438PubMedCrossRefGoogle Scholar
  5. Cuevas P, Carceller F, Angulo J, Gonzblez-Corrochano R, Cuevas-Bourdier A, Giminez-Gallego G (2011) Antiglioma effects of a new, low molecular mass, inhibitor of fibroblast growth factor. Neurosci Lett 491:1–7PubMedCrossRefGoogle Scholar
  6. Dunham-Ems SM, Pudavar HE, Myers JM, Maher PA, Prasad PN, Stachowiak MK (2006) Factors controlling fibroblast growth factor receptor-1's cytoplasmic trafficking and its regulation as revealed by FRAP analysis. Mol Biol Cell 17:2223–2235PubMedCrossRefGoogle Scholar
  7. Geuze HJ, Stoorvogel W, Strous GJ, Slot JW, Bleekemolen JE, Mellman I (1988) Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles. J Cell Biol 107:2491–2501PubMedCrossRefGoogle Scholar
  8. Haugsten EM, Sorensen V, Brech A, Olsnes S, Wesche J (2005) Different intracellular trafficking of FGF1 endocytosed by the four homologous FGF receptors. J Cell Sci 118:3869–3881PubMedCrossRefGoogle Scholar
  9. Haugsten EM, Wiedlocha A, Olsnes S, Wesche J (2010) Roles of fibroblast growth factor receptors in carcinogenesis. Mol Cancer Res 8:1439–1452PubMedCrossRefGoogle Scholar
  10. Hausott B, Schlick B, Vallant N, Dorn R, Klimaschewski L (2008) Promotion of neurite outgrowth by fibroblast growth factor receptor 1 overexpression and lysosomal inhibition of receptor degradation in pheochromocytoma cells and adult sensory neurons. Neuroscience 153:461–473PubMedCrossRefGoogle Scholar
  11. Hausott B, Rietzler A, Vallant N, Auer M, Haller I, Perkhofer S, Klimaschewski L (2011) Inhibition of fibroblast growth factor receptor 1 endocytosis promotes axonal branching of adult sensory neurons. Neuroscience 188:13–22PubMedCrossRefGoogle Scholar
  12. Hausott B, Vallant N, Hochfilzer M, Mangger S, Irschick R, Haugsten EM, Klimaschewski L (2012) Leupeptin enhances cell surface localization of fibroblast growth factor receptor 1 in sensory neurons by increased recycling. Eur J Cell Biol 91:129–138PubMedCrossRefGoogle Scholar
  13. Hopkins CR (1983) Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma A431 cells. Cell 35:321–330PubMedCrossRefGoogle Scholar
  14. Hubbard SR, Miller WT (2007) Receptor tyrosine kinases: mechanisms of activation and signaling. Curr Opin Cell Biol 19:117–123PubMedCrossRefGoogle Scholar
  15. Jovic M, Sharma M, Rahajeng J, Caplan S (2010) The early endosome: a busy sorting station for proteins at the crossroads. Histol Histopathol 25:99–112PubMedGoogle Scholar
  16. Levkowitz G, Waterman H, Zamir E, Kam Z, Oved S, Langdon WY, Beguinot L, Geiger B, Yarden Y (1998) c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev 12:3663–3674PubMedCrossRefGoogle Scholar
  17. Loilome W, Joshi AD, ap Rhys CM, Piccirillo S, Vescovi AL, Gallia GL, Riggins GJ (2009) Glioblastoma cell growth is suppressed by disruption of Fibroblast Growth Factor pathway signaling. J Neurooncol 94:359–366PubMedCrossRefGoogle Scholar
  18. Maxwell M, Naber SP, Wolfe HJ, Hedley-Whyte ET, Galanopoulos T, Neville-Golden J, Antoniades HN (1991) Expression of angiogenic growth factor genes in primary human astrocytomas may contribute to their growth and progression. Cancer Res 51:1345–1351PubMedGoogle Scholar
  19. McLendon et al (2008) Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455:1061–1068CrossRefGoogle Scholar
  20. Mebratu Y, Tesfaigzi Y (2009) How ERK1/2 activation controls cell proliferation and cell death: is subcellular localization the answer? Cell Cycle 8:1168–1175PubMedCrossRefGoogle Scholar
  21. Mineo C, Gill GN, Anderson RG (1999) Regulated migration of epidermal growth factor receptor from caveolae. J Biol Chem 274:30636–30643PubMedCrossRefGoogle Scholar
  22. Morrison RS, Yamaguchi F, Saya H, Bruner JM, Yahanda AM, Donehower LA, Berger M (1994) Basic fibroblast growth factor and fibroblast growth factor receptor I are implicated in the growth of human astrocytomas. J Neurooncol 18:207–216PubMedCrossRefGoogle Scholar
  23. Ong SH, Guy GR, Hadari YR, Laks S, Gotoh N, Schlessinger J, Lax I (2000) FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors. Mol Cell Biol 20:979–989PubMedCrossRefGoogle Scholar
  24. Powers CJ, McLeskey SW, Wellstein A (2000) Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 7:165–197PubMedCrossRefGoogle Scholar
  25. Rand V, Huang J, Stockwell T, Ferriera S, Buzko O, Levy S, Busam D, Li K, Edwards JB, Eberhart C, Murphy KM, Tsiamouri A, Beeson K, Simpson AJ, Venter JC, Riggins GJ, Strausberg RL (2005) Sequence survey of receptor tyrosine kinases reveals mutations in glioblastomas. Proc Natl Acad Sci USA 102:14344–14349PubMedCrossRefGoogle Scholar
  26. Romanelli RJ, Wood TL (2008) Directing traffic in neural cells: determinants of receptor tyrosine kinase localization and cellular responses. J Neurochem 105:2055–2068PubMedCrossRefGoogle Scholar
  27. Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG (1992) Caveolin, a protein component of caveolae membrane coats. Cell 68:673–682PubMedCrossRefGoogle Scholar
  28. Sorensen V, Wiedlocha A, Haugsten EM, Khnykin D, Wesche J, Olsnes S (2006) Different abilities of the four FGFRs to mediate FGF-1 translocation are linked to differences in the receptor C-terminal tail. J Cell Sci 119:4332–4341PubMedCrossRefGoogle Scholar
  29. Sorkin A, Von ZM (2002) Signal transduction and endocytosis: close encounters of many kinds. Nat Rev Mol Cell Biol 3:600–614PubMedCrossRefGoogle Scholar
  30. Stachowiak MK, Maher PA, Joy A, Mordechai E, Stachowiak EK (1996) Nuclear localization of functional FGF receptor 1 in human astrocytes suggests a novel mechanism for growth factor action. Brain Res Mol Brain Res 38:161–165PubMedCrossRefGoogle Scholar
  31. Stachowiak EK, Maher PA, Tucholski J, Mordechai E, Joy A, Moffett J, Coons S, Stachowiak MK (1997) Nuclear accumulation of fibroblast growth factor receptors in human glial cells: association with cell proliferation. Oncogene 14:2201–2211PubMedCrossRefGoogle Scholar
  32. Turner N, Grose R (2010) Fibroblast growth factor signalling: from development to cancer. Nat Rev Cancer 10:116–129PubMedCrossRefGoogle Scholar
  33. Vecchione A, Cooper HJ, Trim KJ, Akbarzadeh S, Heath JK, Wheldon LM (2007) Protein partners in the life history of activated fibroblast growth factor receptors. Proteomics 7:4565–4578PubMedCrossRefGoogle Scholar
  34. Wang J-K, Gao G, Goldfarb M (1994) Fibroblast growth factor receptors have different signaling and mitogenic potentials. Mol Cell Biol 14:181–188PubMedGoogle Scholar
  35. Yamaguchi F, Saya H, Bruner JM, Morrison RS (1994) Differential expression of two fibroblast growth factor-receptor genes is associated with malignant progression in human astrocytomas. Proc Natl Acad Sci USA 91:484–488PubMedCrossRefGoogle Scholar

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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