Sorting of the FGF receptor 1 in a human glioma cell line.
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.
Division of Neuroanatomy, Medical University Innsbruck, Muellerstrasse 59, 6020 Innsbruck, Austria.
Regina
R
Irschick
Tobias
T
Trost
Georg
G
Karp
Barbara
B
Hausott
Maria
M
Auer
Peter
P
Claus
Lars
L
Klimaschewski
D022461:Caveolins / Q000378:metabolism; D045744:Cell Line, Tumor; D048430:Cell Shape; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000201:enzymology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005910:Glioma / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation; D021381:Protein Transport; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D052118:Lysosomal-Associated Membrane Protein 1
D022461:Caveolins; D002966:Clathrin; D048049:Extracellular Signal-Regulated MAP Kinases; D016222:Fibroblast Growth Factor 2; D007976:Leupeptins; D008024:Ligands; D008164:Luminescent Proteins; D051907:Lysosomal Membrane Proteins; D051496:Receptor, Fibroblast Growth Factor, Type 1; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C496348:FGFR1 protein, human; C032854:leupeptin