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First published online 9 August 2005
doi: 10.1242/jcs.02509

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Different intracellular trafficking of FGF1 endocytosed by the four homologous FGF receptors

Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, The University of Oslo, Montebello, 0310 Oslo, Norway

View larger version (39K): [in a new window]   Fig. 1. Binding of FGF1 to cell-surface FGFRs. (A) HeLa cells, not transfected (-) or transiently transfected with FGFR1, 2, 3 or 4 were treated with Cy3-labelled FGF1 and 50 U/ml heparin for 2 hours at 4°C. The cells were then fixed and examined by confocal microscopy. The red channel image was superimposed onto the corresponding interference contrast image. Bar, 5 µm. (B) HeLa cells not transfected (-) or transiently transfected with FGFR1, 2, 3 or 4 (R1, R2, R3, R4, respectively) were incubated at 37°C with [35S]methionine-labelled FGF1 and 50 U/ml heparin for 1 hour. The cells were washed with PBS and lysed. FGF1 was extracted from the lysate by binding to heparin-Sepharose and analysed by SDS-PAGE.

View larger version (73K): [in a new window]   Fig. 2. Colocalization of FGFRs and endocytosed FGF1 after 2 hours. HeLa cells transiently transfected with FGFR1, 2, 3 or 4 were incubated with Cy3-FGF1, 50 U/ml heparin and 0.3 mM leupeptin for 2 hours at 37°C. The cells were then fixed, permeabilized and treated with rabbit anti-FGFR1, anti-FGFR2, anti-FGFR3 or anti-FGFR4 primary antibodies. The cells were further treated with Cy2-conjugated anti-rabbit secondary antibodies and examined by confocal microscopy. Arrows point to colocalization. Bar, 5 µm.

View larger version (83K): [in a new window]   Fig. 3. Colocalization of EEA1 and endocytosed FGF1 after 15 minutes. HeLa cells were transiently transfected with FGFR1, 2, 3 or 4 and kept at 4°C with Cy3-FGF1 and 50 U/ml heparin for 2 hours. The cells were washed and incubated in the presence of 0.3 mM leupeptin for 15 minutes at 37°C. The cells were fixed, permeabilized and treated with mouse anti-EEA1 primary antibody. The cells were further treated with Cy2-conjugated anti-mouse secondary antibodies and examined by confocal microscopy. Arrows point to colocalization. Bar, 5 µm.

View larger version (56K): [in a new window]   Fig. 4. Different colocalization of LAMP1 and endocytosed FGF1 after 2 hours. (A) HeLa cells were transiently transfected with FGFR1, 2, 3 or 4 and kept at 4°C with Cy3-FGF1 and 50 U/ml heparin for 2 hours. They were then washed and incubated in the presence of 0.3 mM leupeptin for 2 hours at 37°C. The cells were fixed, permeabilized and treated with mouse anti-LAMP1 primary antibody. The cells were further treated with Cy2-labelled anti-mouse secondary antibody and examined by confocal microscopy. Arrows point to colocalization in the case of FGFR1-3 and lack of colocalization in the case of FGFR4. Bar, 5 µm. (B) The percentage of FGF1-positive structures within cells that colocalized with LAMP1 was quantitated as described in Materials and Methods. Error bars denote the s.d.; n=15.

View larger version (65K): [in a new window]   Fig. 5. Colocalization of endocytosed EGF, transferrin and FGF1 after 2 hours. (A) HeLa cells were transiently transfected with FGFR1, 2, 3 or 4 and incubated for 2 hours at 37°C with Cy3-FGF1 and Alexa 488-EGF in the presence of 50 U/ml heparin and 0.3 mM leupeptin. Alexa 647-transferrin (Tf) was added after 90 minutes. The cells were fixed and examined by confocal microscopy. Insets show selected areas of the images enlarged (3x). Bar, 5 µm. (B) The percentage of FGF1-positive structures within transfected cells that colocalized with EGF, Tf, neither EGF nor Tf, or both EGF and Tf was quantitated as described in Materials and Methods. Error bars denote the s.d.; n=15.

View larger version (28K): [in a new window]   Fig. 6. Degradation of endocytosed FGFRs. (A) Cell-surface proteins on HeLa cells, not transfected (NT) or transiently transfected with FGFR1, 2, 3 or 4 were biotinylated and the cells were then incubated for the indicated periods of time at 37°C in the presence of 100 ng/ml FGF1 and 20 U/ml heparin. After lysis, the solubilised receptor proteins were adsorbed to streptavidin-Sepharose and analysed by immunoblotting (IB) with appropriate anti-FGFR antibodies. (B) The intensity of the bands at time point zero was set to 100% and the relative amount of receptors at each time point was calculated. The presented values represent the average of three independent experiments. Error bars denote the s.d.

View larger version (25K): [in a new window]   Fig. 7. Degradation of the endocytosed 18 kDa form of FGF1. (A) HeLa cells, transiently transfected with FGFR1, 2, 3 or 4 were incubated with the [35S]methionine-labelled 18 kDa form of FGF1 and 20 U/ml heparin at 37°C for 1 hour to allow endocytosis to occur. The cells were then either lysed immediately (0 h) or incubated further in growth medium with or without chloroquine (chl) at 37°C for 3 or 6 hours before lysis. FGF1 was extracted from the lysate by binding to heparin-Sepharose and analysed by SDS-PAGE. (B) For each receptor the amount of 18 kDa form of FGF1 was calculated at each time point and expressed as a percentage of the amount of 18 kDa form at time zero. Values are averages of five independent experiments for FGFR1 and FGFR4 and of three independent experiments for FGFR2 and FGFR3. Error bars denote the s.d.

View larger version (15K): [in a new window]   Fig. 8. Degradation and recycling of endocytosed FGF1. HeLa cells transfected with FGFR1 (R1) or FGFR4 (R4) were incubated in growth medium with 100 ng/ml [125I]FGF1 and 40 U/ml heparin for 20 minutes at 37°C. Excess and surface-bound [125I]FGF1 was removed by acid/salt wash of the cells, which were incubated further in growth medium at 37°C for the indicated periods of time. (A) Degradation of internalized [125I]FGF1 was measured for each time point as the amount of radioactivity in the TCA-soluble fractions and expressed as a percentage of the total radioactivity in the culture. (B) Recycling of [125I]FGF1 was measured for each time point as the amount of radioactivity in the TCA-insoluble fraction of the medium and of the high salt/low pH buffer and expressed as a percentage of the total radioactivity in the culture. The experiment was carried out twice with similar results.

View larger version (83K): [in a new window]   Fig. 9. Amino acid sequence alignment of the intracellular parts of FGFR1-4. The protein sequence alignment of the intracellular part of FGFR1, FGFR2, FGFR3 and FGFR4 was created using the Vector NTI 9.0 software based on a Clustal W algorithm (Thompson et al., 1994). The protein sequences were obtained from the following DNA sequences at NCBI; M34641, BC039243, NM_000142 and X57205. A dash represents a gap introduced to optimize the alignment. Lysines conserved in all the four receptors are indicated in light grey, whereas other lysines are indicated in dark grey.

View larger version (75K): [in a new window]   Fig. 10. Ubiquitylation of endocytosed FGFRs. HeLa cells were cotransfected with myc-ubiquitin and FGFR1 (R1), FGFR4 (R4) or the empty pcDNA3 vector (-) and starved overnight. Cell-surface proteins were biotinylated and the cells were incubated for the indicated periods of time at 37°C in the presence of 200 ng/ml FGF1, 20 U/ml heparin and 0.3 mM leupeptin. After lysis, solubilised receptor proteins were adsorbed to streptavidin-Sepharose and analysed by immunoblotting (IB) with anti-myc antibody. The membrane was stripped and reprobed with anti-phospho-FGFR antibodies (anti-pFGFR) and anti-transferrin receptor antibodies (anti-TfR).