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First published online 26 September 2006
doi: 10.1242/jcs.03209

Journal of Cell Science 119, 4332-4341 (2006)
Published by The Company of Biologists 2006
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Different abilities of the four FGFRs to mediate FGF-1 translocation are linked to differences in the receptor C-terminal tail

Vigdis Sørensen*, Antoni Wiedlocha, Ellen Margrethe Haugsten, Denis Khnykin, Jørgen Wesche and Sjur Olsnes

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


Figure 1
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  		Fig. 1. Expression of FGFRs and uptake of FGF-1 in COS-1 cells. (A) Untransfected COS-1 cells and COS-1 cells transfected with FGFR1, FGFR2, FGFR3, FGFR4, FGFR1ß, or FGFR2ß, respectively, were lysed and analyzed by SDS-PAGE and immunoblotting. The membrane was probed sequentially with antibodies specific for FGFR1, FGFR2, FGFR3 and FGFR4. (B) To cells transfected with FGFR as indicated, 35S-FGF-1 was allowed to bind at 4°C for 30 minutes and then the cells were washed and lysed immediately (upper panel) or further incubated at 37°C for 30 minutes to allow endocytosis of 35S-FGF-1 to take place and then washed and lysed (lower panel). 35S-FGF-1 was extracted from the lysates by binding to heparin-Sepharose and analyzed by SDS-PAGE and autoradiography. In the names of the receptors, FGFR is abbreviated to R. (C) COS-1 cells, untransfected or transfected with FGFR1, FGFR2, FGFR3, FGFR4, FGFR1ß or FGFR2ß as indicated, were incubated with Cy3-FGF-1 and heparin (except for the first image, which is without heparin) for 2 hours, fixed and analyzed by confocal fluorescence microscopy.

 

Figure 2
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  		Fig. 2. Phosphorylation of exogenous FGF-1 in FGFR-transfected cells. (A) COS-1 cells and (B) HeLa cells were transfected with FGFRs as indicated, pretreated with [33P]phosphate and incubated for 6 hours with FGF-1 or FGF-1 K132E and with heparin where indicated. FGF-1 was extracted from cell lysates as described in Materials and Methods and then separated by SDS-PAGE and blotted onto Immobilon-P membrane. 33P-phosphorylated FGF-1 was detected by autoradiography (upper panel) and the total cell-associated FGF-1 was detected by anti-FGF-1 antibodies (lower panel).

 

Figure 3
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  		Fig. 3. Inhibition of FGF-1 translocation by bafilomycin A1 and FGF-1 accumulation in the nucleus by LMB treatment. (A) COS-1 cells were transfected with FGFR1 or FGFR4, treated with [33P]phosphate and incubated for 6 hours with FGF-1 and heparin in the presence or absence of bafilomycin A1 and monensin, as indicated. FGF-1 was extracted from the cell lysates and assessed for phosphorylation by SDS-PAGE and autoradiography. (B) COS-1 cells were transfected with FGFR as indicated, pretreated with [33P]phosphate and incubated for 6 hours with FGF-1 or FGF-1 K132E and with LMB where indicated. The cells were fractionated into cytoplasmic (C) and nuclear (N) fractions and FGF-1 was extracted as described in Materials and Methods and then separated by SDS-PAGE and blotted onto Immobilon-P membrane. 33P-phosphorylated FGF-1 in the cytoplasmic (upper panel) and nuclear (second panel) fractions was detected by autoradiography and the total cell associated FGF-1 in the cytoplasmic (third panel) and nuclear (bottom panel) fractions was detected by anti-FGF-1 antibodies. (C) Cytoplasmic and nuclear fractions of lysed COS-1 cells were analyzed for their purity by western blotting and antibodies against the cytosolic protein MAPK, the ER resident protein Calnexin and the nuclear protein LaminA. (D) The experiment was performed as described in (B) but only nuclear fractions were analyzed. In addition, known amounts of FGF-1 was loaded on additional lanes of the gel to estimate the amount of FGF-1 that had been extracted from the nuclear fraction. Upper panel, autoradiography. Lower panel, anti FGF-1 immunodetection.

 

Figure 4
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  		Fig. 4. Kinetics of uptake and phosphorylation of FGF-1. (A) COS-1 cells transfected with FGFR1 or FGFR4 were labeled with [33P]phosphate and incubated with FGF-1 and heparin for the time indicated. FGF-1 was then extracted from the cell lysates and analyzed as in Fig. 2. The amount of 33P-phosphorylated FGF-1 (upper panel) and total intracellular FGF-1 (detected by anti-FGF-1, lower panel) was quantified and plotted against time as percent of the maximal values (B).

 

Figure 5
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  		Fig. 5. Ability of chimeric and deleted receptors to translocate FGF-1. (A) Schematic overview of the FGFR mutants. Red indicates FGFR1-derived sequences, blue indicates FGFR2 and yellow indicates FGFR4. Deletions are indicated by a broken line. (B-E) COS-1 cells were transfected with FGFR-constructs as indicated. The cells were labeled with [33P]phosphate and incubated with FGF-1 and heparin for 6 hours. FGF-1 was then extracted from the cell lysates and analyzed as in Fig. 2. Upper panels show phosphorylated FGF-1 detected by autoradiography and the lower panels show the total of endocytosed FGF-1, detected by anti-FGF-1.

 

Figure 6
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  		Fig. 6. Analyses of the C-terminal tails of the FGFRs. (A) ClustalW alignments of the C-terminal regions of FGFRs from various species as indicated. Numbering on top refers to the amino acids numbering here used for human FGFR1. Amino acid positions of special importance for FGF-1 translocation are indicated by *, ** and ***. (B-D) Ability of wild type and mutated receptors to translocate FGF-1. COS-1 cells were transfected with FGFR-constructs as indicated. The cells were labeled with [33P]phosphate and incubated with FGF-1 and heparin for 6 hours. FGF-1 was then extracted from the cell lysates and analyzed as in Fig. 2. Upper panels show phosphorylated FGF-1 detected by autoradiography and the lower panels show the total of endocytosed FGF-1, detected by anti-FGF-1.

 

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© The Company of Biologists Ltd 2006