IntroductionThe large family of fibroblast growth factors (FGFs) comprisesin humans 22 structurally related heparin binding polypeptideswhich are involved in the regulation of many key cellularprocesses (Powers et al., 2000). The FGFs mediate theirbiological effects through binding to high-affinity cell-surfacereceptors, FGFRs. The FGFR family constitutes a variety ofpolypeptides encoded by four related genes (Johnson andWilliams, 1993; Ornitz and Itoh, 2001). The receptors sharecommon structural features and consist of an extracellularligand binding domain, a transmembrane domain and acytoplasmic region. The extracellular domain contains aunique acidic region and two or three immunoglobulin-likedomains (D1-D3), dependent on alternative splicing. Thecytoplasmic region contains a split tyrosine kinase domain(Johnson and Williams, 1993).

Binding of FGFs to FGF receptors (FGFRs) is stabilized byheparan sulfate proteoglycans and results in a dimer receptor-ligand complex that activates the intracellular tyrosine kinasedomain by autophosphorylation. The autophosphorylationtriggers the transient assembly of a large intracellular complex,which activates downstream signalling pathways such asphospholipase C-�/protein kinase C, phosphoinositide 3-kinase/Akt and Ras/MAPK (mitogen-activated protein kinase)(Klint and Claesson-Welsh, 1999; Schlessinger, 2004).

Depending on the target cell type, FGF signalling can inducecell proliferation, differentiation, survival and motility(Basilico and Moscatelli, 1992).

Signalling from activated transmembrane receptors isattenuated by degradation in lysosomes. Lysosomal targetingof tyrosine kinase receptors is best illustrated for the epidermalgrowth factor receptor (EGFR) and involves the attachment ofubiquitin to lysine residues in the cytoplasmic tail of theactivated receptor (Levkowitz et al., 1998). Uponinternalization, the receptors appear in early/sortingendosomes where the receptors destined for degradation in thelysosomes become ubiquitylated. As a result, they arerecognized by Hrs and the ESCRT (endosomal sorting complexrequired for transport) complexes and internalized into theendosomes by membrane invagination (Raiborg et al., 2003).Multivesicular bodies fuse with late endosomes and theendocytosed material is then sorted to lysosomes fordegradation.

Receptors that are not retained in the sorting endosomesrecycle either directly or via the endocytic recyclingcompartment, ERC, back to the cell surface. Most receptorsknown to recycle possess no signalling activity and are oftenassociated with uptake of nutrients. The transferrin receptor,TfR, is known to recycle via the ERC and is often used as amarker for the recycling endocytic pathway (Yamashiro and

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Many growth factors and cytokines bind to more than onereceptor, but in many cases the different roles of theseparate receptors in signal transduction are unclear.Intracellular sorting of ligand-receptor complexes maymodulate the signalling, and we have here studied theintracellular trafficking of ligand bound to receptors forfibroblast growth factors (FGFs). For this purpose, wetransfected HeLa cells with any one of the four tyrosinekinase FGF receptors (FGFR1-4). In cells expressing anyone of these receptors, externally added FGF1 was localizedto sorting/early endosomes after 15 minutes at 37°C. Afterlonger incubation times, FGF1 internalized in cellsexpressing FGFR1 was localized mainly to lateendosomes/lysosomes, similarly to EGF. By contrast, FGF1internalized in cells expressing FGFR4 followed largely thesame intracellular pathway as the recycling ligand,transferrin. In cells expressing FGFR2 or FGFR3, sortingof FGF1 to lysosomes was somewhat less efficient than that

observed for FGFR1. Furthermore, FGF1 was more slowlydegraded in cells expressing FGFR4 than in cellsexpressing FGFR1-3 and in addition, internalized FGFR4as such was more slowly degraded than the other receptors.The data indicate that after endocytosis, FGFR4 and itsbound ligand are sorted mainly to the recyclingcompartment, whereas FGFR1-3 with ligand are sortedmainly to degradation in the lysosomes. Alignment of theamino acid sequence of the intracellular part of the fourFGFRs revealed several lysines conserved in FGFR1-3 butabsent in FGFR4. Lysines are potential ubiquitylation sitesand could thus target a receptor to lysosomes fordegradation. Indeed, we found that FGFR4 is lessubiquitylated than FGFR1, which could be the reason forthe different sorting of the receptors.

Key words: FGF, FGFR, Recycling, Ubiquitylation, Endocytosis

Summary

Different intracellular trafficking of FGF1 endocytosedby the four homologous FGF receptorsEllen Margrethe Haugsten, Vigdis Sørensen, Andreas Brech, Sjur Olsnes and Jørgen Wesche*Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, The University of Oslo, Montebello, 0310 Oslo,Norway*Author for correspondence (e-mail: jorgen.wesche@labmed.uio.no)

Accepted 23 May 2005Journal of Cell Science 118, 3869-3881 Published by The Company of Biologists 2005doi:10.1242/jcs.02509

Research Article

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Maxfield, 1984). The importance of ubiquitin as a signal forlysosomal sorting is illustrated by experiments wheretransferrin receptors were fused to ubiquitin and found to besorted into the degradative pathway (Raiborg et al., 2002).

From what is known about the endocytosis of the differentFGFRs, it appears that they may utilize different mechanismsfor internalization and that this also may vary between differentcell types (Gleizes et al., 1996; Marchese et al., 1998; Citoreset al., 1999; Citores et al., 2001; Reilly et al., 2004). However,irrespective of the mechanism of endocytosis, FGF/FGFRcomplexes have been observed in early endosomes/sortingendosomes approximately 10 minutes after internalization(Gleizes et al., 1996; Citores et al., 1999; Belleudi et al., 2002).Subsequent to their presence in sorting endosomes, KGF(FGF7) and KGFR (a splicing variant of FGFR2), were foundto be sorted to late endosomes in HeLa cells (Belleudi et al.,2002). FGF2 has also been observed in late endosomes andlysosomes in BHK cells (Gleizes et al., 1996). However,FGF1/FGFR4 in COS cells were found to accumulate inintracellular structures identified as the recycling compartment(Citores et al., 1999). It was found that binding of FGF toFGFR1 and FGFR3 induces ubiquitylation of the receptors andthat this contributes to their downregulation (Mori et al., 1995;Monsonego-Ornan et al., 2002; Wong et al., 2002; Cho et al.,2004). Activated FGFR3 has recently been reported to betargeted for lysosomal degradation through c-Cbl-mediatedubiquitylation, whereas FGFR3 harbouring mutationsassociated with skeletal disorders were found to be lessubiquitylated and escape lysosomal targeting (Cho et al.,2004).

To compare the intracellular fate of FGF internalized by thefour related tyrosine kinase FGFRs, HeLa cells transfectedwith any one of the four FGFRs were chosen as a modelsystem, and FGF1, which binds equally well to the four FGFRs(Ornitz et al., 1996), was used as a ligand. The present workshows that FGF1 endocytosed by the four receptors is indeedsorted differently and that different levels of ubiquitylationappear to be the molecular mechanism responsible for thedifferent sorting.

Materials and MethodsMaterialsRabbit anti-FGFR1, anti-FGFR2, anti-FGFR3 and anti-FGFR4antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).Rabbit antibodies against tyrosine 653/654 phosphorylated FGFRswere from Cell Signalling (Beverly, MA). Mouse anti-EEA1antibodies were obtained from Transduction laboratories (Lexington,KY) and mouse anti-LAMP-1 antibodies were from DevelopmentalStudies Hybridoma Bank (Iowa City, IA). Mouse anti-myc antibodieswere from 9E10 hybridoma (Evan et al., 1985). Mouse anti-TfRantibodies were from Boehringer Mannheim (Mannheim, Germany).The secondary antibodies Cy2-conjugated anti-mouse IgG, Cy2-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG andHRP-conjugated anti-rabbit IgG were from Jackson Immuno-Research Laboratories (West Grove, PA).

Cy3-maleimide, heparin-Sepharose, streptavidin-Sepharose, ECLplus western blotting system were from Amersham Biosciences(Buckinghamshire, UK). APS, TEMED, 40% acrylamide/bis-acrylamide and Restore Western Blot Stripping Buffer were fromBIO-RAD (Hercules, CA). Fugene 6 was from Boehringer Mannheim(Indianapolis, IN). Dulbecco’s Modified Eagle Medium (DMEM),streptomycin and penicillin were from GIBCO, Invitrogen (Carlsbad,

CA). Alexa 488 EGF and Alexa 647 Transferrin were from MolecularProbes, Invitrogen. Restriction enzymes were from New EnglandBiolabs, (Beverly, MA). Mowiol was from Novabiochem Corporation(La Jolla, CA). Fetal calf serum was from PAA Laboratories GmbH(Linz, Austria). Easytag Methionine L-[35S] was from Perkin Elmer(Boston, MA). Leupeptin to use on live cells was from PeptideInstitute (Osaka, Japan). Ez-link sulfo-NHS-LC-Biotin was fromPierce (Rockford, IL). Rabbit Reticulocyte Lysate System wasobtained from Promega Corporation (Madison, WI). Complete EDTAfree protease inhibitor cocktail tablets were from Roche Diagnostics(Penzberg, Germany). T3 RNA polymerase and T7 RNA polymerasewere from Stratagene (La Jolla, CA). Other chemicals were fromSigma-Aldrich (St Louis, MO). Alexa 488-labelled FGF2 was agenerous gift from Jedrzej Malecki, this Institute. [125I]FGF1 was agenerous gift from Malgorzata Zakrzewska, Institute of Biochemistryand Molecular Biology, University of Wroclaw.

PlasmidspcDNA3-hFGFR1: cDNA encoding hFGFR1 IIIc was cut out frompSV7d (Wennstrom et al., 1991) with EcoRI and XbaI in twofragments, and ligated into pcDNA3 (Invitrogen, Carlsbad, CA) cutwith the same enzymes. pcDNA3-hFGFR2: cDNA encoding hFGFR2IIIc lacking D1 was cut out from pBluescript (RZPD, Berlin,Germany, Clone ID: IMAGp998N0911701Q3) with NotI and SpeI,and ligated into pcDNA3 cut with NotI and XbaI. The pcDNA3-hFGFR3 IIIc construct was a generous gift from Avner Yayon,ProChon Biotech Ltd, Israel (Adar et al., 2002). The pcDNA3-hFGFR4 and the pB-FGF1 construct has been described previously(Wesche et al., 2000; Klingenberg et al., 2000). The pTriEX-2-FGF1construct was a generous gift from Camilla Skiple Skjerpen, thisInstitute, and the pcDNA3-myc-tagged-ubiquitin was a generous giftfrom Harald Stenmark, this Institute.

CellsHeLa cells were propagated in DMEM, supplemented with 10%(vol/vol) fetal calf serum and 100 U/ml penicillin and 100 �g/mlstreptomycin in a 5% CO2 atmosphere at 37°C.

TransfectionsTransient expression of the different receptors was performed bytransfecting HeLa cells with the plasmid DNA (pcDNA3 withappropriate inserts) by using Fugene 6 transfection reagent accordingto the manufacturer’s protocol. Cells were seeded into plates (BDBiosciences, San Jose, CA and Nalge Nunc International, Rochester,NY) the day preceding the transfection and experiments wereperformed 15-24 hours after transfection.

Laser scanning confocal microscopyFGF1 was labelled with Cy3-maleimide according to themanufacturer’s protocol. Not transfected or transiently transfectedHeLa cells grown on coverslips at 37°C were incubated with Cy3-FGF1 for 2 hours in HEPES medium at 4°C in the presence of 50U/ml heparin. The cells were then washed three times in PBS andincubated for different periods of time in DMEM with 0.3 mMleupeptin at 37°C. The cells were fixed in 3% paraformaldehyde inPBS for 15 minutes, washed three times in PBS and mounted inMowiol. In some cases the cells were in addition to Cy3-FGF1incubated with Alexa 488 EGF and Alexa 647 transferrin in thepresence of 50 U/ml heparin and 0.3 mM leupeptin. When antibodieswere used to visualize structures within the cell, the fixation wasquenched with 50 mM NH4Cl in PBS for 15 minutes and the cellswere permeabilized with 0. 05% saponin in PBS for 5 minutes. Thecells were then incubated with primary antibody in 0. 05% saponin in

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PBS for 20 minutes, washed three times in 0. 05% saponin in PBSand incubated for additional 20 minutes with the secondary antibodycoupled to a fluorophore. After washing once in 0. 05% saponin andtwice in PBS, the cells were mounted in Mowiol and examined witha Zeiss LSM 510 META confocal microscope (Zeiss, Jena, Germany).Images were prepared with Adobe Photoshop 7. 0 (Adobe, San Jose,CA) and Zeiss LSM Image Browser (Version 3).

Quantification of colocalizationImages of transfected, randomly chosen cells were divided intosquares, and every fifth square within the chosen cell was examined.Red structures indicating internalized Cy3-labelled FGF1 werecompared with structures of the different markers and the proportionof red structures that colocalized with structures of the specific markerwas calculated. The mean and s.d. were calculated from 15 cells ineach case.

Degradation of internalized receptorsHeLa cells not transfected or transiently transfected with FGFR1,FGFR2, FGFR3 or FGFR4 were washed three times in PBS and cell-surface proteins were biotinylated with 0.5 mg/ml Ez-link sulfo-NHS-LC-Biotin in PBS for 15 minutes at 4°C. The biotinylationreaction was quenched with 50 mM Tris-HCl pH 8.0. The cells werewashed twice with PBS and then incubated for the indicated periodsof time in DMEM containing 100 ng/ml FGF1 and 20 U/ml heparin.The cells were washed with PBS and lysed on ice in lysis buffer (0.1M NaCl, 10 mM Na2HPO4, 1% Triton X-100, 1 mM EDTA,supplemented with complete protease inhibitors, pH 7.4) for 20minutes. The lysate was centrifuged to remove nuclei and thenbiotinylated proteins were pulled down from the supernatant withstreptavidin-Sepharose beads at 4°C over night. The beads were thenwashed three times in PBS containing 0.1% Tween 20 and finallyresuspended in 15 �l of reducing SDS-PAGE sample buffer. Proteinswere separated by 7% SDS-PAGE as described by Laemmli(Laemmli, 1970) and transferred to Immobilon-P PVDF membrane(Millipore Corporation, Bedford, MA), which was probed with anti-FGFR1, anti-FGFR2, anti-FGFR3 or anti-FGFR4 primary antibodiesand anti-rabbit HRP-conjugated secondary antibody. Immunoactivitywas detected by using ECL plus western blotting system and ChemiGenius Image Acquisition System (Syngene, Cambridge, UK). Tocompare the intensity of bands of interest on the membrane,ImageQuant software (Amersham Biosciences, Buckinghamshire,UK) was used. Background correction was performed by subtractingvalues obtained by scanning adjacent areas of the membrane with thesame size but containing no visible bands from those obtained withthe bands of interest.

Degradation of internalized FGF1The [35S]methionine-labelled 18 kDa, long form of FGF1 and the 16kDa form of FGF1 was produced by transcription using T7 RNApolymerase or T3 RNA polymerase, respectively, and translation in arabbit reticulocyte lysate supplemented with Easytag Methionine L-[35S] according to the manufacturer’s protocol.

HeLa cells, not transfected or transiently transfected with thedifferent FGFRs, were incubated at 37°C with the [35S]methionine-labelled 16 kDa form of FGF1 and 50 U/ml heparin for 1 hour to allowbinding and endocytosis of FGF1. The cells were washed with PBSand lysed. FGF1 was extracted from the lysate by binding to heparin-Sepharose and analysed by 13% SDS-PAGE.

HeLa cells, transiently transfected with the different FGFRs, wereincubated with the [35S]methionine-labelled 18 kDa form of FGF1and 20 U/ml heparin at 37°C for 1 hour to allow endocytosis via high-affinity receptors. Then the cells were washed twice with a high salt,low pH buffer (2 M NaCl, 20 mM NaAc, pH 4. 0) and once with PBS

on ice to remove excess and cell-surface bound FGF1 (Klingenberget al., 2000). The cells were then either lysed immediately in lysisbuffer or incubated further in growth medium with or without 100 �Mchloroquine at 37°C for 3 or 6 hours before lysis. [35S]Methionine-labelled FGF1 was extracted from the lysate by adsorption to heparin-Sepharose and analysed by 15% SDS-PAGE. The proteins on the gelswere fixed in fixative (25% methanol, 7.5% acetic acid) and then thegels were dried. STORM Phosphorimager scanning and Image Quant,Version 5.0 (Molecular Dynamics, Amersham Biosciences,Buckinghamshire, UK) software were used to estimate the relativeamount of radioactive FGF.

To study degradation and recycling of FGF1, HeLa cells weretransfected with FGFR1 or FGFR4 and incubated at 37°C for 20minutes in growth medium containing 100 ng/ml [125I]FGF1 and 40U/ml heparin. To remove surface bound FGF1, the cells were washedtwice with a high salt/low pH buffer and twice in PBS. The cells werefurther incubated in growth medium at 37°C for indicated periods oftime. The medium was then removed and collected. The cells wereincubated for 5 minutes with high salt/low pH buffer and the bufferwas collected. Finally, the cells were collected after solubilisation in0.1 M KOH at 37°C for 30 minutes. The collected medium, thecollected high salt/low pH buffer wash and the collected dissolvedcells were adjusted to 5% trichloroacetic acid (TCA) and kept on icefor 20 minutes and then centrifuged at 4°C for 5 minutes. Theradioactivity present in the supernatants (TCA-soluble fraction) andpellets (TCA-insoluble fraction) was determined in a gamma counter.Degradation was measured for each time point as the amount ofradioactivity in the TCA-soluble fractions and expressed as apercentage of the total radioactivity in the culture. Recycling wasmeasured for each time point as the amount of radioactivity in theTCA-insoluble fractions in the medium and in the high salt/low pHbuffer and expressed as a percentage of the total radioactivity in theculture.

Ubiquitylation of internalized receptorsHeLa cells cotransfected with myc-ubiquitin and FGFR1, FGFR4 orempty vector were starved for 16 hours and then washed three timesin PBS. Cell-surface proteins were biotinylated with 0.5 mg/ml Ez-link sulfo-NHS-LC-Biotin in PBS for 15 minutes at 4°C. Thebiotinylation reaction was quenched with 50 mM Tris-HCl pH 8. 0and the cells were washed twice with PBS. The cells were incubatedfor 2 hours in 200 ng/ml FGF1, 20 U/ml heparin and 0.3 mM leupeptinat 37°C in DMEM without serum. The cells were then washed oncein DMEM without serum and lysed at 95°C for 5 minutes in 1% SDSin PBS. The lysate was decanted into QIAshredder columns andcentrifuged for 2 minutes at 4°C. Equal amounts of lysate and 2� pulldown-buffer (2% Triton X-100, 0.5% sodium deoxycholate, 2 mMEDTA, 40 mM NaF, 1% bovine serum albumine, 2 mM N-ethylmaleimide supplemented with protease and phosphataseinhibitors) were added to streptavidin-Sepharose beads to pull downbiotinylated proteins. After tumbling 1 hour at 4°C, the beads werewashed twice in 1� pull-down buffer (0.5% SDS and 50% 2� pull-down buffer in PBS) and once in 1:10 diluted PBS. The proteins thatremained bound to the streptavidin-Sepharose beads were submittedto 7% SDS-PAGE and then transferred to a PVDF membrane whichwas probed with anti-myc primary antibody and anti-mouse HRP-conjugated secondary antibody to detect the level of ubiquitylation ofinternalized FGFRs. Immunoreactivity was detected using ECL pluswestern blotting system and Chemi Genius Image AcquisitionSystem. The membrane was stripped twice and reprobed with anti-phospho FGFR primary antibody and anti-rabbit HRP-conjugatedsecondary antibody to detect the level of internalized receptors, andwith anti-transferrin receptor primary antibody and anti-mouse HRP-conjugated secondary antibody to verify equal loading of the gel. Toensure equal expression of ubiquitin, the cells were analysed byimmunofluorescence microscopy.

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ResultsCharacterization of the endocytic pathway followed byFGF1/FGFR1-4Upon ligand binding to the FGFRs, the ligand-receptorcomplexes are internalized (Sorokin et al., 1994; Munoz et al.,1997) and transported to various intracellular compartments.Because FGF1 binds equally well to the four high-affinityFGFRs (Ornitz et al., 1996), this ligand was labelled with thefluorescent dye Cy3 and the fluorescent growth factor was thenused as a marker to explore the intracellular trafficking ofFGF1 and the four FGFRs. Cy3-labelled FGF1 has previouslybeen shown to retain its binding capacity towards the FGFRsand heparin sulfate proteoglycans (Citores et al., 1999).

The distribution of fluorescent growth factor-receptorcomplexes was studied in HeLa cells transiently transfectedwith the different FGFRs and incubated with Cy3-FGF1 fordifferent periods of time. HeLa cells do not express detectableamounts of endogenous FGFRs. To avoid FGF1 binding tocell-surface heparan proteoglycans and to facilitate binding tothe high-affinity FGFRs, heparin was added to the extracellularmedium. The data in Fig. 1A show that fluorescent FGF1 bindsto the surface of transfected cells when treated with the growthfactor at 4°C in the presence of heparin. There was nodetectable binding to untransfected cells (Fig. 1A, first panel).Uptake of similar amounts of radiolabelled FGF1 in HeLa cellstransfected with any of the four receptors indicates that thetransfected cells express comparable levels of the four FGFreceptors (Fig. 1B). In addition, very little FGF1 wasassociated with untransfected cells. When the cells wereincubated at 37°C, the fluorescent growth factor appeared asintracellular dots, indicating uptake into vesicles (Fig. 2).

To determine whether FGF1 and the different FGFRs remainin the same compartments after internalization, we carried outdouble-labelling experiments where cells were allowed to takeup Cy3-labelled growth factor for 2 hours at 37°C in thepresence of the inhibitor of lysosomal degradation, leupeptin,and then stained with antibodies against the different FGFRs.There was considerable overlap between internalized FGF1and FGFRs, as visualised in double-staining experiments whenspots labelled with both fluorophores appeared yellow (Fig. 2).

This indicates that the ligand and the receptor are in the sameintracellular compartments as previously reported forKGF/KGFR and FGF1/FGFR4 (Marchese et al., 1998; Citoreset al., 1999; Belleudi et al., 2002).

To follow the endocytic pathway and to identify theintracellular compartments where the different FGF1/FGFRcomplexes are localized upon internalization, the transientlytransfected HeLa cells were allowed to bind Cy3-labelledFGF1 at 4°C and then they were incubated for different periodsof time at 37°C. The cells were subsequently fixed and stainedwith antibodies against markers for different intracellularcompartments. As shown in Fig. 3, incubation for 15 minutesat 37°C resulted in good overlap of EEA1, a protein associatedwith early/sorting endosomes (Mu et al., 1995), andendocytosed Cy3-FGF1. Quantitation of the colocalizationshowed that about 70-80% of the FGF1-positive structures incells transfected with either of the four FGFRs were positivefor EEA1 after 15 minutes of endocytosis. Thus, all the fourFGF1/FGFR complexes reach the sorting endosomalcompartment.

After a 2 hour chase in the presence of leupeptin to inhibitdegradation in the lysosomes, the major part of the internalizedFGF1 in cells transfected with FGFR1-3 was found tocolocalize with antibodies against LAMP-1. LAMP-1 is aprotein associated with late endosomes/lysosomes (Geuze etal., 1988). In the case of FGFR4-transfected cells there wasless colocalization (Fig. 4A). Quantitation showed that about90% of the FGF1-positive structures in FGFR1-transfectedcells were LAMP-1 positive, whereas in the case of FGFR4only about 45% were LAMP-1 positive (Fig. 4B). In the caseof FGFR2 and FGFR3 about 70% of the FGF1-positivestructures were also positive for LAMP-1. These findingsindicate that subsequent to their uptake in early/sortingendosomes the four FGFRs are sorted differently. The majorpart of internalized FGFR1-3 is sorted to lysosomes, whereasthe major part of internalized FGFR4 is not.

To further study the different sorting of the receptors and todetermine the localization of the FGF1/FGFR4 complexsubsequent to its appearance in early/sorting endosomes, theendocytic pathway followed by the fluorescent FGF1 was

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Fig. 1. Binding of FGF1 to cell-surface FGFRs. (A) HeLa cells, not transfected (–) ortransiently transfected with FGFR1, 2, 3 or 4 were treated with Cy3-labelled FGF1and 50 U/ml heparin for 2 hours at 4°C. The cells were then fixed and examined byconfocal microscopy. The red channel image was superimposed onto thecorresponding interference contrast image. Bar, 5 �m. (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 for1 hour. The cells were washed with PBS and lysed. FGF1 was extracted from thelysate by binding to heparin-Sepharose and analysed by SDS-PAGE.

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Fig. 2. Colocalization ofFGFRs and endocytosedFGF1 after 2 hours.HeLa cells transientlytransfected with FGFR1,2, 3 or 4 were incubatedwith Cy3-FGF1, 50U/ml heparin and 0.3mM leupeptin for 2hours at 37°C. The cellswere then fixed,permeabilized andtreated with rabbit anti-FGFR1, anti-FGFR2,anti-FGFR3 or anti-FGFR4 primaryantibodies. The cellswere further treated withCy2-conjugated anti-rabbit secondaryantibodies and examinedby confocal microscopy.Arrows point tocolocalization. Bar,5 �m.

Fig. 3. Colocalization ofEEA1 and endocytosedFGF1 after 15 minutes.HeLa cells weretransiently transfectedwith FGFR1, 2, 3 or 4and kept at 4°C withCy3-FGF1 and 50 U/mlheparin for 2 hours. Thecells were washed andincubated in thepresence of 0.3 mMleupeptin for 15 minutesat 37°C. The cells werefixed, permeabilized andtreated with mouse anti-EEA1 primary antibody.The cells were furthertreated with Cy2-conjugated anti-mousesecondary antibodiesand examined byconfocal microscopy.Arrows point tocolocalization. Bar,5 �m.

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compared with those taken byepidermal growth factor (EGF) andtransferrin (Tf). EGF receptors andits ligand, EGF, progress tolysosomes upon internalization(Futter et al., 1996), whereas thetransferrin receptor and its ligand,Tf, are known to be recycled fromearly/sorting endosomes via theendosomal recycling compartmentback to the cell surface (Hopkins,1983).

HeLa cells transfected with thedifferent FGFRs were incubated for2 hours at 37°C with Alexa 488-labelled EGF and Cy3-labelledFGF1 in the presence of leupeptin.Alexa 647-labelled transferrin wasadded after 90 minutes.Colocalization was shown inoverlay experiments when dotslabelled with Cy3-FGF1 and Alexa488 EGF appeared yellow and dotslabelled with Cy3-FGF1 and Alexa647-labelled transferrin appearedpurple. Fluorescent FGF1endocytosed by FGFR1-3 showedconsiderable overlap withfluorescent EGF, indicating that themajor part of internalizedFGF1/FGFR1-3 complexesaccumulates in lysosomes.However, fluorescent FGF1endocytosed by FGFR4 showed anotable overlap with transferrin,indicating that a great part of theinternalized FGF1/FGFR4complexes accumulate in theendocytic recycling compartment(Fig. 5A).

Quantitation of colocalizationwas performed as described inMaterials and Methods. As shownin Fig. 5B approximately 65% ofthe FGF1-positive structures in cells transfected with FGFR1colocalized with intracellular structures containing EGF,whereas only about 8% of the FGF1-positive structurescolocalized with intracellular structures containing transferrin.In cells transfected with receptor 2 or 3 between 40% and 50%of the FGF1-positive structures also contained EGF, whereasabout 20% of the FGF1-positive structures containedtransferrin. In the case of cells transfected with FGFR4 onlyabout 20% of the FGF1-positive structures contained EGF,whereas about 50% of the FGF1-positive structures containedtransferrin. Between 20% and 30% of the FGF1-positivestructures in the transfected cells contained neither EGF nortransferrin and a small fraction of about 5% of the FGF1-positive structures contained both EGF and transferrin.

To decide whether the observed difference in the sorting ofFGF1 internalized by the four FGFRs is specific for FGF1,cells transfected with FGFR1 or FGFR4 were allowed to

endocytose Cy3-labelled FGF1 and Alexa 488-labelled FGF2for 2 hours in the presence of leupeptin. The two fluorophore-labelled ligands showed almost complete colocalization in bothcases (data not shown). This finding suggests that the differentsorting does not depend on the ligand.

Degradation of internalized FGF1 and FGFRsThe different sorting of the four related FGFRs could result indifferent kinetics of degradation of FGF1 as well as thereceptors. The degradation of the four FGFRs and FGF1 wasanalysed in FGFR-transfected HeLa cells.

To analyse the degradation of internalized receptors, cellstransfected with the different FGFRs were submitted tobiotinylation of proteins exposed at the cell surface. The cellswere then treated with FGF1 for increasing periods of time andthen lysed. The biotinylated proteins in the lysates were

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Fig. 4. Different colocalization of LAMP1 andendocytosed FGF1 after 2 hours. (A) HeLa cells weretransiently transfected with FGFR1, 2, 3 or 4 and kept at4°C with Cy3-FGF1 and 50 U/ml heparin for 2 hours.They were then washed and incubated in the presence of0.3 mM leupeptin for 2 hours at 37°C. The cells werefixed, permeabilized and treated with mouse anti-LAMP1primary antibody. The cells were further treated with Cy2-labelled anti-mouse secondary antibody and examined byconfocal microscopy. Arrows point to colocalization in thecase of FGFR1-3 and lack of colocalization in the case ofFGFR4. Bar, 5 �m. (B) The percentage of FGF1-positivestructures within cells that colocalized with LAMP1 wasquantitated as described in Materials and Methods. Errorbars denote the s.d.; n=15.

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collected by adsorption tostreptavidin-Sepharose followed bySDS-PAGE and immunoblotting withthe appropriate anti-receptorantibodies. We observed for eachFGFR a gradual reduction in theintensity of the bands corresponding tothe receptors, indicating degradationof the internalized receptors (Fig. 6A).There was, however, a large differencein the rate of degradation. Thus, theintensity of the bands corresponding toFGFR1 was strongly reduced after 2hours, whereas that corresponding toFGFR4 was only slightly reduced after6 hours. The degradation of FGFR2and FGFR3 was slower than thedegradation of FGFR1 butconsiderably faster than that ofFGFR4.

The bands representing FGFRs inFig. 6A were quantitated from the gelsand plotted as a percentage of theamount of receptors at time zero. Themean values from three independentexperiments (Fig. 6B) show that theamount of FGFR1 decreased from100% to ~15% in 2 hours, whereas theamount of FGFR4 was only reduced to~80% in 6 hours. In the case of FGFR2and FGFR3, 30-40% of the initialamount remained after 6 hours.

To investigate the degradation of FGF1, transfected cellswere incubated with the radiolabelled 18 kDa form of FGF1for 1 hour to allow endocytosis of the growth factor-receptorcomplex to occur. The cells were then washed to removesurface-bound FGF1 and further incubated for increasingperiods of time. Finally, the cells were lysed and solubilisedproteins were adsorbed to heparin-Sepharose and analysed bySDS-PAGE. The degradation of FGF1 can be seen in Fig. 7Aas a stepwise conversion of the 18 kDa form of FGF1 into theshorter 16 kDa form followed by further degradation.

The further degradation of FGF1 seems to occur more

slowly, indicating that the 16 kDa form of FGF1 is moreresistant to degradation than the 18 kDa form. The degradationof internalized FGF1 was inhibited by the weak basechloroquine (shown only for FGFR1), suggesting that thedigestion occurred in a lysosomal compartment.

After 6 hours only a small fraction of FGF1 remained as the18 kDa form in cells transfected with FGFR1, whereas asignificant amount of the 18 kDa form was present in cellstransfected with FGFR4. The amount of the 18 kDa form ofFGF1 that remained in the cells was calculated for each receptortype and each time point, and expressed as a percentage of theamount of the 18 kDa form at time zero. The values plotted in

Fig. 5. Colocalization of endocytosedEGF, transferrin and FGF1 after 2 hours.(A) HeLa cells were transientlytransfected with FGFR1, 2, 3 or 4 andincubated for 2 hours at 37°C with Cy3-FGF1 and Alexa 488-EGF in the presenceof 50 U/ml heparin and 0.3 mM leupeptin.Alexa 647-transferrin (Tf) was addedafter 90 minutes. The cells were fixed andexamined by confocal microscopy. Insetsshow selected areas of the imagesenlarged (3�). Bar, 5 �m. (B) Thepercentage of FGF1-positive structureswithin transfected cells that colocalizedwith EGF, Tf, neither EGF nor Tf, or bothEGF and Tf was quantitated as describedin Materials and Methods. Error barsdenote the s.d.; n=15.

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the graph in Fig. 7B are average values from five (FGFR1 andFGFR4) and three (FGFR2 and FGFR3) independentexperiments. Approximately 50% of FGF1 was in the 18 kDaform in FGFR4 transfected cells after 3 hours, whereas only15% of FGF1 remained as the 18 kDa form after 3 hours incells transfected with FGFR1. The fraction of the 18 kDa formin cells transfected with FGFR2 or FGFR3 was reduced to about

30% after 3 hours. The results indicate that FGF1 endocytosedby FGFR4 is more slowly degraded than FGF1 endocytosed byFGFR1. FGF1 endocytosed by FGFR2 or FGFR3 seems to bemore slowly degraded than FGF1 endocytosed by FGFR1, butfaster than FGF1 endocytosed by FGFR4.

The degradation and the recycling of FGF1 was furtheranalysed in HeLa cells transfected with FGFR1 or FGFR4. The

transfected cells were allowed tointernalize [125I]FGF1 for 20 minutes,washed to remove excess and cell-surface-associated [125I]FGF1 and incubatedfurther for the periods of time indicated.The degradation of FGF1 was measured asthe percentage of radioactivity in theculture that was TCA soluble. RecycledFGF1 was measured as the percentage of

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Fig. 6. Degradation of endocytosed FGFRs. (A) Cell-surface proteins on HeLa cells, not transfected (NT) ortransiently transfected with FGFR1, 2, 3 or 4 werebiotinylated and the cells were then incubated for theindicated periods of time at 37°C in the presence of100 ng/ml FGF1 and 20 U/ml heparin. After lysis, thesolubilised receptor proteins were adsorbed tostreptavidin-Sepharose and analysed byimmunoblotting (IB) with appropriate anti-FGFRantibodies. (B) The intensity of the bands at time pointzero was set to 100% and the relative amount ofreceptors at each time point was calculated. Thepresented values represent the average of threeindependent experiments. Error bars denote the s.d.

Fig. 7. Degradation of the endocytosed 18 kDa form of FGF1.(A) HeLa cells, transiently transfected with FGFR1, 2, 3 or 4were incubated with the [35S]methionine-labelled 18 kDa formof FGF1 and 20 U/ml heparin at 37°C for 1 hour to allowendocytosis to occur. The cells were then either lysedimmediately (0 h) or incubated further in growth medium withor without chloroquine (chl) at 37°C for 3 or 6 hours beforelysis. FGF1 was extracted from the lysate by binding toheparin-Sepharose and analysed by SDS-PAGE. (B) For eachreceptor the amount of 18 kDa form of FGF1 was calculated ateach time point and expressed as a percentage of the amountof 18 kDa form at time zero. Values are averages of fiveindependent experiments for FGFR1 and FGFR4 and of threeindependent experiments for FGFR2 and FGFR3. Error barsdenote the s.d.

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the total amount of TCA-insoluble radioactivity in the culturethat was present in the medium or released from the cell surfaceby a high salt/low pH wash. About 60% of FGF1 internalized

by FGFR1 was found to be degraded after 5 hours, whereasonly about 30% of FGF1 internalized by FGFR4 was degradedafter 5 hours (Fig. 8A). The rate of recycling for FGF1internalized by FGFR4 was higher than for FGF1 internalizedby FGFR1 (Fig. 8B). Altogether, the data indicate that cellstransfected with FGFR1 degrade FGF1 more efficiently thancells transfected with FGFR4 and that FGF1 endocytosed byFGFR4 shows a higher extent of recycling than FGF1internalized by FGFR1.

Ubiquitylation of internalized FGFRsThe attachment of ubiquitin to lysines in the intracellular partof a membrane protein is thought to function as a signal forlysosomal degradation (Raiborg and Stenmark, 2002; Haglund

et al., 2003). An amino acid sequence

alignment of the intracellular part ofthe four receptors revealed severalconserved lysines in FGFR1-3 thatwere absent in FGFR4 (Fig. 9). Theintracellular domain of FGFR1 andof FGFR2 contains 29 lysines, theintracellular part of FGFR3 contains25 lysines, whereas only 16 lysinesare present in the intracellulardomain of FGFR4. Because lysinesare potential ubiquitylation sites, it ispossible that FGFR4 is lessubiquitylated than FGFR1.

A

B

Fig. 8. Degradation and recycling of endocytosed FGF1. HeLa cellstransfected with FGFR1 (R1) or FGFR4 (R4) were incubated ingrowth medium with 100 ng/ml [125I]FGF1 and 40 U/ml heparin for20 minutes at 37°C. Excess and surface-bound [125I]FGF1 wasremoved by acid/salt wash of the cells, which were incubated furtherin growth medium at 37°C for the indicated periods of time.(A) Degradation of internalized [125I]FGF1 was measured for eachtime point as the amount of radioactivity in the TCA-solublefractions and expressed as a percentage of the total radioactivity inthe culture. (B) Recycling of [125I]FGF1 was measured for each timepoint as the amount of radioactivity in the TCA-insoluble fraction ofthe medium and of the high salt/low pH buffer and expressed as apercentage of the total radioactivity in the culture. The experimentwas carried out twice with similar results.

aa 401 470FGFR1 VIVYKMKSGT KKSDFHSQMA VHKLAKSIPL RRQVTVSADS SASMNSGVLL VRPSRLSSSG TPMLAGVSEYFGFR2 VILCRMKNTT KKPDFSSQPA VHKLTKRIPL RRQVSAESSS SMNSNTPLVR ITTRLSSTAD TPMLAGVSEYFGFR3 VTLCRLRSPP KK..GLGSPT VHKISR.FPL KRQVSLESNA SMSSNTPLVR IAR..LSSGE GPTLANVSELFGFR4 AGLYRGQALH GR.HPRPPAT VQKLSR.FPL ARQFSLESGS SGKSSSSLVR GVR..LSSSG PALLAGLVSL

471 540FGFR1 ELPEDPRWEL PRDRLVLGKP LGEGCFGQVV LAEAIGLDKD KPNRVTKVAV KMLKSDATEK DLSDLISEMEFGFR2 ELPEDPKWEF PRDKLTLGKP LGEGCFGQVV MAEAVGIDKD KPKEAVTVAV KMLKDDATEK DLSDLVSEMEFGFR3 ELPADPKWEL SRARLTLGKP LGEGCFGQVV MAEAIGIDKD RAAKPVTVAV KMLKDDATDK DLSDLVSEMEFGFR4 DLPLDPLWEF PRDRLVLGKP LGEGCFGQVV RAEAFGMDPA RPDQASTVAV KMLKDNASDK DLADLVSEME

541 610FGFR1 MMKMIGKHKN IINLLGACTQ DGPLYVIVEY ASKGNLREYL QARRPPGLEY CYNPSHNPEE QLSSKDLVSCFGFR2 MMKMIGKHKN IINLLGACTQ DGPLYVIVEY ASKGNLREYL RARRPPGMEY SYDINRVPEE QMTFKDLVSCFGFR3 MMKMIGKHKN IINLLGACTQ GGPLYVLVEY AAKGNLREFL RARRPPGLDY SFDTCKPPEE QLTFKDLVSCFGFR4 VMKLIGRHKN IINLLGVCTQ EGPLYVIVEC AAKGNLREFL RARRPPGPDL SPDGPRSSEG PLSFPVLVSC

611 680FGFR1 AYQVARGMEY LASKKCIHRD LAARNVLVTE DNVMKIADFG LARDIHHIDY YKKTTNGRLP VKWMAPEALFFGFR2 TYQLARGMEY LASQKCIHRD LAARNVLVTE NNVMKIADFG LARDINNIDY YKKTTNGRLP VKWMAPEALFFGFR3 AYQVARGMEY LASQKCIHRD LAARNVLVTE DNVMKIADFG LARDVHNLDY YKKTTNGRLP VKWMAPEALFFGFR4 AYQVARGMQY LESRKCIHRD LAARNVLVTE DNVMKIADFG LARGVHHIDY YKKTSNGRLP VKWMAPEALF

681 750FGFR1 DRIYTHQSDV WSFGVLLWEI FTLGGSPYPG VPVEELFKLL KEGHRMDKPS NCTNELYMMM RDCWHAVPSQFGFR2 DRVYTHQSDV WSFGVLMWEI FTLGGSPYPG IPVEELFKLL KEGHRMDKPA NCTNELYMMM RDCWHAVPSQFGFR3 DRVYTHQSDV WSFGVLLWEI FTLGGSPYPG IPVEELFKLL KEGHRMDKPA NCTHDLYMIM RECWHAAPSQFGFR4 DRVYTHQSDV WSFGILLWEI FTLGGSPYPG IPVEELFSLL REGHRMDRPP HCPPELYGLM RECWHAAPSQ

751 820FGFR1 RPTFKQLVED LDRIVALTSN QEYLDLSMPL DQYSPSFPDT RSSTCSSGED SVFSHEPLPE EPCLPRHPAQFGFR2 RPTFKQLVED LDRILTLTTN EEYLDLSQPL EQYSPSYPDT RSS.CSSGDD SVFSPDPMPY EPCLPQYP..FGFR3 RPTFKQLVED LDRVLTVTST DEYLDLSAPF EQYSPGGQDT PSS.SSSGDD SVFAHDLLP. ....PAPP..FGFR4 RPTFKQLVEA LDKVLLAVS. EEYLDLRLTF GPYSPSGGDA SST..CSSSD SVFSHDPLPL GS..SSFP..

821 829FGFR1 LANGGLKRRFGFR2 HINGSVKT.FGFR3 .SSGGSRT.FGFR4 .FGSGVQT.

Fig. 9. Amino acid sequence alignmentof the intracellular parts of FGFR1-4.The protein sequence alignment of theintracellular part of FGFR1, FGFR2,FGFR3 and FGFR4 was created usingthe Vector NTI 9.0 software based on aClustal W algorithm (Thompson et al.,1994). The protein sequences wereobtained from the following DNAsequences at NCBI; M34641,BC039243, NM_000142 and X57205. Adash represents a gap introduced tooptimize the alignment. Lysinesconserved in all the four receptors areindicated in light grey, whereas otherlysines are indicated in dark grey.

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To test this, HeLa cells co-expressing myc-tagged ubiquitinand either FGFR1, FGFR4 or empty vector were incubatedwithout serum overnight. This was included to avoidstimulation and possibly ubiquitylation of surface proteins byfactors in the serum.

Cell-surface proteins were then biotinylated and the cellswere treated with FGF1 for 2 hours. Leupeptin was added toprevent lysosomal degradation of the receptors. The cells werethen lysed and biotinylated proteins were collected andanalysed by western blotting using anti-myc antibody (Fig.10). Ubiquitylation of the receptors was detected as a smear ofbands migrating more slowly than the nonubiquitylatedreceptors. The signal was much stronger for FGFR1 than forFGFR4, indicating that FGFR1 is ubiquitylated to a higherextent than FGFR4. Little ubiquitylation was detected in cellstransfected with the empty vector.

To test whether equal amounts of FGFR were present at themembrane in the two cases, the membrane was stripped andreprobed with an anti-phospho FGFR antibody that was raisedby immunizing rabbits with a synthetic peptide correspondingto residues surrounding tyrosine 653/654 of human FGFR1,which are conserved in FGFR1 and FGFR4. The resultsindicate the presence of similar amounts of FGFR1 and FGFR4(Fig. 10). The membrane was stripped and reprobed with anti-transferrin receptor antibodies to verify approximately equalloading on the gel (Fig. 10). Similar expression of the myc-ubiquitin construct was confirmed by immunofluorescencemicroscopy (data not shown).

Altogether, the observations show that FGFR4 is lessubiquitylated than FGFR1 and they suggest that different levelsof ubiquitylation is the molecular mechanism determining thedifferent sorting of the receptors.

DiscussionThe present work shows that FGF1 internalized by FGFR1 istargeted for lysosomal degradation whereas the majority ofFGF1 internalized by FGFR4 escapes into a recycling pathway.In cells expressing FGFR2 or FGFR3, somewhat less FGF1 issorted to degradation than in cells expressing FGFR1.Furthermore, FGF1 endocytosed by FGFR4 was more slowlydegraded than FGF1 endocytosed by FGFR1, FGFR2 orFGFR3. Also, FGFR4 as such was more slowly degraded thanthe other receptors.

Targeting of receptors for lysosomal degradation has beenassociated with the attachment of mono-ubiquitin to multiplelysines in the intracellular part of the receptors (Haglund et al.,2003). Consistent with the observed different sorting of theFGFRs, FGFR4 has fewer potential ubiquitylation sites(lysines) in its intracellular part than the other FGFRs. Thepresent work shows that FGFR4 is indeed less ubiquitylatedthan FGFR1. This indicates that different levels ofubiquitylation of the FGFRs determine their intracellularsorting.

The present findings are in accordance with previous dataconcerning the trafficking of endocytosed FGFRs.Endocytosed KGF and KGFR in HeLa cells and FGF2 andFGFR3 in RCJ cells have previously been reported to enter thelysosomes (Belleudi et al., 2002; Cho et al., 2004), whereasinternalized FGF1 and FGFR4 were found to accumulate in therecycling compartment in COS cells (Citores et al., 1999). Ithas also been reported that binding of FGF to FGFR1 in HeLacells (Wong et al., 2002) and PAE cells (Mori et al., 1995) andbinding of FGF to FGFR3 in COS-7 cells (Cho et al., 2004)and 293T cells (Monsonego-Ornan et al., 2002) inducesubiquitylation of the receptors and that this contributes to theirdownregulation. Taken together, these findings indicate that thedistinct sorting of the FGFRs depends on receptor type ratherthan cell type or ligand.

Late endosomes and lysosomes contain large amounts ofglycoproteins such as the lysosome-associated membraneprotein-1 (LAMP-1) (Geuze et al., 1988). The staining of thesecompartments for laser scanning confocal microscopy analysiswith primary antibodies against LAMP-1 and fluorophore-conjugated secondary antibodies gave a dense pattern ofLAMP-1-positive structures. The dense pattern may havecaused an overestimation of the degree of colocalizationbetween FGF1-positive structures and LAMP-1-positivestructures. In FGFR4-transfected cells approximately 45% ofthe FGF1-positive structures were positive for LAMP-1.However, when the distribution of fluorophore-labelled FGF1internalized via FGFR4 was compared with the distribution ofinternalized fluorophore-labelled EGF, a marker for lysosomaltrafficking, only 20% of the FGF1-positive structurescontained EGF.

When continuous uptake of fluorophore-labelled FGF1 wasallowed in cells transfected with the different receptors,between 20% and 30% of the FGF1-positive structures insidethe cells colocalized neither with the marker for lysosomaltrafficking (EGF) nor with transferrin, the marker for therecycling pathway. It is likely that the amount of overexpressedFGFRs exceeds the amount of the other receptors at the cellsurface. Therefore, free FGFRs could still be present at the cellsurface, ready to bind and internalize ligand when most of theEGF and transferrin receptors are already located in

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Fig. 10. Ubiquitylation of endocytosed FGFRs. HeLa cells werecotransfected with myc-ubiquitin and FGFR1 (R1), FGFR4 (R4) orthe empty pcDNA3 vector (–) and starved overnight. Cell-surfaceproteins were biotinylated and the cells were incubated for theindicated 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, solubilisedreceptor proteins were adsorbed to streptavidin-Sepharose andanalysed by immunoblotting (IB) with anti-myc antibody. Themembrane was stripped and reprobed with anti-phospho-FGFRantibodies (anti-pFGFR) and anti-transferrin receptor antibodies(anti-TfR).

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intracellular vesicles. Some of the FGF1-positive structuresinside the cells also contained both fluorophore-labelled EGFand transferrin. This is probably material in early endosomes.

The kinetics of degradation of FGF1 as well as that of thereceptors were analysed in FGFR-transfected HeLa cells andthe results correlate with the different sorting observed forFGF1 endocytosed by the four FGFRs –that is, degradationwas faster for receptor/ligand sorted to lysosomes (particularlyFGFR1) than for recycling receptor/ligand. The degradation ofinternalized FGF1 appeared first as a conversion of the 18 kDaform of FGF1 to the shorter 16 kDa form. Further degradationof the short form of FGF1 occurred more slowly, indicatingthat the 16 kDa form of FGF1 is more resistant to degradationthan the 18 kDa form. This seems to be the case for FGF2 also.Internalized FGF2 in BCE cells has earlier been reported to berapidly cleaved from an 18 kDa form to a 16 kDa form, andthe 16 kDa form was then found to be more slowly degraded,with a half-life of approximately 8 hours (Moscatelli, 1988). Itis not clear which FGFR were expressed on the cells in thatcase.

Furthermore, if the recycled ligand is allowed to detach fromthe receptor at the cell surface, the observed degradation ofFGF1 might be slightly overestimated. It is therefore possible,particularly in the case of FGFR4, that FGF1 is actually moreslowly degraded than apparent from Fig. 7.

All the four FGFRs have been found to have distinct patternsof distribution in many human tissues. The most widespreadexpression has been observed for FGFR1, whereas FGFR4 wasfound to have a more limited distribution (Hughes, 1997). Genedeletion and mutation studies in mice have implicated FGFR1-3 in numerous developmental events, whereas FGFR4 seemsto play a more modest role in development (Goldfarb, 1996;Xu et al., 1999; Ornitz and Marie, 2002). It is possible that thisis due to the different sorting of the FGFRs. The recycling ofFGFR4 could prolong the signalling. It is therefore possiblethat FGFR4 is less suited for processes where rapiddownregulation is necessary. On the other hand, the recyclingof FGFR4 could provide a mechanism for gradient formationduring developmental processes. A simple model of gradientformation implies that morphogens dilute as they diffusebetween cells. Recent data, however, suggest that movement ofmorphogens could also occur by transcytosis whereendocytosed morphogens can be re-secreted and move forwardinto the target tissue (Entchev et al., 2000). FGFs couldtherefore, after binding and activation of FGFR4 in one cell,be recycled and activate neighbouring cells and then spreadthrough the tissue.

FGFRs have been found overexpressed or mutated toconstitutively active forms in numerous human cancers(Powers et al., 2000). FGFR1 is the most studied FGFR inhuman cancer and several reports closely link abnormalexpression of FGFR1 to cancer progression (Becker et al.,1992; Yamaguchi et al., 1994; Giri et al., 1999). But also anincreasing number of reports links overexpression of FGFR4with poor prognosis in several human tumour types (Jaakkolaet al., 1993; Facco et al., 1998; Yamada et al., 2002;Gowardhan et al., 2005). Several mechanisms are involved inthe attenuation of signalling from activated receptors. One ofthem is the degradation of the receptors in lysosomes. Accuratedownregulation of activated signalling receptors is importantto prevent increased signalling and enhanced cancer

progression (Bache et al., 2004). It is therefore possible thatelevated levels or constitutively active forms of recyclingreceptors could predispose cancer patients for accelerateddisease progression because the receptors are not efficientlydownregulated. A recent study of the four FGFRs in humanthyroid cancer indicates that FGFR4, in contrast to the otherFGFRs, may promote the progression of these tumours. Thus,FGFR4 was found to be strongly expressed in the moreaggressive tumour types and in the most rapidly proliferatingcells (St Bernard et al., 2004). A mechanism for FGFR4 toinduce cell proliferation in breast tumour cells throughregulation of cyclin D1 translation in cooperation with ErbB2has recently been proposed (Koziczak and Hynes, 2004;Koziczak et al., 2004).

The expression of ErbB2/HER2, a member of the EGFreceptor family, is frequently elevated in human cancers(Yarden and Sliwkowski, 2001). ErbB2 does not bind ligand,but instead acts as a heterodimerization partner for ligand-activated members of the EGF receptor family, thus amplifyingthe signalling. As FGFR4, ErbB2 is rapidly recycled uponendocytosis. Overexpression of ErbB2 potentiates EGFRsignalling by diverting EGFR from EGF-induceddownregulation (Worthylake et al., 1999). The ability of ErbB2to shunt ligand-activated receptors to recycling may explain itsoncogenic potential. It is possible that overexpression ofFGFR4 followed by possible heterodimerization could in asimilar way lead to impaired downregulation of the otherFGFRs, thereby increasing their oncogenic potential.

Several monoclonal antibodies that inhibit ErbB2-transformation in vitro and have antitumour properties in vivoare proposed to trigger surface downregulation and endocyticdegradation of ErbB2 (Klapper et al., 2000; Austin et al.,2004). Similar therapeutic strategies might be introduced toinhibit FGFR and particularly FGFR4 signalling in tumourswhere elevated levels of FGFR signalling are associated withpoor prognosis.

However, examination of constitutively activated derivativesof FGFR1, FGFR3 and FGFR4 in which a myristylation signalwas substituted for the extracellular and transmembranedomains, thereby targeting the kinase domain to the plasmamembrane, revealed that FGFR4 was much less transformingthan activated FGFR1 and FGFR3 (Hart et al., 2000). BecauseFGFR1 also exhibits higher signalling activity than FGFR4 ithas been suggested that FGFR1 is the most potent mutagenicmember of the FGFR family (Vainikka et al., 1994; Wang etal., 1994). Other mechanisms for attenuating signals maytherefore play a role to limit the signalling from the FGFR4.

The present work shows that endocyotosed FGF1 is sortedto recycling or degradation depending on the receptor type.Many growth factors can bind to more than one receptor, butin many cases the different roles of the separate receptors areunclear. The sorting of ligands and receptors to recycling ordegradation affects their half-lives and may thus regulate theirsignalling. Although the exact biological role of the differenttrafficking of the FGFRs remains to be elucidated, differentintracellular sorting of the ligand-receptor complexes is likelyto be important for their signalling properties.

E.M.H. is a pre-doctoral fellow and V.S. is a post-doctoral fellowof the Norwegian Cancer Society. This work has been supported bythe Research Council of Norway, the Novo Nordisk Foundation, the

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Blix Fund for the Promotion of Medical Research, the Rachel andOtto Kr. Bruuns Fund, Torsteds Fund and the Jahre Foundation.

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