The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor Jacky Bonaventure1,2, Linda Gibbs2, William C Horne3 and Roland Baron3 ´ Institut Curie, Universite Paris Sud, Orsay, France ˆ Department of Medical Genetics INSERM U393, Hopital Necker, Paris, France Department of Cell Biology and Orthopaedics, Yale University School of Medicine, New Haven, CT, USA Keywords Cbl; FGFR3; mutation; phosphorylation; ubiquitylation Correspondence J Bonaventure, Institut Curie, CNRS UMR ´ 146, Bat 110, Universite Paris Sud, 91400 Orsay, France Fax: +33 69 86 53 01 Tel: +33 69 86 71 80 E-mail: jacky.bonaventure@curie.u-psud.fr R Baron, Department of Cell Biology and Orthopaedics, Yale University School of Medicine, PO Box 208044, New Haven, CT 208044, USA Fax: +1 203 785 2744 Tel: +1 203 785 4150 E-mail: roland.baron@yale.edu (Received February 2007, revised 16 April 2007, accepted 18 April 2007) doi:10.1111/j.1742-4658.2007.05835.x Recurrent missense fibroblast growth factor receptor (FGFR3) mutations have been ascribed to skeletal dysplasias of variable severity including the lethal neonatal thanatophoric dysplasia types I (TDI) and II (TDII) To elucidate the role of activating mutations causing TDI on receptor trafficking and endocytosis, a series of four mutants located in different domains of the receptor were generated and transiently expressed The putatively elongated X807R receptor was identified as three isoforms The fully glycosylated mature isoform was constitutively but mildly phosphorylated Similarly, mutations affecting the extracellular domain (R248C and Y373C) induced moderate constitutive receptor phosphorylation By contrast, the K650M mutation affecting the tyrosine kinase (TK2) domain produced heavy phosphorylation of the nonglycosylated and mannose-rich isoforms that impaired receptor trafficking through the Golgi network This resulted in defective expression of the mature isoform at the cell surface Normal processing was rescued by tyrosine kinase inhibitor treatment Internalization of the R248C and Y373C mutant receptors, which form stable disulfide-bonded dimers at the cell surface was less efficient than the wild-type, whereas ubiquitylation was markedly increased but apparently independent of the E3 ubiquitin-ligase casitas B-lineage lymphoma (c-Cbl) Constitutive phosphorylation of c-Cbl by the K650M mutant appeared to be related to the intracellular retention of the receptor Therefore, although mutation K650M affecting the TK2 domain induces defective targeting of the overphosphorylated receptor, a different mechanism characterized by receptor retention at the plasma membrane, excessive ubiquitylation and reduced degradation results from mutations that affect the extracellular domain and the stop codon Fibroblast growth factor receptor (FGFR3) belongs to a family of four genes (FGFR1–4) encoding receptors with tyrosine kinase activity (RTK) These struc- turally related proteins exhibit an extracellular domain (ECD) composed of three immunoglobin-like domains, an acid box, a single transmembrane domain and a Abbreviations ACH, achondroplasia; BFA, brefeldin A; Cbl, casitas B-lineage lymphoma; ECD, extracellular domain; EGFR, epidermal growth factor receptor; endo H, endopeptidase H; ER, endoplasmic reticulum; FGF, fibroblast growth factor; FGFR3, fibroblast growth factor receptor 3; HRP, horseradish peroxidase; PDGFR, platelet-derived growth factor receptor; PDI, peptidyl disulfide isomerase; PNGase F, peptidyl N-glycosidase F; RTK, receptor tyrosine kinase; TDI, thanatophoric dysplasia type I; TK, tyrosine kinase 3078 FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS J Bonaventure et al split tyrosine kinase (TK) domain Binding of of the 22 fibroblast growth factor (FGF) ligands in the presence of cell-surface heparan sulfate proteoglycans acting as coreceptors, induces receptor dimerization and trans-autophosphorylation of key tyrosine residues in the cytoplasmic domain Phosphorylated residues serve as docking sites for the adaptor proteins and effectors that propagate FGFR signals via different signalling pathways resulting in the regulation of many cellular processes including proliferation, differentiation, migration and survival [1–4] Dominant mutations in three members of the FGFR family (FGFR1–3) have been shown to account for two groups of skeletal disorders, namely short-limb dwarfisms and craniosynostoses [5,6] Mutations in FGFR3 are mostly responsible for long-bone dysplasias including achondroplasia (ACH), the most common form of dwarfism in humans, the milder form hypochondroplasia and the neonatal lethal form thanatophoric dysplasia (TD) types I and II [7,8] Interestingly, whereas TDII is exclusively accounted for by a single recurrent K650E missense mutation in the TK2 domain, TDI has been ascribed to a series of mutations creating cysteine residues in the ECD (R248C, S249C, G370C, S371C, Y373C) and to base substitutions eliminating the termination codon (X807R ⁄ C ⁄ G ⁄ S ⁄ W) [9] Likewise, substitution of Lys650 by methionine (K650M) can give rise to TDI [10,11] or to a less severe phenotype called severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN) [12], whereas replacement of lysine by asparagine or glutamine (K650N ⁄ Q) is associated with hypochondroplasia [13] Based on several in vitro and in vivo studies, FGFR3 mutations have been assumed to induce constitutive activation of the receptor either via a ligand-independent process in TD [14] or by stabilizing ligand-induced dimers resulting in prolonged signalling at the cell surface in ACH [15,16] In recent years, numerous efforts have been devoted to elucidate how FGFR3 mutations of the highly conserved Lys650 lead to constitutive receptor phosphorylation and can produce three different phenotypes of increasing severity depending on the substituting amino acid [13,17–23] However, little attention has been paid to mutations creating unpaired cysteine residues in the ECD and the consequences of the stop codon mutation on receptor function remain unknown In addition, the mechanisms by which FGFR3 mutants are endocytosed and targeted for degradation to attenuate signalling are far from being elucidated Thorough analyses of other RTKs such as epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor (PDGFR) have convincingly shown that these recep- Variable phosphorylation of FGFR3 mutants in TDI tors become ubiquitylated through recruitment of the E3 ubiquitin ligase casitas B-lineage lymphoma (c-Cbl) [24–26] This adaptor protein binds to multiple sites in the intracellular domain of the EGF or PDGF receptors ensuring their monoubiquitylation rather than polyubiquitylation after ligand-induced activation [27,28] This allows receptor endocytosis and subsequent degradation in the lysosome [27,29] By contrast, no direct interaction between FGFR3 and c-Cbl [30] or FGFR1 and c-Cbl [31] has been detected by coimmunoprecipitation, even though constitutive phosphorylation of c-Cbl in COS-7 cells stably expressing the FGFR3 K650E mutant has been described [21] In this study, four FGFR3 mutations causing TDI and affecting the extracellular or intracellular domains of the receptor were generated and used for biochemical and immunocytochemical studies in transiently transfected cells Mutations creating cysteine residues or disrupting the termination codon had mild effects on receptor phosphorylation and glycosylation, whereas conversion of Lys650 into methionine induced strong constitutive phosphorylation of the native nonglycosylated form of the receptor Such hyperphosphorylation markedly hampered receptor glycosylation at the Golgi level resulting in reduced levels of fully glycosylated receptors at the cell surface of transfected cells Reversal of this situation following treatment with the FGFR tyrosine kinase inhibitor SU5402 indicated that hyperphosphorylation adversely affected trafficking of the mutant receptor through the Golgi system Endocytosis and ubiquitylation of the different TDI mutants were also investigated, as was the putative involvement of c-Cbl in this process Ubiquitylation of the R248C, Y373C and X807R mutant receptors was stronger than the wild-type and apparently independent of c-Cbl Constitutive phosphorylation of c-Cbl in cells transiently expressing the K650M mutant was shown to affect Tyr731 which lies outside the ubiquitin-conjugating enzyme-binding RING finger domain that is required for E3 ubiquitin ligase activity [25,26,32] Our results indicate that receptors are constitutively phosphorylated to variable extents and are differentially processed at the intracellular level depending on the domain in which the mutation arises and the level of phosphorylation Receptors with mutations in the ECD or stop codon are weakly phosphorylated, retained at the cell surface, and strongly ubiquitylated By contrast, the highly phosphorylated but moderately ubiquitylated K650M mutant is retained intracellularly and unlike other mutants induces constitutive phosphorylation of c-Cbl which, nonetheless, does not seem to directly regulate FGFR3 ubiquitylation FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3079 Variable phosphorylation of FGFR3 mutants in TDI J Bonaventure et al We first tested whether the different mutations causing TDI affected receptor biosynthesis and post-translational processing Twenty-four hours after transient transfection of 293-VnR cells with the wild-type, R248C and Y373C cDNAs, three isoforms with respective molecular masses of 130, 115 and 105 kDa were visible (Fig 1A,C) When cells were transfected for 48 h, the relative level of the 105 kDa isoform was slightly reduced (Fig 1A) Transient expression of the X807R mutation gave rise to three isoforms with higher molecular masses than the wild-type and other mutants, ranging from 144 to 119 kDa, in good agreement with the predicted 141 additional residues separating the regular stop codon from the next inframe stop codon (supplementary Fig S1) This additional domain apparently decreased the affinity of the anti-FGFR3 serum for the receptor, so that a higher amount of total protein had to be loaded onto the gel in order to obtain a signal equivalent to wild-type and Results TDI mutations differentially affect receptor processing A series of four mutants (R248C, Y373C, K650M and X807R) reproducing mutations identified in TDI patients and located in different domains of the receptor (Fig S1) was created by site-directed mutagenesis of the full-length human FGFR3 cDNA and subcloning into the pcDNA3.1 vector Based on the cDNA sequence of FGFR3 including the 5¢-UTR, the X807R mutation that eliminates the regular stop codon was expected to produce an elongated protein of 947 amino acids and containing a highly hydrophobic domain rich in cysteine [9] (Fig S1) An extensive search in databases failed to reveal significant homology of the additional 141 amino acid C-terminal tail with other proteins A B IB: FGFR3 WT Y373C K650M WT K650M WT K650M IP: FGFR3 IB: FGFR3 130 115 105 Hrs : 24 48 TCL 24 48 24 48 C PNGase: - - D IP: FGFR3 IB: FGFR3 130 115 105 WT X807R Y373C R248C IP: FGFR3 IB: FGFR3 160 144 129 119 PNGase: IB: FGFR3 (non reduced) (reduced) Dimer + X807R Endo H: E IP: FGFR3 + 105 - + - - + F kDa 250 IB: FGFR3 WT Y373C K650M K650N X807R 160 160 130 115 105 105 Y373C WT WT Y373C WT WT - + - + FGF9: - 105 TCL ATDC5 cells Fig Immunoblot analysis of different FGFR3 mutations causing TDI in transiently transfected 293-VnR and ATDC5 cells (A) 293-VnR cells were transfected for 24 or 48 h and total cell lysates (TCL) were immunoblotted with anti-FGFR3 serum (B) 293-VnR cells transfected with the wild-type or K650M mutant cDNAs were immunoprecipitated with an anti-FGFR3 serum, treated with PNGase for h and blotted with an anti-FGFR3 serum (C) 293-VnR cells were transfected with the wild-type or X807R, Y373C or R248C mutant cDNAs for 24 h then immunoprecipitated and immunoblotted with an anti-FGFR3 serum Because of the lower affinity of the antibody, the amount of total protein used for immunoprecipitation of the X807R mutant was three times that used for wild-type and other mutants (D) Lysates of 293-VnR cells transfected with the X807R mutant were immunoprecipitated with an anti-FGFR3 serum and the immune complexes were treated with endo H or PNGase as indicated prior to immunoblotting with an anti-FGFR3 serum (E) Immune complexes immunoprecipitated from lysates of 293-VnR cells transfected with the wild-type and Y373C mutant were separated using SDS ⁄ PAGE under nonreducing (left) or reducing (right) conditions and blotted with an anti-FGFR3 serum The upper arrow indicates the location of the receptor dimer Cells transfected with the wild-type cDNA were stimulated (or not) with 100 ngỈmL)1 FGF9 and heparin for 10 (F) ATDC5 cells were transiently transfected for 24 h with different mutants and cell lysates were analysed by immunoblot with an anti-FGFR3 serum of proteins separated on SDS ⁄ PAGE run under reducing conditions 3080 FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS J Bonaventure et al other mutants (Fig 1C) The 130 kDa isoform of the K650M mutant was only weakly and variably detected in immunoblots Scanning densitometry of the gel further indicated that the intensity of the 105 kDa band was greatly increased in this mutant at 24 h post transfection (31% of the total signal in K650M versus 10% in wild-type) Similar results were obtained when the same mutants were transiently transfected in chondrogenic ATDC5 cells (Fig 1F) In order to confirm that the 130 and 115 kDa bands (or 144 and 129 kDa bands in the X807R mutant) corresponded to differently glycosylated forms of the receptor, immunoprecipitated wild-type and mutant receptors were digested with peptidyl N-glycosidase F (PNGase), which completely eliminates glycosyl groups from N-glycosylated proteins, and endopeptidase H (endo H) which cleaves mannose residues from mannose-rich intermediates Both the 130 and 115 kDa (or 144 and 129 kDa) bands were converted into the nonglycosylated 105 (or 119) kDa isoform by PNGase treatment (Fig 1B,D) Endo H specifically eliminated the 115 (or 129) kDa band in the wild-type and mutant receptors (Fig 1D and not shown), indicating that this band represented a partially processed mannose-rich form of the receptor To verify that mutations creating cysteine residues in the ECD of the receptor induced formation of disulfide-bonded dimers, lysates from 293-VnR cells transfected with the Y373C mutant were immunoprecipitated with an anti-FGFR3 serum and separated by electrophoresis under nonreducing and reducing conditions The Y373C mutant, in the absence of ligand, formed dimeric receptors (260 kDa) that disappeared upon dithiothreitol treatment As expected, no dimer was visible with the wild-type receptor (Fig 1E) No dimer was detected in cells transfected with the X807R mutant (data not shown) The degree of constitutive phosphorylation of the mutant receptor is mutation specific Because several FGFR3 mutations have been reported to variably induce constitutive phosphorylation of the receptor [13,20,33], the extent of receptor phosphorylation and the relationship with glycosylation in 293VnR cells was assessed by immunoprecipitation of the receptor and immunoblotting with an anti-phosphotyrosine serum Both the R248C and Y373C mutants showed moderate phosphorylation of the fully glycosylated isoform (130 kDa) in the absence of ligand, whereas FGF was required to induce phosphorylation of the wild-type receptor (Fig 2A) By contrast, the 105 kDa nonglycosylated isoform of the K650M Variable phosphorylation of FGFR3 mutants in TDI mutant, and to a lesser extent the 115 kDa mannoserich intermediate, were heavily phosphorylated 24 h post transfection, whereas the 130 kDa band was not detectably phosphorylated (Fig 2B) The identity of the phosphorylated bands was confirmed by PNGase treatment of the immunoprecipitated K650M receptor (Fig 2D) Forty-eight hours after transfection, phosphorylation of the K650M receptor was significantly reduced, but the 105 kDa band remained preferentially phosphorylated (Fig 2B) Finally, the X807R mutant showed mild constitutive phosphorylation of the 144 kDa mature isoform (Fig 2C) indicating that this mutant behaved similarly to receptors with mutations in the ECD Immunofluorescent staining of 293-VnR and ATDC5 cells expressing the Y373C mutant with antiFGFR3 and anti-phosphotyrosine sera showed both intracellular and cell-surface phosphotyrosine staining (Figs 2Eb,c and supplementary Fig S2A) A similar pattern was observed with the FGF9-activated wildtype (Fig 2Ed) and the R248C and X807R mutants (not shown), whereas both 293-VnR and ATDC5 cells expressing the K650M mutant had a round morphology and exhibited strong phosphotyrosine signal in the cytoplasm with no detectable cell surface staining (Figs 2Ee,f and supplementary Fig S2A) These results were further supported by labelling the plasma membrane with fluoresceine-conjugated cholera toxin and an anti-FGFR3 serum Marked colocalization of cholera toxin with wild-type FGFR3 was observed, whereas the K650M mutant showed very little overlap (not shown) Subcellular distribution of wild-type and mutant FGFR3 molecules To determine more precisely the subcellular localization of the mutant receptors, cells were stained with anti-(peptidyl disulfide isomerase) (PDI) and antiGM130, markers of the endoplasmic reticulum (ER) and Golgi system, respectively Costaining with FGFR3 and PDI showed only partial colocalization of the two proteins in cells transfected with the Y373C, R248C and X807R mutants (Fig 2Eh,j and not shown) The K650M mutant was much more colocalized with PDI than the other mutants (Fig 2Ei) suggesting that most of the receptor was present in the ER Costaining with calnexin (another marker of the ER) and Ptyr antibodies gave similar results (not shown) Colocalization of FGFR3 and the cis-Golgi marker GM130 was mostly visible in cells expressing the wild-type and Y373C mutant and to a lesser extent in those expressing the X807R mutant (supplementary FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3081 Variable phosphorylation of FGFR3 mutants in TDI J Bonaventure et al Time (hrs) : 24 B A FGF9: C 105 WT Y373C R248C WT + 130 115 105 K650M K650M WT WT 160 IP FGFR3 IB FGFR3 X807R 144 129 119 D PNGase: - a c b WT Y373C e d 115 105 WT+FGF h i K650M Y373C WT Y373C K650M K650M g f + IP FGFR3 IB: Ptyr IP FGFR3 IB: FGFR3 Ptyr E kDa 115 105 IP FGFR3 IB FGFR3 105 f 24 48 IP FGFR3 IB: Ptyr kDa 160 IP FGFR3 IB: Ptyr 48 j K650M X807R Fig FGFR3 mutations causing TDI induce variable constitutive phosphorylation of the receptor, which partially colocalizes with the ER marker PDI (A) Constitutive phosphorylation in the absence of ligand of the Y373C and R248C FGFR3 mutants transiently expressed in 293-VnR cells for 24 h Stimulation of the wild-type receptor with 100 ngỈmL)1 FGF9 and heparin for 10 induced phosphorylation of the 130 kDa isoform (B) Constitutive phosphorylation of the K650M mutant 24 or 48 h after transfection of 293-VnR cells After 24 h, both the 105 and 115 kDa isoforms were heavily phosphorylated in the absence of ligand Phosphorylation decreased after 48 h (C) Constitutive phosphorylation of the X807R mutant in 293-VnR cells transfected for 24 h Protein lysate was immunoprecipitated with an anti-FGFR3 serum, then immunoblotted with anti-FGFR3 (left) and anti-phosphotyrosine (right) sera (D) PNGase treatment converts the 115 kDa phosphorylated isoform of the K650M mutant to the 105 kDa isoform (E) Immunocytochemical staining of wild-type and TDI-causing FGFR3 mutants with anti-FGFR3 (green) and anti-phosphotyrosine (P-Tyr, red) sera in transiently transfected 293-VnR (a,b,d,e) and ATDC5 (c,f) cells (g–j) Immunostaining of the wild-type and three TDI FGFR3 mutants with anti-FGFR3 (green) and anti-PDI (red) sera in transiently transfected 293-VnR cells Magnification: 100· In a–f, nuclei were counterstained with 4¢,6-diamidino-2-phenylindole (blue) FGF9 was added at 100 ngỈmL)1 for 10 in (d) Fig S2B) There was little colocalization of the K650M mutant and GM130, indicating that transfer of this receptor from the ER to the Golgi compartment was less efficient than that of the wild-type receptor and other mutants Immunostaining of K650M-transfected cells with GM130 and FGFR3 following fragmentation of the Golgi network into ministacks by nocadazole treatment showed colocalization of the two proteins in scattered puncta (Fig 3Ba), confirming that some K650M FGFR3 molecules were present in the cis-Golgi By contrast, very little overlap was seen between K650M FGFR3 and the trans-Golgi marker p230 (Fig 3Bb) suggesting that K650M mutant molecules were inefficiently transferred from the cis- to the trans-Golgi compartments Effect of brefeldin A treatment on the processing of wild-type and mutant FGFR3 molecules To further characterize trafficking of the wild-type and mutant FGFR3 molecules through the Golgi appa3082 ratus, cells were treated for h with brefeldin A (BFA), a molecule that reversibly disrupts Golgi assembly by inhibiting anterograde transport from the ER to the Golgi [34] Western blot analysis with an anti-FGFR3 serum of BFA-treated cells expressing the wild-type or Y373C mutant revealed a significant decrease in the 130 kDa fully glycosylated isoform together with an increase in the 115 kDa isoform (Fig 3A, left), indicating that glycosylation that normally occurs within the Golgi system was prevented by blocking transport from the ER to the Golgi BFA had no effect on the relative lack of the 130 kDa isoform of the K650M mutant Endo H digestion of the immunoprecipitated wild-type and Y373C receptors after BFA treatment revealed a partial conversion of the 115 kDa mannose-rich isoform into an endo H-resistant intermediate form (Fig 3A, left) This was in keeping with previous reports that BFA treatment induces Golgi enzymes (mannosidase II and thiamine pyrophosphatase) to redistribute into the ER, leading to partially processed endo H-resistant glycosylated proteins [34,35] FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS J Bonaventure et al Variable phosphorylation of FGFR3 mutants in TDI A WT Y373C K650M WT Y373C IP: FGFR3 IB: FGFR3 K650M kDa - 130 - 115 - 105 Endo H BFA: B c IP: FGFR3 IB: Ptyr d e K650M mutant K650M f + Nocodazole a b GM130 + FGFR3 (merge) p230 + FGFR3 (merge) BFA GM130 + FGFR3 (merge) BFA p230 + FGFR3 (merge) Fig Effect of BFA and nocodazole treatment on the processing of wild-type and mutant FGFR3 (A) 293-VnR cells transiently transfected with wild-type or mutant FGFR3 cDNAs as indicated, were treated or not for h with BFA Total cell lysates were immunoprecipitated with an anti-FGFR3 serum and treated or not with endo H, then separated by SDS ⁄ PAGE under reducing conditions and immunoblotted with anti-FGFR3 (left) or anti-phosphotyrosine (right) sera The phosphorylated 115 kDa isoform was partially resistant to endo H in both the presence and absence of BFA (B) Immunostaining of 293-VnR cells transfected with the K650M mutant and treated or not with nocodazole or BFA (a,b) Cells treated with nocadazole for h before staining with antibodies; (c,d) nontreated cells; (e,f) cells treated with BFA for h Cells were stained with anti-GM130 (red) and anti-FGFR3 (green) sera or with anti-p230 (red) and anti-FGFR3 (green) sera Nuclei were counterstained with 4¢,6-diamidino-2-phenylindole Magnification: 40· Unexpectedly, the phosphorylated 115 kDa band of the K650M mutant was partially resistant to endo H digestion in both untreated and BFA-treated cells (Fig 3A, right) This suggests that some hyperphosphorylated K650M molecules undergo partial processing at the cis ⁄ medial-Golgi level to become endo H resistant without being fully glycosylated in the transGolgi compartment, and are either retained in the cis ⁄ medial-Golgi compartment or sent back to the ER through retrograde transport Consistent with this possibility, colocalization of FGFR3 K650M with the cis-Golgi marker GM130 was observed in BFA-treated cells (Fig 3Be), whereas little overlap was detected with the trans-Golgi marker p230 (Fig 3Bf) Cell-surface expression and endocytosis of wild-type and mutant receptors To investigate whether TDI FGFR3 mutations affected cell-membrane localization of the receptor, total 293VnR cell-surface proteins were first labelled with NHSbiotin, immunoprecipitated with an anti-FGFR3 serum then separated on nonreducing or reducing gels and blotted with avidin D (Fig 4A) Although the wildtype receptor showed a single 130-kDa band corresponding to the mature monomer, both the R248C and Y373C mutants showed the presence of a 260-kDa dimer in addition to the monomer The K650M mutant gave only a faint signal with avidin D, consistent with its intracellular retention We then examined endocytosis of the wild-type and mutant receptors Cell-surface proteins were labelled by incubating cells with cleavable sulfo-NHS-S-S-biotin for 30 on ice [36] Cells were then warmed to 37 °C for increasing times to allow receptor internalization, and the biotin remaining on the cell surface was stripped by washing with glutathione Biotinylated cells were lysed, the receptors were immunoprecipitated, and the immune complexes were blotted with avidin D to reveal endocytosed molecules As expected, no biotinylated FGFR3 molecules (wildtype or mutant) were detected when cells were kept at °C (Fig 4C and not shown) A substantial amount of the biotinylated receptor (130 kDa) was found after h in the absence of ligand, indicating that wild-type FGFR3 is constitutively endocytosed The signal reached a peak after h then decreased progressively to become undetectable after h (Fig 4B) The Y373C mutant gave two bands corresponding to the mature 130 kDa monomer and the disulfide-bonded dimer Internalization was slower than the wild-type, as attested by the delay in reaching the maximum amount of protected biotinylated receptor and the presence of FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3083 Variable phosphorylation of FGFR3 mutants in TDI J Bonaventure et al A kDa 250 dimer IP: FGFR3 IB: avidin D 160 130 dimer 250 160 130 115 105 IP: FGFR3 IB: FGFR3 105 (non reduced) WT Y373C R248C WT B 130 WT K650M (reduced) C Y373C dimer IP: FGFR3 IB: Avidin D WT K650M IP: FGFR3 IB: Avidin D dimer IP: FGFR3 IB: FGFR3 Time (hours): IP: FGFR3 IB: FGFR3 Fig Cell-surface expression and endocytosis of wild-type and mutant FGF receptors (A) Cells were surface biotinylated (NHS-biotin) for 30 at °C, then washed extensively with 15 mM glycine in NaCl ⁄ Pi Total cell lysates were immunoprecipitated with an anti-FGFR3 serum Immunoprecipitates were separated on nonreducing gels to visualize dimers (left) or under reducing conditions (right) Blots were sequentially probed with HRP-conjugated avidin D and anti-FGFR3 serum (B) Endocytosis of wild-type and the Y373C mutant receptor was analysed using cleavable biotin Cells were treated with sulfo-NHS-SS-biotin for 30 at °C, then reincubated with serum-supplemented DMEM for increasing times at 37 °C to allow endocytosis of the receptor At the indicated times, incubation was stopped, remaining cell surface biotin was cleaved and total cell lysates were immunoprecipitated with an anti-FGFR3 serum Immunoprecipitates were separated on nonreducing acrylamide gels Blots were sequentially probed with HRP-conjugated avidin D and anti-FGFR3 serum (C) Endocytosis of wild-type and the K650M mutant receptor was analysed as in (B) A faint biotinylated band is visible with the K650M mutant after and h significant amounts of biotinylated receptor after h Similar results were obtained with the R248C mutant (not shown) Much less biotinylated K650M mutant was detected at any time point because of the reduced amount of mature receptor at the cell surface (Fig 4C) Blocking constitutive receptor phosphorylation restores normal maturation and distribution of the K650M mutant The kinase activity of FGFRs, including FGFR3 [37,38], is inhibited by SU5402, which binds to the kinases’ ATP-binding site [39] We therefore determined whether SU5402 prevented constitutive phosphorylation of FGFR3 mutants, and if so, whether inhibiting receptor phosphorylation altered trafficking of the mutant receptors between different membrane compartments Cells expressing the Y373C or K650M mutants were treated with different doses of SU5402 for increasing periods A 25 lm concentration for 16 h was sufficient to totally abolish receptor phosphorylation in cells expressing the Y373C mutant (not shown) Phosphorylation of the K650M mutant, although dramatically reduced, was not completely abrogated 3084 (Fig 5A,B) Increased inhibitor concentrations had no further effect on phosphorylation but affected cell viability (not shown) Immunoblot analysis of the wild-type and K650M mutant receptors following SU5402 treatment and immunoprecipitation with an anti-FGFR3 serum showed the presence of the mature 130 kDa isoform both in the wild-type and mutant (Fig 5A), indicating that inhibiting the constitutive phosphorylation restored full maturation of the K650M receptor to a significant degree To firmly establish that SU5402 allowed the K650M receptor to be transported to the plasma membrane and endocytosed, sulfobiotinylation of the mutant receptor with cleavable sulfobiotin was performed after SU5402 treatment Large amounts of endocytosed receptors were detected after 2–3 h confirming the ability of the mutant receptor to traffic efficiently to the cell surface and be internalized with a kinetic resembling that of the wild-type receptor when hyperphosphorylation was prevented (Fig 5B) Excessive ubiquitylation of mutant receptors Internalized Rtk are usually committed to degradation through ubiquitylation of lysine residues We therefore FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS J Bonaventure et al Variable phosphorylation of FGFR3 mutants in TDI A WT K650M WT K650M IP: FGFR3 IB: FGFR3 IP: FGFR3 IB: Ptyr 105 SU5402: B phosphorylated TDI mutant receptors (R248C, Y373C and X807R) were higher than the wild-type By contrast, the heavily phosphorylated K650M mutant was less ubiquitylated than the wild-type, consistent with its poor expression at the cell surface kDa 130 115 105 SU5402: IP FGFR3 IB Avidin D - - + - + + c-Cbl does not mediate the ubiquitylation of FGFR3, but it is constitutively phosphorylated by the K650M mutant + - + - + 130 130 115 105 IP FGFR3 IB FGFR3 Time (hrs): K650M 1 2 SU 5402 Biotin 16 hrs 30’ 3 DMEM 0-3 hrs (37°C) Fig Effect of the tyrosine kinase inhibitor SU5402 on phosphorylation, processing and internalization of the K650M mutant (A) Immunoblot analysis of the K650M mutant before and after SU5402 treatment Transfected cells were immunoprecipitated with an antiFGFR3 serum then blotted with anti-phosphotyrosine or anti-FGFR3 sera (B) SU5402 treatment increases the surface expression of the K650M mutant Transfected cells were treated or not with SU5402, followed by sulfobiotinylation of cell-surface proteins and re-incubation in serum-supplemented DMEM for the indicated times After immunoprecipitation with anti-FGFR3 serum, proteins were separated on a nonreducing gel then blotted and visualized by hybridization with HRP-conjugated avidin D and anti-FGFR3 serum studied ubiquitylation of wild-type and mutant receptors by cotransfecting cells with wild-type or mutant FGFR3 and HA-tagged ubiquitin cDNAs Ubiquitylated receptors identified by blotting with anti-ubiquitin sera appeared as a smear of bands with a lower mobility than the nonubiquitylated receptors The Y373C mutant gave a stronger signal than the wild-type and the intensity was increased slightly in both cases by treatment with the proteasome inhibitor MG132 (Fig 6A) indicating that partial degradation of the receptor could occur at the proteasome level We also analysed ubiquitylation of the Y373C and K650M mutants both in the presence and absence of chloroquine, a lysosomal inhibitor Unlike Y373C, the K650M mutant was less ubiquitylated than the wildtype receptor and the amounts of ubiquitylated wildtype and mutant FGFR3 were slightly increased by chloroquine treatment (Fig 6B), suggesting that the lysosomal pathway may also participate to their degradation The X807R mutant also exhibited an increased ubiquitylation compared with wild-type (not shown) confirming that ubiquitylation levels of the weakly c-Cbl is an adaptor protein and an E3-ubiquitin ligase that is phosphorylated downstream of several growth factor receptors and contributes to their downregulation by mediating their ubiquitylation [40], suggesting that it may be involved in the ubiquitylation of FGFR3 and ⁄ or be phosphorylated by FGFR3 in a basal or ligand-dependent process [21] We therefore first examined whether c-Cbl might mediate the ubiquitylation of the TDI FGFR3 mutants Overexpression of c-Cbl with wild-type (stimulated by FGF9) or Y373C mutant FGFR3 did not significantly affect receptor ubiquitylation (Fig 6C), and the ubiquitinylation of wild-type, Y373C and K650M FGFR3 mutants was not significantly different when either c-Cbl or the oncogenic mutant 70Z-Cbl, which lacks E3-ligase activity and dominant-negatively inhibits ligand-induced EGFR ubiquitylation [25], were coexpressed with the receptors (Fig 6D) Consistent with the absence of an effect of c-Cbl or 70Z-Cbl on the ubiquitylation of FGFR3 receptors, myc-tagged c-Cbl failed to coimmunoprecipitate with wild-type FGFR3 (treated or not by FGF9) and FGFR3 mutants (supplementary Fig S3C and not shown), indicating that in our cell system, c-Cbl apparently does not directly interact with wildtype FGFR3 or the TDI FGFR3 mutants To determine if c-Cbl is phosphorylated downstream of wild-type or mutated FGFR3, we examined lysates from 293-VnR cells coexpressing c-Cbl and wild-type or mutant FGFR3 using immunoblotting or immunofluorescence analysis with an anti-phosphotyrosine serum or an antibody against phospho-Tyr731, a c-Cbl tyrosine residue that is phosphorylated downstream of several receptor and nonreceptor TKs to form a binding site for phosphatidylinositol 3-kinase No phosphorylation of c-Cbl was seen in cells that expressed the wild-type receptor, the Y373C or the X807R mutant receptors (Figs 7A,B and supplementary Fig S3A,B) Stimulation of the wild-type receptor with FGF9 failed to induce c-Cbl phosphorylation (supplementary Fig S3B) By contrast, marked c-Cbl tyrosine phosphorylation occurred in cells expressing the K650M mutant (Figs 7A and supplementary Fig S3A) Tyrosine 731 was one of the residues phosphorylated FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3085 Variable phosphorylation of FGFR3 mutants in TDI J Bonaventure et al B WT Y373C WT Y373C IP : FGFR3 IB : Ubiquitin WT Y37 3C K65 0M WT Y37 3C K65 0M A kDa 250 kDa IP : FGFR3 IB : Ubiquitin 250 160 160 IP : FGFR3 IB : FGFR3 160 160 IP : FGFR3 IB : FGFR3 105 105 Chloroquine: + IP : FGFR3 IB : Ubiquitin WT FGFR3: WT WT C - - + + + + D kDa 250 160 FGFR3: IP : FGFR3 IB : Ubiquitin WT WT Y37 3C Y37 3C K65 0M K65 0M - Y37 3C Y37 3C MG132: - kDa 250 160 IP : FGFR3 IB : FGFR3 c-Cbl: c-Cbl70Z: c-Cbl: FGF9: IB: c-Cbl TCL Fig Effect of proteasome and lysosome inhibitors on ubiquitylation of wild-type and mutant FGFR3 (A) Ubiquitylation of wild-type and Y373C FGFR3 in the absence or presence of the proteasome inhibitor MG132 (50 lM for h) 293-VnR cells were cotransfected with HA-tagged ubiquitin and wild-type FGFR3 or FGFR3 Y373C Protein lysates were immunoprecipitated with an anti-FGFR3 serum and sequentially blotted with anti-ubiquitin and anti-FGFR3 sera (B) Ubiquitylation of wild-type, Y373C and K650M FGFR3 in the absence and presence of the lysosomal inhibitor chloroquine (500 lM for h) Cells transfected with the indicated cDNAs were treated with chloroquine as indicated Lysates were immunoprecipitated and processed for immunoblotting with anti-ubiquitin and anti-FGFR3 sera (C) Ubiquitylation of the wild-type receptor is increased by FGF9 treatment but cotransfection of c-Cbl with wild-type or FGFR3 Y373C does not affect ubiquitylation of the receptor Transfected cells were exposed to FGF9 (50 ngỈmL)1) and heparin (1 lgỈmL)1) for h Cell lysates were immunoprecipitated with an anti-FGFR3 serum then immunoblotted with anti-ubiquitin and anti-FGFR3 sera (D) Disabling the c-Cbl ubiquitylating activity does not affect the ubiquitylation of the wild-type, Y373C and K650M mutant receptors Total cell lysates (TCL) of 293-VnR cells cotransfected with the wild-type, Y373C or K650M mutant receptors and c-Cbl or 70Z-Cbl were either immunoblotted with an anti-(c-Cbl) serum or immunoprecipitated with an anti-FGFR3 serum followed by blotting with an anti-ubiquitin serum in the K650M-expressing cells (Figs 7B and supplementary Fig S3A, left) Cbl phosphorylation in the K650M-expressing cells was not detectably affected by deleting (70Z-Cbl) or mutating (c-CblY371F) Tyr371 (Fig 7A,C), whose phosphorylation is required for ubiquitylation [32,41] In fact, phosphorylation of 70ZCbl appeared slightly higher than the wild-type c-Cbl This suggests either that multiple tyrosines in addition to Tyr371 are phosphorylated downstream of FGFR3 K650M or that Tyr371 is not a major site of phosphorylation Discussion In this study, the effects of TDI-inducing missense mutations on receptor processing, endocytosis and ubiquitylation were investigated by using transiently transfected 293-VnR and ATDC5 cells Although primary cultured chondrocytes from affected patients 3086 would be representative of a more physiological model, the difficulty of efficiently transfecting human chondrocytes and maintaining their differentiated phenotype prompted us to use established cell lines, keeping in mind that overexpression of the receptor in transiently transfected cells may affect their physiological properties We first demonstrated that replacement of the stop codon by an arginine residue resulted in a stable elongated receptor, which appeared on western blotting as a combination of three bands including the nonglycosylated, mannose-rich and fully glycosylated isoforms, indicating that this elongated receptor underwent the same maturation process as the Y373C and R248C mutants However, under nonreducing conditions, these two mutants with an additional cysteine in the ECD gave rise to a disulfide-bonded mutant dimer, thus confirming constitutive activation of the receptor [14] Consistent with previous studies [13,17,20,23], we found that substitution of Lys650 by methionine FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS J Bonaventure et al Variable phosphorylation of FGFR3 mutants in TDI - WT K650M FGFR3: IP: myc IB: Ptyr FGFR3: Cbl + + K650M kDa - - IB: Cbl 120 TCL + C IB: FGFR3: Phospho-CblY731 120 IP : Myc 160 Cbl 105 c-Cbl: + 105 - + - W T Y3 73 C K6 50 M B WT Phospho IB: FGFR3 -Cbl IP: myc IB: Cbl c-Cbl: c-CblY371F: - Y3 73 C K6 50M FGFR3: WT A + IB: Phospho-Cbl Y731 IP: Myc 160 105 + FGFR3 Cbl c-Cbl70Z: + + + TCL Fig The FGFR3 K650M mutant phosphorylates the adaptor protein c-Cbl (A) 293-VnR cells were cotransfected with wild-type or K650M FGFR3 and myc–tagged c-Cbl or c-CblY371F constructs Aliquots of total cell lysates (TCL) were used for western blotting with anti-FGFR3 and anti-Cbl sera Cell lysates were also immunoprecipitated with anti-myc sera, then immunoblotted with anti-phosphotyrosine (P-Tyr) and anti-Cbl sera (B) Western blot analysis of c-Cbl phosphorylation in 293-VnR cells transiently cotransfected with myc-tagged c-Cbl and wildtype or mutant FGFR3 cDNAs Immunoprecipitation of c-Cbl with an anti-myc serum was followed by immunoblotting with an antibody specific for phosphorylated Cbl Tyr731 or an anti-Cbl serum Total cell lysates (TCL) were immunoblotted with an anti-FGFR3 antibody (C) Cells were cotransfected with 70Z-Cbl (a mutant lacking 17 amino acids in the linker and RING finger domain of c-Cbl) and wild-type or mutant FGFR3 cDNAs as indicated, then immunoprecipitated and blotted as in (B) resulted in a different electrophoretic pattern characterized by a variable but marked reduction in the fully glycosylated isoform and a significant increase in the nonglycosylated and partially glycosylated isoforms This defective maturation of the receptor resulted in inefficient targeting to the plasma membrane and strong constitutive tyrosine phosphorylation of the nonglycosylated isoform Similar observations have been reported previously in PC12 cells expressing K650E and K650M chimeric receptors [17] Inhibition of receptor phosphorylation with SU5402 restored proper receptor maturation and trafficking to the cell surface, suggesting that intracellular retention was a direct consequence of receptor hyperphosphorylation Support for this hypothesis is provided by the report that eliminating constitutive mouse Fgfr3 phosphorylation by mutating the mechanistically critical Tyr718 in the Fgfr3 activation loop restores normal Fgfr3 receptor maturation [20] However, we cannot exclude that abnormal constitutive phosphorylation of proteins involved in the trafficking of the receptor, including c-Cbl, could account for its intracellular retention By contrast, TDI mutations in the ECD or disruption of the termination codon induced a much lower level of phosphorylation of only the fully glycosylated isoform, which did not hamper its maturation, suggesting that factors other than constitutive FGFR3 autophosphorylation are involved in the severity of mutant-associated skeletal disorders It is noteworthy that tyrosine phosphorylation of at least four members of the RTK family (e.g Kit, PDGFRb, Ros and FLT-3) has been recently reported to lead to defective expression of the mature receptors at the cell surface [42] Although mechanisms regulating maturation arrest of phosphorylated receptors have not been clearly elucidated, our coimmunolocalization studies pointed to a role for components of the ER–Golgi vesicle transport Through the use of markers for the ER (PDI) and the Golgi apparatus (GM130, p230), the phosphorylated isoforms of the K650M mutant were identified in both the ER and cisGolgi compartments but were hardly detectable in the trans-Golgi These observations differ from those of Lievens et al [20] who concluded that mouse mutant K644E ⁄ M molecules were trapped in the ER Disrupting the Golgi apparatus with BFA or nocodazole provided evidence that at least some of the mutant receptors were transported to the Golgi Nocodazole induces reversible scattering of the juxtanuclear Golgi to peripheral sites via microtubule depolymerization FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3087 Variable phosphorylation of FGFR3 mutants in TDI J Bonaventure et al [43] Colocalization of mutant K650M molecules with the Golgi marker GM130 in mini-stacks dispersed throughout the cytosol indicated that these molecules had reached the cis ⁄ medial-Golgi compartment This conclusion was further supported by the demonstration that mannose-rich K650M receptors showed partial resistance to endo H treatment in the absence as well as in the presence of BFA, whereas other FGFR3 mutants exhibited resistance to endo-H digestion only after BFA treatment Our observation is consistent with the previous demonstration that cis-Golgi oligosaccharide-modifying enzymes (mannosidase II and thiamine pyrophosphatase) undergo retrograde transport to the ER after BFA treatment [35] We propose that in the absence of BFA, some heavily phosphorylated K650M molecules were able to reach the cis ⁄ medial-Golgi compartment where they were partially processed into endo H-resistant molecules However, they failed to be efficiently routed to the trans-Golgi network, as documented by the poor colocalization with p230 These molecules were finally recycled to the ER through retrograde transport in a manner similar to Golgi-resident glycosylation enzymes involved in the modification of transiting proteins [43,44] Direct evidence of defective processing and trafficking of the K650M mutant was provided by labelling wild-type and mutant receptors with membrane-impermeant NHS-biotin Reduced amounts of the biotinylated K650M mutant receptor were found, whereas the Y373C and R248C mutants were biotinylated at levels similar to the wild-type and formed stable dimers, thus confirming that disulfide-bonded receptors were properly processed and expressed at the cell surface Whether disulfide bonding between two mutant receptors occurred intracellularly or at the plasma membrane remains to be elucidated Analysis of receptor endocytosis through the use of cleavable biotin indicated that internalization of disulfide-bonded mutant receptors was slower than the wild-type A small amount of the biotinylated K650M mutant was detected, in keeping with its defective expression at the cell surface Treatment with SU5402 was able to at least partially restore trafficking of the K650M mutant receptor to the cell surface and its subsequent endocytosis Retention of the disulfide-bonded dimers at the cell surface was indicative of defective receptor internalization, allowing ligand-independent prolonged signalling to target molecules Mechanisms that control receptor endocytosis are multiple and complex [45] Ubiquitylation is considered one of the critical signals for endocytosis and degradation in the lysosome or the proteasome [27,46] Consistent with data from Monsonego-Ornan et al [30] on 3088 the G380R ACH mutant, ubiquitylation of the TDI mutants (R248C, Y373C and X807R) was found to be higher than wild-type, but these results differed from those of Cho et al [21] who reported reduced ubiquitylation of the ACH mutant in stably transfected cells Discrepancies between these studies may be due to the two different cell types (HEK293 versus COS-7 cells) and the use of retroviruses for stable transfection of cDNA constructs versus transient transfection of plasmids Polyubiquitylation is responsible for the internalization and proteasomal degradation of several plasma membrane proteins [46], but monoubiquitylation has been recently identified as the main mechanism regulating RTK endocytosis and degradation [27,28] and is associated in yeast with proteasome-independent functions including protein trafficking [47] Although it is not known whether FGFR3 mutants are monoubiquitylated, polyubiquitylated or both, it is tempting to speculate that highly ubiquitylated R248C, Y373C and X807R receptors could be preferentially monoubiquitylated on their 26 lysine residues lying in the intracellular domain [48] (supplementary Fig S1) and transferred to early endosomes From this compartment, part of the ubiquitylated mutant molecules could be sorted for degradation with a lesser efficiency than moderately ubiquitylated wild-type receptors, whereas a higher number of mutant molecules than wild-type would be recycled back to the plasma membrane The E3-ubiquitin ligase c-Cbl is directly involved in the ubiquitylation of several RTKs [24–26,32] and may participate in the downregulation of FGFR1 via an indirect interaction with the phosphorylated docking protein FRS2a [3,31]; but definitive evidence that c-Cbl is responsible for ubiquitylation of FGFR3 is still missing We conclude that c-Cbl does not play a key role in the ubiquitylation process of TDI FGFR3 mutants in our cell system because: (a) the extent of ubiquitylation of wild-type and TDI FGFR3 mutants was similarly unaffected by cotransfecting c-Cbl or the dominant-negative ubiquitylation-deficient 70Z-Cbl (Fig 6C,D); and (b) c-Cbl failed to coimmunoprecipitate with wild-type and TDI FGFR3 mutants, consistent with previous observations on ACH and TDII mutants [30] However, the possible involvement of the adaptor proteins FRS2 and Grb2 in the ubiquitinylation process cannot be excluded [31,40] Alternatively, other E3 ubiquitin ligases such as the von Hippel–Lindau protein, which regulates surface localization of FGFR1 [49], might be involved in FGFR3 ubiquitylation Phosphorylation of Tyr731, one of several phosphorylated tyrosine residues located in the C-terminal half of c-Cbl, most likely resulted from intracellular FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS J Bonaventure et al retention of the K650M FGFR3 mutant, even though it did not involve a direct interaction between the two proteins Because several Src-like kinases including Src, Fyn and Yes have been shown to phosphorylate c-Cbl on Tyr731 [50–52], we hypothesized that c-Cbl phosphorylation would be mediated via a tripartite complex involving K650M FGFR3 and a Src-like kinase The observation that c-Cbl was able to interact with FGFR2 and Fyn or Lyn in osteoblastic cells [53] and the demonstration, using a phosphoproteomic approach, that FGFR1, when phosphorylated, induced phosphorylation of both Cbl-b and Fyn [54], are consistent with this hypothesis Hence, unlike other TDI mutants, the K650M mutant could elicit signalling via an alternative internal pathway involving c-Cbl and a Src-like kinase Taken together, the data reported here provide evidence that TDI is caused by mutations affecting the receptor in at least two different ways Conversion of Lys650 into methionine in the TK2 domain induces hyperphosphorylation and marked intracellular retention of the mutant receptor leading to phosphorylation of target signalling molecules including c-Cbl By contrast, mutations creating cysteine residues in the ECD or elongating the receptor result in delayed endocytosis, excessive ubiquitylation and reduced degradation of the mutant proteins, but have lower impact on FGFR3 phosphorylation Experimental procedures DNA constructs and plasmids Full-length wild-type human FGFR3 cDNA cloned into pLNCX was kindly provided by M Hayman (State University, New York, NY) and subcloned into pBSII Two different strategies were used to obtain point mutations in different subdomains of the receptor Total RNA extracted from cultured cells of TDI patients carrying the R248C or Y373C mutations were reverse transcribed with two different set of primers (supplementary Table S1) RT-PCR products of the mutant allele were cloned into TOPO TA cloning vector (Invitrogen, Carlsbad, CA) then digested with RsrII and PmlI (for R248C) or with PmlI and MluI (for Y373C) DNA fragments were subcloned into the FGFR3 pBSII vector at the RsrII ⁄ PmlI sites or PmlI ⁄ MluI sites Wild-type and mutant FGFR3 cDNAs were then transferred from pBSII to pcDNA3.1 at the HindIII ⁄ EcoRI restriction sites Single-point mutations in the intracellular domain, namely K650M and X807R were generated by site-directed mutagenesis (Quick ChangeÒ site-directed mutagenesis, Stratagene, La Jolla, CA) according to the manufacturer’s Variable phosphorylation of FGFR3 mutants in TDI instructions Sequences of the primers used for mutagenesis are shown in supplementary Table S1 Mutagenesis for the K650M mutant was performed on the BsaBI ⁄ SphI fragment of FGFR3 in pBSII For X807R mutagenesis, the SpeI ⁄ SphI fragment of FGFR3 in pBSII was used The mutant FGFR3 cDNA was then transferred to pCDNA3.1 The presence of mutations was confirmed by sequencing on an ABI prism 3100 (Applied Biosystems, Foster City, CA) Generation of plasmids containing full-length myc-tagged c-Cbl and c-Cbl mutants (c-70Z-Cbl and c-CblY371F) has been described previously [41,55] Cell lines and transfection Human embryonic kidney cells stably expressing the vitronectin receptor (293-VnR) were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics These cells rather than HEK293 cells were used as they attach more tightly to plastic surfaces The patterns of expression and post-translational processing of wild-type and mutant FGFR3, determined by western blot, were comparable in the two cell lines, indicating that the presence of elevated levels of the VnR did not affect the pathways studied in these experiments ATDC5 cells were cultured in a : mixture of DMEM and Ham’s F12 medium containing 5% fetal bovine serum, insulin (10 lgỈmL)1), ferritin (10 lgỈmL)1), selenium (1 ngỈmL)1) and antibiotics Cells at 60% confluency were transiently transfected with wild-type or mutant FGFR3 cDNAs in the presence of Fugene (Roche, Indianapolis, IN) according to the manufacturer’s instructions Cells were collected after 24 or 48 h In some experiments, BFA (Epicentre Technologies, Madison, WI) was added to transfected cells for h at a final concentration of lgỈmL)1 The tyrosine kinase inhibitor SU5402 (a gift from G McMahon, SUGEN, San Francisco, CA) was dissolved in dimethylsulfoxide and added to transfected cells for 16 h at a final concentration of 25 lm Control cells were incubated with dimethylsulfoxide alone at a final concentration of 1% Nocodazole treatment (10 lgỈmL)1) was performed 24 h post transfection, for h at 37 °C Immunoblotting and immunoprecipitation Transfected cells were washed in NaCl ⁄ Pi and lysed in radioimmune precipitation assay buffer (50 mm Tris HCl pH 7.6, 150 mm NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, mgỈmL)1 pepstatin A, mgỈmL)1 leupeptin, mgỈmL)1 aprotinin, mm phenylmethanesulfonyl fluoride, mgỈmL)1 sodium orthovanadate), then clarified by centrifugation for 30 at 12 000 g Aliquots of lysates were reserved for immunoblotting and the rest of the lysates were immunoprecipitated for h at °C with an antiFGFR3 serum raised against the cytoplasmic domain FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3089 Variable phosphorylation of FGFR3 mutants in TDI J Bonaventure et al (Sigma, St Louis, MO) Immune complexes were bound to Protein G agarose beads and washed three times with radioimmune precipitation assay buffer, then heated at 95 °C for 10 in 4· loading buffer (Invitrogen) Total cell lysates or immunoprecipitates were resolved by electrophoresis on 4–12% gradient NU-PAGE gels (Invitrogen) Proteins were transferred to poly(vinylidene) difluoride membranes (Immobilon, Millipore, Bedford, MA), incubated with primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies and the bands detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) The following primary antibodies were used for immunoprecipitation and immunoblotting: rabbit anti-FGFR3 (Sigma), mouse anti-(phosphotyrosine P-Tyr-102) (Cell Signaling Technology, Beverly, MA); mouse anti-myc from 9E10 hybridoma (Roche Molecular Biochemicals); mouse anti-Cbl, mouse anti-GM130 and mouse anti-p230 (BD Biosciences, Franklin Lakes, NJ), rabbit anti-(phospho-Cbl tyrosine 731) (Cell Signaling), mouse anti-(peptidyl disulfide isomerase) (Affinity Bioreagents, Golden, CO) and mouse anti-ubiquitin (Chemicon, Temecula, CA) Deglycosylation of FGFR3 isoforms FGFR3 was immunoprecipitated from cell lysates using anti-FGFR3 serum Immune complexes bound to protein G–agarose beads were resuspended in 50 mm sodium citrate, pH 5.5, supplemented with 1% SDS and 1% bmercaptoethanol and heated for 10 at 95 °C Endo H (Roche) was added at a final concentration of 50 mU and the mixture was incubated at 37 °C for h Peptidyl N-glycosidase F (PNGase F) treatment was achieved by diluting vol of the sodium citrate ⁄ SDS ⁄ b-mercaptoethanol solution with vol of sodium citrate 50 mm, pH 5.5, containing 1% NP-40 Then U of PNGase F solution (Roche) were added followed by incubation for h at 37 °C Enzymatic activities were blocked by adding 4· loading buffer Immunocytochemistry 293-VnR cells were seeded in Labtek chambers (BD Biosciences) at a density of 15 000 cellsỈwell)1 Cells were allowed to reach 60% confluency, then transfected with wild-type or mutant FGFR3 cDNAs using Fugene (0.5 lLỈwell)1) After 24 h, cells were fixed with 4% paraformaldehyde, permeabilized for 15 with 0.1% Triton X-100 in NaCl ⁄ Pi and incubated for 30 with 10% sheep serum in NaCl ⁄ Pi The following sera were used for immunostaining: rabbit anti-FGFR3 (1 : 400), mouse anti(phosphotyrosine P-Tyr102) (1 : 200), mouse anti-GM130 (1 : 100), mouse anti-p230 (1 : 100), mouse anti-(peptidyl disulfide isomerase) (1 : 100) Appropriate second sera: anti-(rabbit Alexa fluor green 458), anti-(mouse Alexa fluor red 561) (Molecular Probes, Eugene, OR) were added at a 3090 ⁄ 400 dilution and incubated at room temperature for h 4¢,6-Diamidino-2-phenylindole was used for nuclear counterstaining Glass slides were mounted and photographed using an inverted Olympus microscope Surface biotinylation 293-VnR cells transiently transfected with wild-type or mutant FGFR3 cDNAs were washed twice with cold NaCl ⁄ Pi then incubated at °C for 30 with either NHS-biotin or cleavable sulfo-NHS-S-S-biotin (Uptima, Montlucon, France) at a 0.5 mgỈmL)1 concentration in ¸ NaCl ⁄ Pi Coupling of NHS-biotin was blocked by washing with 15 mm glycine in NaCl ⁄ Pi When cells were treated with sulfo-NHS-S-S-biotin, a previously described procedure for analysis of endocytosis was used [36] Briefly, after biotinylation of cell surface proteins, excess biotin was quenched by incubating cells for 10 with 50 mm Tris ⁄ HCl, pH 7.5 at °C Cells were re-incubated at 37 °C in fresh DMEM for various times (0–6 h) to allow receptor endocytosis Biotin was then cleaved from proteins on the cell surface by washing with 50 mm glutathione, 75 mm NaCl, 75 mm NaOH, 10% fetal bovine serum Cells were then washed with 50 mm iodoacetamide in 1% BSA to quench residual glutathione Cells were lysed with RIPA buffer and lysates were immunoprecipitated with antiFGFR3 sera Precipitated proteins were separated on NuPAGE gels under nonreducing conditions, transferred to poly(vinylidene difluoride) membranes and probed with HRP-conjugated avidin D (Vector Laboratories, Burlingame, CA) Ubiquitylation 293-VnR cells were cotransfected with wild-type or mutant FGFR3 cDNAs and HA-tagged-ubiquitin cDNA (a gift of D Bohmann, Rochester, NY) In some experiments cells were also cotransfected with c-Cbl or the mutant 70Z-Cbl At 24 h post transfection, cells were treated for h with the proteasome inhibitor MG132 (Biomol Research Laboratories, Plymouth Meeting, PA) at a final concentration of 50 lm in 0.1% dimethylsulfoxide or with the lysosome inhibitor chloroquine (Sigma) at a final concentration of 500 lm Cell lysates were immunoprecipitated with antiFGFR3 or anti-HA sera (Sigma) and analysed by immunoblotting with anti-HA, anti-ubiquitin or anti-FGFR3 sera Treatment with 10 lgỈmL)1 cycloheximide for h followed by incubation in fresh cyclohexamide-free medium was performed when required to block protein synthesis Acknowledgements We are grateful to Dr M Hayman (State University, New York, NY) and Dr D Bohmann (University of FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS J Bonaventure et al Rochester, NY) for providing plasmids and to Dr G McMahon (SUGEN, San Francisco, CA) for providing the SU5402 TK inhibitor We thank Dr Archana Sanjay for helpful suggestions Part of this work was supported by the European Skeletal Dysplasia Network (grant QLG1-CT-2001-02188) and by the Philip Foundation References Schlessinger J (2000) Cell signalling by receptor tyrosine kinases Cell 103, 211–225 Ornitz DM (2000) FGFs, heparan sulfate and 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supplementary material is available online: Variable phosphorylation of FGFR3 mutants in TDI Fig S1 Schematic representation and predicted amino acid sequence of the elongated FGFR3 receptor (947 aa) resulting from an X807R mutation Fig S2 (A) Immunocytochemical staining of 293-VnR cells transfected with Y373C and K650M mutant cDNAs with an anti-phosphotyrosine (P-Tyr, red) serum (B) Immunocytochemical staining of 293-VnR cells transfected with wild-type or mutant cDNAs Cells were stained sequentially with anti-GM130 (red) and anti-FGFR3 (green) sera Fig S3 (A) Immunocytochemical analysis of 293-VnR cells transiently cotransfected with c-Cbl and FGFR3 K650M or FGFR3 X807R mutant cDNAs (B) Activation of wild-type FGFR3 by FGF9 does not induce c-Cbl phosphorylation (C) wild-type and mutant FGFR3 fail to coimmunoprecipitate with c-Cbl Table S1 Sequence of primers used to generate FGFR3 mutants This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3093 ... immunoprecipitated with an anti -FGFR3 serum then immunoblotted with anti-ubiquitin and anti -FGFR3 sera (D) Disabling the c-Cbl ubiquitylating activity does not affect the ubiquitylation of the wild -type, ... proteasome and lysosome inhibitors on ubiquitylation of wild -type and mutant FGFR3 (A) Ubiquitylation of wild -type and Y373C FGFR3 in the absence or presence of the proteasome inhibitor MG132 (50 lM... with chloroquine as indicated Lysates were immunoprecipitated and processed for immunoblotting with anti-ubiquitin and anti -FGFR3 sera (C) Ubiquitylation of the wild -type receptor is increased by