Tài liệu Báo cáo khoa học: The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor pptx

The localization of FGFR3 mutations causing
thanatophoric dysplasia type I differentially affects
phosphorylation, processing and ubiquitylation
of the receptor
Jacky Bonaventure
1,2
, Linda Gibbs
2
, William C. Horne
3
and Roland Baron
3
1 Institut Curie, Universite
´
Paris Sud, Orsay, France
2 Department of Medical Genetics INSERM U393, Ho
ˆ
pital Necker, Paris, France
3 Department of Cell Biology and Orthopaedics, Yale University School of Medicine, New Haven, CT, USA
Fibroblast growth factor receptor 3 (FGFR3) belongs
to a family of four genes (FGFR1–4) encoding recep-
tors 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
Keywords
Cbl; FGFR3; mutation; phosphorylation;
ubiquitylation
Correspondence
J. Bonaventure, Institut Curie, CNRS UMR
146, Bat. 110, Universite

´
Paris Sud, 91400
Orsay, France
Fax: +33 1 69 86 53 01
Tel: +33 1 69 86 71 80
E-mail:
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:
(Received 5 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 3 (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 traffick-
ing 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 gly-
cosylated mature isoform was constitutively but mildly phosphorylated.
Similarly, mutations affecting the extracellular domain (R248C and
Y373C) induced moderate constitutive receptor phosphorylation. By con-
trast, the K650M mutation affecting the tyrosine kinase 2 (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 sur-

face. Normal processing was rescued by tyrosine kinase inhibitor treatment.
Internalization of the R248C and Y373C mutant receptors, which form sta-
ble 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.
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
split tyrosine kinase (TK) domain. Binding of 1 of the
22 fibroblast growth factor (FGF) ligands in the pres-
ence of cell-surface heparan sulfate proteoglycans act-
ing 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 dwarf-
isms and craniosynostoses [5,6]. Mutations in FGFR3
are mostly responsible for long-bone dysplasias inclu-
ding achondroplasia (ACH), the most common form of
dwarfism in humans, the milder form hypochondropla-
sia and the neonatal lethal form thanatophoric dyspla-
sia (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 ter-
mination codon (X807R ⁄ C ⁄ G ⁄ S ⁄ W) [9]. Likewise, sub-
stitution 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 replace-
ment 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 result-
ing 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 con-
served Lys650 lead to constitutive receptor phosphory-
lation 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-
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 subse-
quent 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 coimmu-
noprecipitation, even though constitutive phosphoryla-
tion 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 biochemi-
cal 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 non-
glycosylated form of the receptor. Such hyperphospho-
rylation markedly hampered receptor glycosylation at
the Golgi level resulting in reduced levels of fully gly-
cosylated receptors at the cell surface of transfected
cells. Reversal of this situation following treatment
with the FGFR tyrosine kinase inhibitor SU5402 indi-
cated 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 puta-
tive involvement of c-Cbl in this process. Ubiquityla-
tion of the R248C, Y373C and X807R mutant
receptors was stronger than the wild-type and appar-
ently independent of c-Cbl. Constitutive phosphoryla-
tion 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 differen-
tially 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 phos-
phorylation of c-Cbl which, nonetheless, does not seem
to directly regulate FGFR3 ubiquitylation.
J. Bonaventure et al. Variable phosphorylation of FGFR3 mutants in TDI
FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3079
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 recep-
tor (Fig. S1) was created by site-directed mutagenesis
of the full-length human FGFR3 cDNA and subclon-
ing 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.
We first tested whether the different mutations caus-
ing TDI affected receptor biosynthesis and post-trans-
lational 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 agree-
ment with the predicted 141 additional residues separ-
ating 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
TCL
WT Y373C K650M WT K650M WT K650M
24 48 24 48 24 48
Hrs :
PNGase:

+ +
ATDC5 cells
WT Y373C K650M K650N X807R
115
250
Y373C WT WT Y373C WT WT
160
105
Dimer
105
130
115

kDa
130
105
IB: FGFR3
IP: FGFR3
IB: FGFR3
IP: FGFR3
IB: FGFR3
WT X807R Y373C R248C
105
130
115
IP: FGFR3
IB: FGFR3
Endo H: - + -
PNGase:

+
129
119
144
160
105
D
C
AB
X807R
E
F
IB: FGFR3

160
105
IP: FGFR3
IB: FGFR3
TCL
(non reduced) (reduced)
FGF9:
- - + - - +
Fig. 1. 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 2 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 ngÆmL
)1
FGF9 and heparin for 10 min. (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.
Variable phosphorylation of FGFR3 mutants in TDI J. Bonaventure et al.
3080 FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS
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 fur-
ther indicated that the intensity of the 105 kDa band
was greatly increased in this mutant at 24 h post trans-

fection (31% of the total signal in K650M versus 10%
in wild-type). Similar results were obtained when the
same mutants were transiently transfected in chondro-
genic 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 differ-
ently glycosylated forms of the receptor, immunopre-
cipitated wild-type and mutant receptors were digested
with peptidyl N-glycosidase F (PNGase), which com-
pletely 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 immunopre-
cipitated with an anti-FGFR3 serum and separated by
electrophoresis under nonreducing and reducing condi-
tions. 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 phosphoryla-
tion and the relationship with glycosylation in 293-
VnR cells was assessed by immunoprecipitation of the
receptor and immunoblotting with an anti-phospho-
tyrosine serum. Both the R248C and Y373C mutants
showed moderate phosphorylation of the fully glycos-
ylated 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
mutant, and to a lesser extent the 115 kDa mannose-
rich 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, phos-
phorylation 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 anti-
FGFR3 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 wild-
type (Fig. 2Ed) and the R248C and X807R mutants
(not shown), whereas both 293-VnR and ATDC5 cells
expressing the K650M mutant had a round morpho-
logy 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 mem-
brane 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 localiza-
tion of the mutant receptors, cells were stained with
anti-(peptidyl disulfide isomerase) (PDI) and anti-
GM130, 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
J. Bonaventure et al. Variable phosphorylation of FGFR3 mutants in TDI
FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3081
Fig. S2B). There was little colocalization of the
K650M mutant and GM130, indicating that transfer
of this receptor from the ER to the Golgi compart-
ment was less efficient than that of the wild-type
receptor and other mutants. Immunostaining of
K650M-transfected cells with GM130 and FGFR3 fol-
lowing fragmentation of the Golgi network into mini-
stacks by nocadazole treatment showed colocalization
of the two proteins in scattered puncta (Fig. 3Ba), con-
firming that some K650M FGFR3 molecules were pre-
sent 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 appa-
ratus, cells were treated for 1 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), indica-
ting 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 proc-
essed endo H-resistant glycosylated proteins [34,35].
f
WT
WT+FGF
Y373C
Y373C
K650M
K650M
Y373C
WT
K650M X807R
WT Y373C R248C WT

FGF9: - - - +
160
105
160
IP FGFR3
IB: Ptyr
IP FGFR3
IB FGFR3
A
105
K650M K650M WT WT
Time (hrs) :
IP FGFR3
IB: Ptyr
IP FGFR3
IB FGFR3
105
115
115
105
130
X807R
Ptyr
IP FGFR3
IB: FGFR3
129
119
C
K650M
PNGase: - +

IP FGFR3
IB: Ptyr
D
105
115
144
kDa
E
B
kDa
24 48 24 48
a
d
e
fij
b
cg
h
Fig. 2. 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 ngÆmL
)1
FGF9 and heparin for 10 min 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 phos-
phorylated 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 ngÆmL
)1
for 10 min in (d).
Variable phosphorylation of FGFR3 mutants in TDI J. Bonaventure et al.
3082 FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS
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 hyperphos-
phorylated K650M molecules undergo partial process-
ing at the cis ⁄ medial-Golgi level to become endo H
resistant without being fully glycosylated in the trans-
Golgi 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 293-
VnR cell-surface proteins were first labelled with NHS-
biotin, immunoprecipitated with an anti-FGFR3 serum
then separated on nonreducing or reducing gels and
blotted with avidin D (Fig. 4A). Although the wild-
type receptor showed a single 130-kDa band corres-
ponding 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 endocyto-
sis of the wild-type and mutant receptors. Cell-surface
proteins were labelled by incubating cells with cleavable
sulfo-NHS-S-S-biotin for 30 min 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 glutathi-
one. 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 (wild-
type or mutant) were detected when cells were kept at
4 °C (Fig. 4C and not shown). A substantial amount of
the biotinylated receptor (130 kDa) was found after 1 h
in the absence of ligand, indicating that wild-type
FGFR3 is constitutively endocytosed. The signal
reached a peak after 2 h then decreased progressively
to become undetectable after 5 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 attes-
ted by the delay in reaching the maximum amount of
protected biotinylated receptor and the presence of
GM130 + FGFR3 p230 + FGFR3
(merge) (merge)
B
BFA BFA

GM130 + FGFR3 p230 + FGFR3
(merge) (merge)
+ Nocodazole
IP: FGFR3
IB: FGFR3
-
-
A
115
-
BFA:
Endo H
130
105
WT Y373C K650M WT Y373C K650M
kDa
K650M
IP:
FGFR3
IB:
Ptyr
K650M mutant
a
d
e
f
b
c
Fig. 3. 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 1 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 pres-
ence 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 2 h before staining with antibodies; (c,d) nontreated cells; (e,f) cells treated with BFA for 1 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·.
J. Bonaventure et al. Variable phosphorylation of FGFR3 mutants in TDI
FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3083
significant amounts of biotinylated receptor after 6 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 kin-
ases’ ATP-binding site [39]. We therefore determined
whether SU5402 prevented constitutive phosphoryla-
tion 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 phosphoryla-
tion in cells expressing the Y373C mutant (not shown).
Phosphorylation of the K650M mutant, although dra-
matically reduced, was not completely abrogated

(Fig. 5A,B). Increased inhibitor concentrations had no
further effect on phosphorylation but affected cell viab-
ility (not shown). Immunoblot analysis of the wild-type
and K650M mutant receptors following SU5402 treat-
ment and immunoprecipitation with an anti-FGFR3
serum showed the presence of the mature 130 kDa iso-
form both in the wild-type and mutant (Fig. 5A), indi-
cating 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 con-
firming the ability of the mutant receptor to traffic effi-
ciently 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
Time (hours): 1 2 3 4 5 6 1 2 3 5 6 0 1 3 4 0 1 3
WT
Y373C
IP: FGFR3
IB: Avidin D
IP: FGFR3
IB: FGFR3
dimer

dimer
WT K650M
IP: FGFR3
IB: Avidin D
IP: FGFR3
IB: FGFR3
160
115
IP: FGFR3
IB: avidin D
IP: FGFR3
IB: FGFR3
130
130
105
160
WT Y373C R248C WT K650M (reduced)
dimer
dimer
250
105
kDa
250
A
B
C
130
(non reduced)
Fig. 4. Cell-surface expression and endocytosis of wild-type and mutant FGF receptors. (A) Cells were surface biotinylated (NHS-biotin) for
30 min at 4 °C, then washed extensively with 15 m

M glycine in NaCl ⁄ P
i
. 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 min at 4 °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 1 and 3 h.
Variable phosphorylation of FGFR3 mutants in TDI J. Bonaventure et al.
3084 FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS
studied ubiquitylation of wild-type and mutant recep-
tors by cotransfecting cells with wild-type or mutant
FGFR3 and HA-tagged ubiquitin cDNAs. Ubiquitylat-
ed receptors identified by blotting with anti-ubiquitin
sera appeared as a smear of bands with a lower mobi-
lity 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 chloro-
quine, a lysosomal inhibitor. Unlike Y373C, the
K650M mutant was less ubiquitylated than the wild-
type receptor and the amounts of ubiquitylated wild-
type and mutant FGFR3 were slightly increased by
chloroquine treatment (Fig. 6B), suggesting that the

lysosomal pathway may also participate to their degra-
dation. The X807R mutant also exhibited an increased
ubiquitylation compared with wild-type (not shown)
confirming that ubiquitylation levels of the weakly
phosphorylated TDI mutant receptors (R248C, Y373C
and X807R) were higher than the wild-type. By con-
trast, the heavily phosphorylated K650M mutant was
less ubiquitylated than the wild-type, consistent with its
poor expression at the cell surface.
c-Cbl does not mediate the ubiquitylation
of FGFR3, but it is constitutively phosphorylated
by the K650M mutant
c-Cbl is an adaptor protein and an E3-ubiquitin ligase
that is phosphorylated downstream of several growth
factor receptors and contributes to their downregula-
tion 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 lig-
and-dependent process [21]. We therefore first exam-
ined 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 ubiquityla-
tion (Fig. 6C), and the ubiquitinylation of wild-type,
Y373C and K650M FGFR3 mutants was not signifi-
cantly 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 coimmunopre-
cipitate 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 wild-
type 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 immuno-
fluorescence 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 bind-
ing site for phosphatidylinositol 3-kinase. No phos-
phorylation 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 (supple-
mentary 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
WT K650M WT K650M
SU5402: - - + +
IP: FGFR3
IB: FGFR3
IP: FGFR3

IB: Ptyr
130
115
105
kDa
K650M
130
IP FGFR3
IB Avidin D
SU5402: - + - + - + - +
Time (hrs): 0 0 1 1 2 2 3 3
IP FGFR3
IB FGFR3
130
115
A
B
SU 5402
16 hrs
Biotin
30’
DMEM
0-3 hrs (37°C)
105
105
Fig. 5. Effect of the tyrosine kinase inhibitor SU5402 on phosphory-
lation, 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 anti-
FGFR3 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-incuba-
tion in serum-supplemented DMEM for the indicated times. After
immunoprecipitation with anti-FGFR3 serum, proteins were separ-
ated on a nonreducing gel then blotted and visualized by hybridiza-
tion with HRP-conjugated avidin D and anti-FGFR3 serum.
J. Bonaventure et al. Variable phosphorylation of FGFR3 mutants in TDI
FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3085
in the K650M-expressing cells (Figs 7B and supple-
mentary 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 70Z-
Cbl 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 phos-
phorylation.
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 pri-
mary cultured chondrocytes from affected patients
would be representative of a more physiological model,
the difficulty of efficiently transfecting human chondro-
cytes 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 proper-
ties. We first demonstrated that replacement of the
stop codon by an arginine residue resulted in a stable
elongated receptor, which appeared on western blot-
ting as a combination of three bands including the
nonglycosylated, mannose-rich and fully glycosylated
isoforms, indicating that this elongated receptor under-
went the same maturation process as the Y373C and
R248C mutants. However, under nonreducing condi-
tions, 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
250
105
IP : FGFR3
IB : FGFR3
WT Y373C WT Y373C
MG132: - - + +
IP : FGFR3
IB : Ubiquitin
IP : FGFR3
IB : FGFR3
160
105
250
160
A

kDa
FGFR3:
IP : FGFR3
IB : Ubiquitin
IP : FGFR3
IB : FGFR3
250
160
c-Cbl:
FGF9:
kDa
B
WT
IP : FGFR3
IB : Ubiquitin
160
160
Chloroquine
: + + +
Y
3
7
3
C
K
6
5
0
M
W

T
Y
3
7
3
C
K
6
5
0
M
kDa
WT
W
T
Y
3
7
3
C
Y
3
7
3
C
W
T
C
WT
Y

3
7
3
C
K
6
5
0
M
W
T
Y
3
7
3
C
K
6
5
0
M
FGFR3:
IP : FGFR3
IB : Ubiquitin
160
kDa
250
c-Cbl
:
c-Cbl70Z:

IB: c-Cbl
TCL
D
Fig. 6. 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 l
M for 1 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 sequen-
tially 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 l
M for 1 h). Cells transfected with the indicated cDNAs were treated with chloroquine as indica-
ted. 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 ngÆmL
)1
) and heparin (1 lgÆmL
)1
) for 4 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 immu-
noprecipitated with an anti-FGFR3 serum followed by blotting with an anti-ubiquitin serum.
Variable phosphorylation of FGFR3 mutants in TDI J. Bonaventure et al.
3086 FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS
resulted in a different electrophoretic pattern charac-
terized 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 phosphoryla-
tion by mutating the mechanistically critical Tyr718 in
the Fgfr3 activation loop restores normal Fgfr3 recep-
tor 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 autophosphory-
lation 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 regula-
ting maturation arrest of phosphorylated receptors
have not been clearly elucidated, our coimmunolocali-
zation studies pointed to a role for components of the

ER–Golgi vesicle transport. Through the use of mark-
ers for the ER (PDI) and the Golgi apparatus
(GM130, p230), the phosphorylated isoforms of the
K650M mutant were identified in both the ER and cis-
Golgi 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. Disrup-
ting the Golgi apparatus with BFA or nocodazole pro-
vided 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
FGFR3:
W
T
Y
3
7
3
C
K
6
5
0
M
c-Cbl:
IP : Myc
Phospho-CblY731
Cbl

160
105
IB:
B
FGFR3
TCL
IB:
Phospho-Cbl
Y731
Cbl
FGFR3:
+ + +c-Cbl70Z:
IP: Myc
W
T
Y
3
7
3
C
K
6
5
0
M
C
+ + +
120
160
105

FGFR3: - WT K650M
c-Cbl: + + +
-
c-CblY371F: +
IP: myc
IB: Ptyr
IP: myc
IB: Cbl
IB: FGFR3
IB: Cbl
105
120
kDa
Phospho
-Cbl
Cbl
TCL
FGFR3: - WT K650M
A
Fig. 7. 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 wild-
type or mutant FGFR3 cDNAs. Immunoprecipitation of c-Cbl with an anti-myc serum was followed by immunoblotting with an antibody spe-
cific 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).
J. Bonaventure et al. Variable phosphorylation of FGFR3 mutants in TDI
FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3087
[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 oligo-
saccharide-modifying enzymes (mannosidase II and
thiamine pyrophosphatase) undergo retrograde trans-
port to the ER after BFA treatment [35]. We propose
that in the absence of BFA, some heavily phosphoryl-
ated 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 evi-
dence 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 pro-
perly processed and expressed at the cell surface. Whe-
ther 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 disul-
fide-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 sub-
sequent 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 degrada-
tion in the lysosome or the proteasome [27,46]. Consis-
tent with data from Monsonego-Ornan et al. [30] on
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 ubiquity-
lation 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 plas-
mids. Polyubiquitylation is responsible for the internal-
ization and proteasomal degradation of several plasma
membrane proteins [46], but monoubiquitylation has
been recently identified as the main mechanism regula-
ting RTK endocytosis and degradation [27,28] and is
associated in yeast with proteasome-independent func-
tions including protein trafficking [47]. Although it
is not known whether FGFR3 mutants are monoubi-
quitylated, polyubiquitylated or both, it is tempting to
speculate that highly ubiquitylated R248C, Y373C and
X807R receptors could be preferentially monoubi-
quitylated on their 26 lysine residues lying in the intra-
cellular domain [48] (supplementary Fig. S1) and
transferred to early endosomes. From this compart-
ment, 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 coimmunoprecipi-
tate 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 phos-
phorylated tyrosine residues located in the C-terminal
half of c-Cbl, most likely resulted from intracellular
Variable phosphorylation of FGFR3 mutants in TDI J. Bonaventure et al.
3088 FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS
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 kin-
ase. 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 con-
sistent 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 evi-
dence 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 retent-
ion of the mutant receptor leading to phosphorylation
of target signalling molecules including c-Cbl. By con-
trast, mutations creating cysteine residues in the ECD
or elongating the receptor result in delayed endocyto-
sis, 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 Univer-
sity, New York, NY) and subcloned into pBSII. Two dif-
ferent 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 differ-
ent 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
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 frag-
ment 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 vitro-
nectin receptor (293-VnR) were cultured in DMEM supple-
mented 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 1 : 1
mixture of DMEM and Ham’s F12 medium containing

5% fetal bovine serum, insulin (10 lgÆmL
)1
), ferritin
(10 lgÆmL
)1
), selenium (1 ngÆmL
)1
) and antibiotics. Cells at
60% confluency were transiently transfected with wild-type
or mutant FGFR3 cDNAs in the presence of Fugene 6
(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 1 h at a final concentra-
tion of 5 lgÆmL
)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 lgÆmL
)1
) was performed
24 h post transfection, for 2 h at 37 °C.
Immunoblotting and immunoprecipitation
Transfected cells were washed in NaCl ⁄ P
i
and lysed in

radioimmune precipitation assay buffer (50 mm Tris HCl pH
7.6, 150 mm NaCl, 1% Nonidet P40, 0.5% sodium deoxy-
cholate, 1 mgÆ mL
)1
pepstatin A, 1 mgÆmL
)1
leupeptin,
1mgÆmL
)1
aprotinin, 2 mm phenylmethanesulfonyl fluoride,
1mgÆmL
)1
sodium orthovanadate), then clarified by centrif-
ugation for 30 min at 12 000 g. Aliquots of lysates were
reserved for immunoblotting and the rest of the lysates
were immunoprecipitated for 4 h at 4 °C with an anti-
FGFR3 serum raised against the cytoplasmic domain
J. Bonaventure et al. Variable phosphorylation of FGFR3 mutants in TDI
FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3089
(Sigma, St Louis, MO). Immune complexes were bound to
Protein G agarose beads and washed three times with radio-
immune precipitation assay buffer, then heated at 95 °C for
10 min 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 (Immo-
bilon, Millipore, Bedford, MA), incubated with primary
antibodies followed by horseradish peroxidase (HRP)-conju-
gated secondary antibodies and the bands detected by
enhanced chemiluminescence (Amersham Pharmacia Bio-

tech, Piscataway, NJ).
The following primary antibodies were used for immuno-
precipitation 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-(phos-
pho-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 pro-
tein G–agarose beads were resuspended in 50 mm sodium
citrate, pH 5.5, supplemented with 1% SDS and 1% b-
mercaptoethanol and heated for 10 min at 95 °C. Endo H
(Roche) was added at a final concentration of 50 mU and
the mixture was incubated at 37 °C for 2 h. Peptidyl N-gly-
cosidase F (PNGase F) treatment was achieved by diluting
1 vol. of the sodium citrate ⁄ SDS ⁄ b-mercaptoethanol solu-
tion with 1 vol. of sodium citrate 50 mm, pH 5.5, contain-
ing 1% NP-40. Then 5 U of PNGase F solution (Roche)
were added followed by incubation for 2 h at 37 °C. Enzy-
matic activities were blocked by adding 4· loading buffer.
Immunocytochemistry
293-VnR cells were seeded in Labtek chambers (BD Bio-
sciences) at a density of 15 000 cellsÆwell
)1
. Cells were

allowed to reach 60% confluency, then transfected with
wild-type or mutant FGFR3 cDNAs using Fugene 6
(0.5 lLÆwell
)1
). After 24 h, cells were fixed with 4%
paraformaldehyde, permeabilized for 15 min with 0.1% Tri-
ton X-100 in NaCl ⁄ P
i
and incubated for 30 min with 10%
sheep serum in NaCl ⁄ P
i
. 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
1 ⁄ 400 dilution and incubated at room temperature for 2 h.
4¢,6-Diamidino-2-phenylindole was used for nuclear count-
erstaining. 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 ⁄ P
i
then incubated at 4 °C for 30 min with either
NHS-biotin or cleavable sulfo-NHS-S-S-biotin (Uptima,
Montluc¸ on, France) at a 0.5 mgÆmL

)1
concentration in
NaCl ⁄ P
i
. Coupling of NHS-biotin was blocked by washing
with 15 mm glycine in NaCl ⁄ P
i
. When cells were treated
with sulfo-NHS-S-S-biotin, a previously described proce-
dure for analysis of endocytosis was used [36]. Briefly, after
biotinylation of cell surface proteins, excess biotin was
quenched by incubating cells for 10 min with 50 mm
Tris ⁄ HCl, pH 7.5 at 4 °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 anti-
FGFR3 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, Burlin-
game, 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 1 h with
the proteasome inhibitor MG132 (Biomol Research Labor-
atories, 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 anti-
FGFR3 or anti-HA sera (Sigma) and analysed by immuno-
blotting with anti-HA, anti-ubiquitin or anti-FGFR3 sera.
Treatment with 10 lgÆmL
)1
cycloheximide for 1 h followed
by incubation in fresh cyclohexamide-free medium was per-
formed 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
Variable phosphorylation of FGFR3 mutants in TDI J. Bonaventure et al.
3090 FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS
Rochester, NY) for providing plasmids and to Dr
G. McMahon (SUGEN, San Francisco, CA) for provi-
ding the SU5402 TK inhibitor. We thank Dr Archana
Sanjay for helpful suggestions. Part of this work was
supported by the European Skeletal Dysplasia Net-
work (grant QLG1-CT-2001-02188) and by the Philip
Foundation.
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Supplementary material
The following supplementary material is available
online:
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) Acti-
vation 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
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 corres-
ponding author for the article.
J. Bonaventure et al. Variable phosphorylation of FGFR3 mutants in TDI
FEBS Journal 274 (2007) 3078–3093 ª 2007 The Authors Journal compilation ª 2007 FEBS 3093