The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor

The localization of FGFR3 mutations causingthanatophoric dysplasia type I differentially affectsphosphorylation, processing and ubiquitylationof the receptorJacky Bonaventure1,2, Linda Gibbs2, William C. Horne3 and Roland Baron3

1 Institut Curie, Universite Paris Sud, Orsay, France

2 Department of Medical Genetics INSERM U393, Hopital 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: [email protected]

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: [email protected]

(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 48Hrs : PNGase: - - + +

ATDC5 cells

WT Y373C K650M K650N X807R

115

250

Y373C WT WT Y373C WT WT

160

105

Dimer

105

130115

kDa

130

105

IB: FGFR3IP: FGFR3IB: FGFR3

IP: FGFR3IB: FGFR3

WT X807R Y373C R248C

105

130115

IP: FGFR3IB: FGFR3

Endo H: - + -PNGase: - - +

129119

144 160

105

DC

A B

X807R

EF IB: FGFR3

160

105

IP: FGFR3IB: 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.


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



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

Y373CWT

K650M X807R

WT Y373C R248C WTFGF9: - - - +

160

105

160IP FGFR3IB: Ptyr

IP FGFR3IB FGFR3

A

105

K650M K650M WT WT

Time (hrs) :

IP FGFR3IB: Ptyr

IP FGFR3IB FGFR3

105115

115105

130

X807R

PtyrIP FGFR3

IB: FGFR3

129119

C

K650M

PNGase: - +

IP FGFR3IB: Ptyr

D

105115

144

kDa

E

B kDa24 48 24 48

a

d e f i j

b c g 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).



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 BFAGM130 + FGFR3 p230 + FGFR3

(merge) (merge)

+ Nocodazole

IP: FGFR3IB: FGFR3 --

A

115-

BFA:Endo H

130

105

WT Y373C K650M WT Y373C K650M kDa K650MIP:FGFR3IB: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·.



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: FGFR3IB: Avidin D

IP: FGFR3IB: FGFR3

dimer

dimer

WT K650M

IP: FGFR3IB: Avidin D

IP: FGFR3IB: FGFR3

160

115

IP: FGFR3IB: avidin D

IP: FGFR3IB: FGFR3

130

130

105

160

WT Y373C R248C WT K650M (reduced)

dimer

dimer 250

105

kDa250

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 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 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.



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: FGFR3IB: FGFR3

IP: FGFR3IB: Ptyr

130115105

kDa

K650M

130

IP FGFR3IB Avidin D

SU5402: - + - + - + - +

Time (hrs): 0 0 1 1 2 2 3 3

IP FGFR3IB 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.



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 : FGFR3IB : FGFR3

WT Y373C WT Y373C

MG132: - - + +

IP : FGFR3IB : Ubiquitin


160

105

250

160

A

kDa

FGFR3:



250

160

c-Cbl: FGF9:

kDa

B

WT


160

160

Chloroquine: - - - + + +

Y37

3CK

650M

WT

Y37

3CK

650M

kDa

WT

WT

Y37

3CY

373C

WT

C

WT

Y37

3C

K65

0M

WT

Y37

3C

K65

0M

FGFR3:


160

kDa250

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 lM 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 lM 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.



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: WT

Y373

CK

650M

c-Cbl:

IP : Myc

Phospho-CblY731

Cbl160

105

IB:

B

FGFR3

TCL

IB:

Phospho-CblY731

Cbl

FGFR3:

+ + +c-Cbl70Z:

IP: Myc

WT

Y373

CK

650M

C

+ + +

120

160

105

FGFR3: - WT K650M

c-Cbl: + + + -c-CblY371F: - - - +

IP: mycIB: Ptyr

IP: mycIB: Cbl

IB: FGFR3

IB: Cbl

105

120

kDaPhospho-Cbl

Cbl

TCL

FGFR3: - WT K650MA

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).



[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



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 Change� 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 ⁄Pi 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 mgÆmL)1 pepstatin A, 1 mgÆmL)1 leupeptin,

1 mgÆmL)1 aprotinin, 2 mm phenylmethanesulfonyl fluoride,

1 mgÆmL)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



(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 ⁄Pi and incubated for 30 min 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

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 ⁄Pi then incubated at 4 �C for 30 min with either

NHS-biotin or cleavable sulfo-NHS-S-S-biotin (Uptima,

Montlucon, France) at a 0.5 mgÆmL)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 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 �Cin 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



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 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 corres-

ponding author for the article.



The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor

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