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..
Trang 1thanatophoric dysplasia type I differentially affects
phosphorylation, processing and ubiquitylation
of the receptor
Jacky Bonaventure1,2, Linda Gibbs2, William C Horne3and Roland Baron3
1 Institut Curie, Universite´ Paris Sud, Orsay, France
2 Department of Medical Genetics INSERM U393, Hoˆpital 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: jacky.bonaventure@curie.u-psud.fr
R Baron, Department of Cell Biology and
Orthopaedics, Yale University School of
Medicine, PO Box 208044, New Haven,
CT 208044, USA
Fax: +1 203 785 2744
Tel: +1 203 785 4150
E-mail: roland.baron@yale.edu
(Received 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.
Trang 2split 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 signallresult-ing 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 immunocytochemibiochemi-cal 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
Trang 3TDI 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
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
129 119
144
160 105
D C
X807R
E
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)1FGF9 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.
Trang 4other 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 relaphosphoryla-tionship with glycosylaphosphoryla-tion 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 (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
Trang 5Fig 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 WT
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
115 105 130
X807R
Ptyr
IP FGFR3 IB: FGFR3
129
C
K650M
PNGase: - +
IP FGFR3 IB: Ptyr
D
105 115 144
kDa
E
B 24 48 24 48 kDa
a
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)1FGF9 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)1for 10 min in (d).
Trang 6Unexpectedly, 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 37C 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
4C (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
GM130 + FGFR3 p230 + FGFR3 (merge) (merge)
+ Nocodazole
IP: FGFR3
-A
115
-BFA:
Endo H
130 105
WT Y373C K650M WT Y373C K650M
kDa K650M IP:
FGFR3 IB:
Ptyr
K650M mutant
a
d
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 abspres-ence 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·.
Trang 7significant 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
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
105
kDa 250
A
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.
Trang 8studied 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 identifiUbiquitylat-ed 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
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 ubiquityladownregula-tion [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 ubiquitinylaubiquityla-tion 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-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.
Trang 9in 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-70Z-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
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
3 C
K 6
0 M
W T Y 7 C
K 6
0 M kDa
WT W T Y 3
3 C
Y 3
3 C
W T
C
WT Y 3
3 C
K 6
0 M
7 C
K 6
0 M FGFR3:
IP : FGFR3
IB : Ubiquitin
160
kDa 250
c-Cbl : c-Cbl70Z:
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.
Trang 10resulted 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
3 C
K 6
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
3 C
K 6
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).