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INTRODUCTION |
The fibroblast growth factor receptor
(FGFR)1 family of
receptor tyrosine kinases mediates growth, differentiation, and
cell migration in a wide range of cell types (1, 2). FGFR3 is one of
four distinct members of the FGFR family that serve as high affinity
receptors for more than 20 different fibroblast growth factors (FGFs)
(3). Binding with a specific ligand together with heparan sulfate
induces receptor dimerization, trans phosphorylation, and activation
followed by receptor internalization and down-regulation (4-6). This
leads to the controlled activation of specific signal transduction
pathways (7). In particular, the membrane-linked docking proteins
FRS2
and FRS2
function as major mediators of signaling by FGF
(8). Recently, it has been shown that several human congenital skeletal
disorders result from point mutations in the FGFR3. Indeed, mutations
in different domains of FGFR3 are implicated in several clinically
related forms of short-stature with graded severity including
achondroplasia, hypochondroplasia, and the neonatal lethal syndrome
thanatophoric dysplasia (TD) (9). TDII is one form of neonatal lethal
dwarfism and results from a lysine (K) to glutamate (E) substitution at
position 650 (644 in mouse) of FGFR3 (10). The K650E mutation is
located in the activation loop of the kinase domain and is associated to strong, ligand-independent constitutive receptor autophosphorylation (11). In in vitro studies and in mice carrying the TDII
mutation it was shown that the signal transduction and activator of
transcription STAT1 is activated and translocated into the nucleus
(12). This, together with activation of the cell cycle inhibitor
p21waf1 was proposed as the molecular mechanism responsible
for the TDII pathology (13). Furthermore, ligand-independent activation
of the STAT signaling pathway was demonstrated in cultured TD cells and
confirmed by immunodetection of activated STAT1 associated to apoptosis
of chondrocytes in TD fetus (14, 15). However, the underlying mechanism
of how the activated TDII-FGFR3 causes disrupted proliferation and
organization of the cartilaginous growth plate of long bones in the
TDII remains unclear.
In this study we have investigated whether additional means by TDII
mutation in FGFR3 could affect receptor signaling. Biosynthesis of
FGFR3 is characterized by three isoforms with various degrees of
N-glycosylation: a 98-kDa unglycosylated native protein, a 120-kDa intermediate membrane-associated glycoprotein, and a 130-kDa mature glycoprotein (16). In particular, we have questioned whether
TDII-FGFR3 intermediates played a role in the signal transduction. We
show herein that the K644E mutation causes the immature phosphorylated TDII-FGFR3 intermediate glycomers to activate STAT1 from the ER. As far
as we know, this is the first report of a tyrosine kinase receptor that
signals from within the cell in its immature form. We discuss that a
novel intracellular signal transduction pathway may be relevant in the
pathogenesis of FGFR3-mediated TDII.
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MATERIALS AND METHODS |
FGFR3 Constructs--
Murine FGFR3 cDNA (17) was kindly
provided by C. Basilico. The cDNA was subcloned into pBSKII
(Stratagene) and the K644E (TDII) mutation was obtained by
polymerase chain reaction (PCR) using the following primers:
5'-GAGGAACAGCTCACCTGCA-3' (mFR3F1), 5'-CATTTGTGGTCTCCTTGTAGTA-3'
(mFR3644R), 5'- TACTACAAGGAGACCACAAATG-3' (mFR3644F), and
5'-CAGGAACAGCTCACCTGCA-3' (mFR3R1). The 5'-half of the wild type mFR3
was amplified with primers mFR3F1 and mFR3644R, whereas the 3'-half of
the same cDNA was amplified with primers mFR3644F and mFR3R1. 5'
and 3' PCR products were then mixed and amplified again with primers
mFR3F1 and mFR3R1. After cleavage with BstEII and
BglII enzymes the secondary PCR product was cloned into
pBSKIImFR3 that had been cleaved with the same two enzymes. The
structure of the DNA construct was confirmed by sequencing, and both
the wild type and the K644E mFGFR3 cDNAs were then subcloned into
the expression vector pRcCMV (Invitrogen). A double HA sequence was
introduced in frame at the 3' end of both the wt and the TDII mutant
mFR3 cDNAs as now described. A first PCR product was obtained by
combining 5'-GAGGAACAGCTCACCTGCA-3' (mFR3F1) and
5'-GGGAGCGTAATCTGGAACATCGTATGGGTACGTCCGAGGTCCCCCGTT-3' (mFR3HaR,
containing the first HA sequence and the half-site of a SmaI
restriction enzyme sequence), and the obtained amplified DNA was
digested with BstEII. The second HA sequence was
obtained by excising it from the yeast vector pYX (R&D System) using
SmaI and NheI. Both inserts were purified and
ligated to the pRcCMVmFR3 digested with BstEII and
XbaI. Positive clones were verified by sequencing analysis.
Finally, mFGFR3-GFP were obtained by subcloning the cDNAs into the
vector pEGFPN1 (Clontech).
Cell Culture and Transfections--
Cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum (Euroclone), 2 mM L-glutamine, and
penicillin/streptomycin. Transient transfection of HEK293, NIH3T3 and
Cos7 cells was performed with FuGENE (Roche) according to
manufacturer's protocols. RCS cells were transfected with
calcium-phosphate technique. KMS11 cells (kindly provided by A. Neri)
were maintained in RPMI supplemented with 10% fetal bovine serum. For
stimulation with FGF9 (R&D), cells were serum-deprived for 12 h,
and then FGF9 was added for 10 min at 100 ng/ml in the presence of
Heparin (Sigma).
Immunoprecipitation and Western Blot--
Transfected cells were
lysed in radioimmune precipitation assay buffer buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) in the presence of a mixture of protease and phosphatase inhibitors. Cell lysates were clarified by centrifugation and then subjected to specific
immunoprecipitation using the following antibodies: anti-HA (Roche),
anti-FGFR3 C-15, anti-STAT1 E-23, and anti-FRS2
H-91 (Santa Cruz).
Immunocomplexes were collected with protein A-Sepharose (Amersham
Biosciences), washed three times with radioimmune precipitation assay
buffer, once with Tris-buffered saline buffer and resuspended in 2×
gel loading buffer. STAT1 and FRS2
proteins were separated by 7.5% SDS-PAGE, whereas FGFR3 proteins were resolved by 7% Tricine gels (18). Proteins were transferred onto polyvinylidene difluoride membranes (ImmobilonP, Millipore), blocked with 5% milk in
Tris-buffered saline, pH 7.5, and incubated with desired antibodies.
Phosphorylated STAT1 proteins were detected by specific
anti-phospho-STAT1 antibodies (Cell Signaling), whereas
phosphorylated FGFR3 proteins were revealed by anti-phosphotyrosine
PY99 (Santa Cruz). Phosphorylated FRS2
proteins were detected by
anti-phosphotyrosine-RC20:HRPO antibodies (Transduction Lab).
Deglycosylation--
Where indicated, immunoprecomplexes were
resuspended in 50 mM sodium citrate, pH 5.5, containing 1%
SDS and 1%
-mercaptoethanol and boiled for 10 min. Endoglycosidase
H (Endo-H) was added directly to the recovered samples. Concerning the
PNGase F (New England Biolabs) reactions, samples were diluted
1:1 with 50 mM sodium citrate, and 1% of Nonidet P-40 was
added prior to adding the enzyme. Both enzymatic reactions were carried
out at 37 °C for 2 h.
Metabolic Labeling--
Confluent cultures of HEK293 cells were
starved in methionine/cysteine-free Dulbecco's modified
Eagle's medium medium (ICN) for one hour prior to labeling with
200 µCi/ml of [35S]methionine/cysteine (Pro-mix,
Amersham Biosciences). Where indicated, cells were labeled in the
presence of 5 µg/ml brefeldin A (BFA) or 5 µg/ml tunicamycin.
Immunocytochemistry--
Cells were grown on glass coverslips
surfaced with 10 µg/ml of polylisine and transfected with wt and TDII
GFP-conjugated constructs for 24, 48, or 72 h. Cells were then
rinsed with phosphate-buffered saline and fixed with 4%
paraformaldeide, permeabilized with 0.4% of Triton X-100, and
incubated with anti-calreticulin antibodies diluted 1:100 for 1 h
at room temperature. The specific labeling was revealed by using
secondary rhodamine-conjugated antibodies (Alexa 594, Molecular
Probes). Cell membranes where visualized with fluorescent conjugates
cholera toxin B subunit from Molecular Probes. To detect apoptosis, we
used antibodies directed to the cleaved p85-poly(ADP)ribose polymerase
(PARP) (19) (Promega) at 1:100 dilution. Analyses were performed with
confocal microscopy (Zeiss).
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RESULTS |
Biosynthesis of wt-FGFR3--
To study the biosynthesis of the
FGFR3, a double HA epitope-sequence was inserted at the 3' end of
murine wt-FGFR3 cDNA. HEK293 cells were transfected with wt-FGFR3
and several G418-resistant cell clones were isolated. The FGFR3
biosynthetic pattern was studied upon immunoprecipitation and Western
blot with anti-HA antibodies. Two specific bands of ~120
(II) and 130 (III) kDa, were observed in the
wt-FGFR3 (Fig. 1, lane 4) as
detected by anti- HA antibodies and according to what was previously
described (16). Enzymatic digestion with PNGase F caused the
disappearance of both 120 and 130 kDa species that were reduced to an
intense 98-kDa band, thus showing that the higher molecular mass
species are N-linked glycosylated isoforms (Fig. 1,
lane 6). Differently, Endo-H treatment resulted in the
disappearance only of the intermediate 120 kDa species, therefore
indicating their presence as high-mannose glycomers still residing into
the ER/cis-Golgi compartments (Fig. 1, lane 5). An identical
protein profile was detected for the endogenous FGFR3 in the multiple
myeloma KMS11 human cells that present the G373C FGFR3 mutant (20)
(Fig. 1, lanes 1-3). Endogenous wt-FGFR3 from
RCS chondrocytic cells presented the same pattern (not shown). The
identity of the FGFR3 protein profiles ruled out any interference by
the HA epitope on the normal processing of the FGFR3-HA protein.

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Fig. 1.
Biosynthesis of FGFR3. Cell lysates from
KMS11 and 293wtFR3-HA cell lines were immunoprecipitated as indicated
and analyzed by Western blot with anti-FGFR3 (lanes 1-3) or
anti-HA (lanes 4-6) antibodies. Bands I,
II, and III correspond to the 98-, 120-, and
130-kDa forms, respectively. Untreated (lanes 1 and
4) or treated with Endo-H (lanes 2 and
5) or PNGase F (lanes 3 and 6) as
indicated. HA-tagged FGFR3 and endogenous FGFR3 from KMS11 cells show
the same protein profile.
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Biosynthetic Pattern of TDII-FGFR3--
A comparative analysis
between wt and mutated TDII-FGFR3 biosynthesis was performed by
transient transfection of the FGFR3 HA-tagged molecules into HEK293
cells followed by metabolic labeling with
[35S]methionine/cysteine. wt-FGFR3 exhibited the three
isoforms described above (Fig.
2A, lane 2).
Surprisingly, only the 98-kDa and a predominant 120-kDa bands
(I and II) were detected in TDII (Fig.
2B, lane 2). To determine whether the different
pattern between wt and TDII receptors was ascribed to an anomalous
glycosylation or to uncompleted maturation of the TDII receptor,
analysis with specific enzymes and drugs were performed. Because sugar
composition of glycoprotein reflects their location in the secretory
pathway (21), Endo-H was considered as diagnostic for the position of the band II intermediates. As already mentioned for wt-FGFR3, also the
120-kDa TDII proteins were Endo-H-sensitive, thus confirming their
nature as immature intermediates located within the ER/cis-Golgi compartments (Fig. 2, A and B, lanes
3). We further confirmed the location of the 120-kDa TDII-FGFR3 by
performing experiments with the fungal drug BFA, which enhances
retrograde traffic from Golgi to ER (22). BFA inhibits the formation of
the mature form (III) of wt-FGFR3 (Fig. 2A,
lanes 4 and 5). In TDII, BFA does not affect the
protein profile thus confirming that the 120-kDa species reside in the
ER (Fig. 2B, lanes 4 and 5).
Inhibition of glycosylation by tunicamycin blocks wt and TDII
biosynthesis at the immature unglycosylated band I (Fig. 2,
A and B, lanes 6). We further
investigated the possibility that the predominance of the band II in
TDII was due to a peculiarity of the HEK293 cells. For this purpose,
transfection experiments were performed in NIH3T3 as well in Cos7
cells. In both cell types, TDII-FGFR3 biosynthetic pattern was
characterized by the absence of the mature form and by Endo-H-sensitive
intermediate glycomers (not shown). In addition, considering that
chondrocytes are the cells mostly affected by FGFR3 mutation in the
growth plate, we tested whether TDII-FGFR3 showed the same biosynthetic
pattern in the RCS cells. As expected, only the 120-kDa form was
observed in RCS cells transfected with TDII-FGFR3 molecules (not
shown). A kinetic of [35S]methionine/cysteine
incorporation was performed to test whether TDII-FGFR3 required a
longer time to be processed into the mature form. After 5 h of
labeling, the mature 130-kDa forms in TDII were still undetected,
whereas the wt mature forms were clearly detected after 90 min of
labeling (Fig. 2C). In addition, after 15 min of labeling,
we consistently observed a comparable 1:1 ratio between the 98 and the
120-kDa species in TDII (Fig. 2C). This suggests a possible
delay in the processing of the native isoforms into the following
maturation step, whereas, in the wt-FGFR3, the native forms were
rapidly glycosylated (Fig. 2C).

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Fig. 2.
Biosynthesis of wt- versus
TDII-FGFR3. Metabolic labeling of HEK293 cells transfected
with wt-FGFR3 or the mutant TDII-FGFR3. Cells were immunoprecipitated
with anti-HA antibodies followed by autoradiography. A,
synthesis of wt-FGFR3. Three forms are detected: form I is
unglycosylated and forms II and III represent
different sugar compositions. The form II is sensitive to
Endo-H, whereas form III is resistant to Endo-H. BFA
inhibits formation of the mature form III. Tunicamycin reduces to a
unique unglycosylated 98 kDa form I. B, synthesis
of TDII-FGFR3. Two bands are detected: unglycosylated form I
and immature, Endo-H-sensitive form II. The mature form
III is undetected. BFA does not alter band pattern as for
wt-FGFR3. C, kinetic labeling of wt- and TDII-FGFR3. Cells
are exposed to [35S]methionine for different time
intervals as indicated. At 90 min the mature form III
appears in wt-FGFR3. In TDII-FGFR3, the mature form III is
absent after 300 min labeling. The asterisk shows a low
molecular weight band recognized only by anti-carboxyl-terminal FGFR3
antibodies. It probably belongs to a truncated form of the receptor
(seen by Western blot, not shown).
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Intracellular Localization of TDII Mutant Receptor--
To
visualize the localization of the TDII intermediates within the cell,
the GFP protein was fused at the C terminus of wt and TDII receptors.
48 h after transfection, HEK293 cells were fixed and stained with
antibodies that recognize calreticulin, which is the major
calcium-binding protein found in the ER (23). Merging analysis by
confocal microscopy showed that TDII-GFP co-localize with calreticulin
in the ER (Fig. 3). The same result was
obtained when cells were analyzed at 72 h post transfection (not
shown). Furthermore, by staining the cells with rhodamine-conjugated
cholera toxin peptides, which specifically recognize membrane lipid
raft, we were able to confirm the absence of TDII-FGFR3 from the cell surface (Fig. 4). On the contrary, the
wt-FGFR3-GFP was clearly exposed on the cell surface (Fig. 4).

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Fig. 3.
TDII-FGFR3 co-localizes with
calreticulin in the ER. Confocal microscopy analysis of
HEK293 cells transfected with wtFGFR3-GFP or
TDIIFGFR3-GFP molecules (green). Following
transfection, cells were fixed and incubated with calreticulin
antibodies that were revealed by rhodamine-conjugated secondary
antibodies (red). Merge analysis (yellow) showed
co-localization of TDII-FGFR3 with calreticulin in the ER. Bar
magnification is indicated.
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Fig. 4.
FGFR3 with the TDII mutation is
not processed on the cell membrane. Confocal microscopy
analysis of HEK293 cells transfected with wtFGFR3-GFP or
TDIIFGFR3-GFP molecules (green). At 48 h following
transfection, cells were fixed and incubated with
fluorescent-conjugated cholera toxin B peptides (red). Merge
analysis showed the presence of the wt-FGFR3 on the cell surface. On
the contrary, the TDIIFGFR3-GFP molecules where not present on the cell
surface as indicated by arrows. Bar magnification is
indicated.
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STAT1 but Not FRS2
Is Activated in the Presence of the
Intermediate TDII-phosphorylated Glycomers--
As previously reported
(13), the human K650E mutation strongly activates FGFR3, which,
in turn, activates STAT1. We have investigated whether the
intracellular intermediate TDII receptor was phosphorylated and, most
importantly, whether STAT1 was activated in cells transiently
transfected with TDII-FGFR3. For this purpose, cell lysates from HEK293
transfected with the wt- or TDII-FGFR3 were first immunoprecipitated
with anti-HA and subsequently with anti-STAT1 antibodies. The collected
immunocomplexes were then analyzed by immunoblot with
anti-phosphotyrosine and anti-phospho-STAT1 antibodies. Interestingly,
the immature 120-kDa TDII-FGFR3 glycomers were phosphorylated (Fig.
5A). Accordingly, STAT1 was
phosphorylated in TDII but not in wt-FGFR3 cells (Fig. 5B).
In addition, when TDII cells were treated with BFA for 5 h, STAT1
was still detected in its phosphorylated form, thus confirming that
such an activation occurred even if the traffic out the ER was
inhibited (not shown). Furthermore, we have investigated whether the
docking protein FRS2
, which is anchored on the cell membrane, could
be activated by TDII intermediates. We did not detect phosphorylated
FRS2
in the TDII cells (not shown).

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Fig. 5.
STAT1 is activated in the presence of
phosphorylated TDII-FGFR3 immature forms. A, wt-FGFR3
presents forms II and III, whereas TDII-FGFR3
shows only the intermediate forms II. Anti-phosphotyrosine
antibodies recognize the intermediate forms (II) in
TDII-FGFR3 but not of wt-FGFR3. B, cells transfected with
TDII or wt-FGFR3 or not transfected (mock) were
immunoprecipitated with anti-STAT1 antibodies. HeLa cells were
stimulated with IFN as positive control for STAT1 activation. As
shown, STAT1 appeared phosphorylated only in TDII-FGFR3 but not
in wt-FGFR3 cells. Antibodies used for IP
(immunoprecipitation) or WB (Western blot) are
indicated.
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STAT1 Activation Is Abrogated in Stable Cell Clones Selected to
Express TDII-FGFR3--
We obtained stable TDII-FGFR3 cell clones,
although the number of the positive TDII colonies was ~50% less
compared with the number of wt-FGFR3 selected cell clones.
Analysis of FGFR3 showed that the 120-kDa intermediate glycomers were
highly represented in all TDII cell clones although, to a lesser
extent, the mature 130-kDa form was also present (Fig. 6A). Of note, in independent
clones showing different levels of FGFR3 expression, the intermediate
120-kDa forms were always favored. Both forms of the receptor were
constitutively phosphorylated. Interestingly, the phosphorylation level
of 120 and 130 kDa bands were comparable in intensity, suggesting a
higher degree of phosphorylation of the mature forms versus
the intermediates (Fig. 6A). Alternatively, the
phosphorylated 130-kDa mature proteins include endogenous co-immunoprecipitated FGFR3. Remarkably, in all the cell clones analyzed, we were unable to detect phosphorylated STAT1 protein (Fig.
6B). To determine whether failure in STAT1 activation was due to a cellular defect in the activation of the STAT1 pathway, cells
were stimulated with
interferon. STAT1 was phosphorylated in
wt and TDII clones as well as in the parental cells treated with
interferon (Fig. 6C). We deduced that only the cells that had lost the ability to activate STAT1 through the TDII mutant would be
favorably selected to become permissive for the TDII-FGFR3. This
indicates that the selection process favored cells defective of a
specific factor(s) linked to TDII-FGFR3 and playing a major role in
mediating STAT1 activation. The situation was different for FRS2
docking protein. Indeed, FRS2
was present as a constitutively phosphorylated protein only in the TDII cell clones (Fig.
6D). Finally, wt and TDII cell clones were treated with
FGF9, a specific ligand for FGFR3. This stimulation resulted in the
phosphorylation of FRS2
in the wt clones, whereas in TDII the
FRS2
remained unchanged in its constitutively phosphorylated form.
Differently, FGF9 did not induce STAT1 phosphorylation in both wt and
TDII cell clones (not shown).

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Fig. 6.
Analysis of TDII proteins in stable
TDII-HEK293 cell clones. A, both immature
(II) and mature (III) species were expressed in
TDII stable cell lines. Although the TDII intermediate isoforms
(II) were predominant, anti-phosphotyrosine antibodies
showed a comparable degree of phosphorylation for both II and III
species (showed in the three independent clones 1, 2, and 3). In the wt
stable cell lines species II and III were equally represented and not
phosphorylated. B, STAT1 was not phosphorylated in
TDII-FGFR3 clones despite phosphorylation of TDII receptors.
C, interferon induces phosphorylation of STAT1 in
TDII and wt clones, thus showing the integrity of that pathway of
activation. D, FRS2 is constitutively activated in
the TDII but not in wt clones. Antibodies used for IP
(immunoprecipitation) and WB (Western blot) are
indicated.
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Apoptosis in TDII-FGFR3 Cells--
To determine whether TDII-FGFR3
triggered apoptosis, cells were transfected with the wt or the TDII-GFP
receptor constructs. At 72 h cells were fixed, incubated with
antibodies that recognize only the cleaved p85-PARP, followed by
rhodamine-conjugated secondary antibodies. As shown in Fig.
7, apoptosis was observed in ~40% of
TDII cells as measured by the number of cells showing double fluorescence.

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Fig. 7.
Apoptosis in TDII-FGFR3-GFP cells.
HEK293 cells were transfected with wt or TDII-FGFR3-GFP molecules or
GFP alone. 72 h later cells were fixed and incubated with
anti-p85-PARP antibodies as indicators of apoptosis.
Rhodamine-conjugated secondary antibodies were used. Several fields
were examined with confocal microscopy. Green (GFP) and red (cleaved
PARP) cells were counted, and only cells with the double stain were
considered apoptotic. The data showed here represent three independent
transfection experiments that gave similar results.
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DISCUSSION |
TDII mutation in FGFR3 strongly activates receptor signaling, as
measured by tyrosine kinase activity and induction of cell proliferation in several cell lines (e.g. BaF3, NIH3T3)
(24). However, the TDII disease is characterized by disrupted
proliferation of chondrocytes in the growth plate, and ultimately the
clinical severity parallels the profound kinase activation of the
receptor (25). This paradox was reconciled by demonstrating
that FGFR3 signaling can operate along two different pathways. One, the
Ras-MAPK (mitogen-activated protein kinase) pathway, leads to cell
proliferation (26), whereas the other pathway, operating through STAT1,
induces cell cycle inhibition (27). Therefore, we have reasoned that the intrinsic tyrosine kinase activity of the TDII receptor, per se, was not sufficient to justify the devastating effects observed in TDII. Our first approach in this study was to determine whether biosynthesis of the TDII-FGFR3 presented anomalies. For this purpose we
have generated FGFR3 (mutant and wt) tagged with the HA epitope. This
allowed us to follow the biosynthesis of the receptor upon transfection
in HEK293 cells. At first we ruled out any interference by HA in
relation to both synthesis and activation of the FGFR3 by comparing the
biosynthetic pattern of wt-FGFR3-HA with endogenous FGFR3 from
different cell lines.
Interestingly, we observed that the K644E substitution hampers the
intermediate glycosylated 120 kDa forms to exit the ER. This is based
on the following observations: first, the mature 130-kDa form was not
detected; second, the TDII 120-kDa intermediates were sensitive to
Endo-H, which is specific for high-mannose sugars on glycoproteins
resident in the ER/cis-Golgi compartments. Indeed, Endo-H is considered
as diagnostic for the position of a glycoprotein in the secretory
pathway. The same results were obtained when TDII-FGFR3 was transiently
transfected into NIH3T3 or chondrocytic RCS cells, thus confirming that
accumulation of the immature TDII form was due to a property of the
mutated protein rather than to a defect of HEK293 cell surveillance on
protein synthesis. Furthermore, the results indicate that the switch
from a basic 644 lysine to the negatively charged glutamic acid, rather
than the tissue specificity, plays a pivotal role in the retention of
TDII receptor within the ER. In addition, the molecular weight of the
protein species and the profiles obtained with specific enzymes do not
indicate a defect in the glycosylation of TDII-FGFR3.
Most importantly, we show that the immature 120-kDa forms of TDII
receptor were phosphorylated. Furthermore, STAT1 was activated upon
TDII transfection, and this coincided with a certain level of
apoptosis, in line with a previous report (15). An important, yet
unanswered, question is whether TDII-FGFR3 is retained in the ER in a
monomeric or dimeric form. Nonetheless, no traces of the endogenous
mature 130-kDa FGFR3 were observed after five hours of metabolic
labeling followed by immunoprecipitation with anti-HA antibodies. We
can not exclude, however, the possibility that the endogenous 120-kDa
immature FGFR3 forms dimerize with TDII and therefore are retained in
the ER. However, it has been previously shown (28) that
Xenopus FGFR2 with the corresponding K652E mutation does not
require dimerization for signaling. Further evidence that confirms the
signaling by TDII-FGFR3 from the ER was obtained with BFA. Indeed, by
blocking the traffic out of the ER, STAT1 was still activated upon transfection.
A different situation was recorded in the stable TDII-FGFR3 cell clones
in which both 120 and 130 kDa receptor forms were present in their
phosphorylated state. At present, we cannot yet explain why the p130
form is present in stable TDII cell clones, however, we can speculate
on two possibilities. The first one is that the defect in p130
processing may be bypassed, and over time some mature forms accumulate
in stable transfectants. The second hypothesis, which is under study,
is that a factor, missing in the stable TDII clones, interacts with the
120-kDa receptor forms in the ER, thus inhibiting, somehow, the regular
TDII-FGFR3 processing. It remains to be determined whether STAT1 plays
a direct role in such a mechanism. Indeed, surprisingly, it was not
detected in its active configuration in stable TDII cells. The fact
that STAT1 is activated only by the immature TDII-FGFR3 but not upon
stimulation of stable clones with FGF9 supports the hypothesis that
STAT1 plays a critical role in the TD (13, 15). In line with the above
observations are the data obtained with human KMS11 and OPM-2 myeloma
cells in which with a strong auto-phosphorylation of FGFR3, carrying
the G373C and K650E mutations, respectively, there is not correspondent
STAT1 activation (29). Treatment of stable TDII cell lines with
the
interferon showed activation of STAT1, thus indicating that
this activation cascade was not affected and that STAT1 itself has not
lost the ability to be phosphorylated. We hypothesize that the
selection process favored cells in which some critical protein(s)
involved in the FGFR3 signaling was missing, thus leaning toward an
oncogenic potential. To verify if a switch in the signaling occurred in
TDII clones, phosphorylation of FRS2
protein was studied.
Interestingly, we have not found phosphorylated FRS2
upon
transfection of the TDII molecules. On the contrary, we detected
phosphorylation of FRS2
in all the stable TDII cell clones analyzed.
Considering that FRS2
is a myristylated protein, which is anchored
on the cytoplasmic membrane, the failure in its activation represents
further evidence that TDII-FGFR3 signals from the ER. Moreover, the
fact that FRS2
fails to be activated upon TDII transfection
indicates that the TDII receptor does not phosphorylate the endogenous FGFR3.
In conclusion, we show that phosphorylation per se may not
be sufficient to explain the severity of the disease. Indeed, we suggest that critical roles are played by phosphorylated immature receptor glycomers that can activate specific substrate(s) from within
the cell.