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INTRODUCTION |
The Wnt proteins comprise a highly conserved, multimember ligand
family, which play important roles in patterning and cell fate
determination (1, 2). A number of downstream components of Wnt
signaling have been identified by a combination of genetic and
biochemical approaches. Wnts act through the cytoplasmic protein Dishevelled to inhibit the activity of the serine-threonine kinase GSK3. GSK3 appears to bind through a bridging molecule, Axin, to the
-catenin-APC1 complex and
phosphorylate
-catenin causing its rapid degradation. Wnt-induced
inhibition of GSK3 leads to
-catenin stabilization resulting in an
increased level of the uncomplexed soluble form (3-5). The latter form
can interact with TCF/LEF transcription factors and, after
translocation to the nucleus, activate target genes (6).
There is evidence that activation of Wnt signaling can contribute to
the neoplastic process (7, 8). Inappropriate expression of these
ligands due to promoter insertion of the mouse mammary tumor virus (1)
or targeted expression in transgenic mice causes mammary tumor
formation (9). Moreover, in cell culture, several Wnt family members
have been shown to induce altered morphology and increased saturation
density of certain epithelial (10, 11) and fibroblast (12, 13) cell
lines. Finally, genetic alterations affecting APC or
-catenin,
associated with increased uncomplexed
-catenin levels, have been
observed in human colon cancers (14), melanomas (15), and
hepatocellular carcinomas (16), indicating that aberrations of Wnt
signaling pathways are critical to the development of these and
possibly other human cancers.
Recent studies have identified the products of the multi-member
frizzled family as Wnt receptors (17). These proteins are characterized
by a large cysteine-rich extracellular domain
(CRD), a seven-transmembrane spanning
domain, and a cytoplasmic tail. Exogenous expression of
Drosophila frizzled 2 (Dfz2) in a
suitable recipient insect cell line conferred to the
Drosophila Wnt prototype, Wingless (Wg), the ability to bind
to the cell surface and to signal by increasing intracellular levels of
-catenin. Furthermore, transfection of Dfz2 or several
different mammalian fz cDNAs into human 293T cells conferred the
ability to bind Wg. The fz CRD has been shown to be its Wnt binding
domain by the ability of Wg to bind to a
glycosylphosphatidylinositol-anchored Dfz CRD in the absence of the
transmembrane portion (17). More evidence implicating fz as the Wnt
receptor has come from experiments performed in the Xenopus
embryo system in which Wnt induced axis duplication when coinjected
with members of the fz family (18).
A family of related molecules, which contain N-terminal CRDs highly
similar to the fz CRD but then diverge and lack any transmembrane domain, has recently been identified. When coexpressed with Wnt family
members in Xenopus embryos, such proteins including human frizzled related protein (FRP) and Frzb-1 were shown to antagonize Wnt-induced duplication of the dorsal axis (19-21), indicating that
these proteins may function as inhibitors of Wnt action during development. Frzb-1 has been further shown to coimmunoprecipitate with
Wnt proteins in vitro and after cotransfection of COS7 cells (20). Moreover soluble Frzb-1 was shown to bind to 293 cells expressing
a WNT-1 transmembrane chimera (19). In addition, mouse members of this
family expressed as glycosylphosphatidylinositol-anchored fusion
proteins conferred to Wg the ability to bind 293 cells (22). More
recently Lin et al. (23) have provided evidence that the
bovine Frzb-1 can prevent WNT-1-induced cytosolic accumulation of
-catenin in 293 cells and have localized sites within the CRD, which
are critical for WNT-1 binding and inhibition of WNT-1-mediated axis
duplication in Xenopus embryos. The present studies were undertaken to investigate the mechanisms by which FRP antagonizes Wnt
function. The results have implications with respect to how FRP
modulates the complex functions of the Wnt multimember family of
developmentally regulated signaling molecules.
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MATERIALS AND METHODS |
Plasmid Construction--
pCEV/WNT-1-HFc and
pCEV/WNT-2-HFc, in which the IgGHFc open reading frame (24)
was fused in frame downstream of the wnt coding region, were
previously described (12). The pcDNA/WNT-2-HFc was
constructed by inserting WNT-2-HFc into pcDNA3
(Invitrogen). pBabe/WNT-2-HFc containing the puromycin
resistance gene was constructed by inserting the WNT-2-HFc
fragment into pBabe, under the control of the cytomegalovirus
transcriptional unit. pLNCX/Wnt-1-HA was kindly provided by
Dr. J. Kitajewski (Columbia University, New York). In
pMMT/FRP, the FRP coding region was introduced
into MMTneo (24). To construct pcDNA/FRP-HA or
pcDNA/FRP-FLAG, the FRP coding region lacking
its stop codon (21) was PCR-amplified using the Expand High Fidelity
PCR system (Roche Molecular Biochemicals) as described previously (12),
in the presence of a forward BamHI-flanked primer,
GGAAGGATCCGCCGGCATGGGCATCGGGCGCA, that included a Kozak consensus
sequence and a reverse EcoRI-flanked primer,
GGCCGAATTCCTTAAACACGGACTGAAAGGTGGG. The FRP PCR product was
inserted in frame into pcDNA3, upstream of an
EcoRI-flanked HA (YPYDVPDYA) or M2 (DYKDDDDK) (Eastman Kodak Co.) epitope-encoding sequence. To construct
pcDNA/CRD-FRP-HA or pcDNA/CRD-FRP-FLAG,
the N-terminal fragment of FRP was PCR-amplified using the
above mentioned forward BamHI-flanked primer and a reverse EcoRI-flanked primer, GGAAGAATTCGGCGATGCAGACGTCCCCCTCCGG,
and the PCR product was introduced in frame upstream of the HA- or M2-encoding sequence, as detailed above. To construct
pcDNA/CRD-Hfz6-FLAG, the N-terminal domain of
Hfz6 (GenBankTM accession number AF072873) was
PCR-amplified using the forward HindIII-flanked primer,
GGAAAAGCTTGCCACCATGGAGATGTTTACATTTTTGTTG, that included a Kozak
consensus sequence and a reverse EcoRI-tagged primer,
GGAAGAATTCGCATGGAGGCGCACACTGGTCAAT. The PCR product was introduced in
frame upstream of M2-encoding sequence as detailed above. Sequence
analysis was performed to ascertain the authenticity of the
PCR-generated products. pGST-E-cadherin was described previously (12).
Two TCF/Luc reporter constructs, pGL3-OT containing TCF-responsive domains and pGL3-OF containing mutant-binding sites, were kindly provided by B. Vogelstein (Johns Hopkins Oncology Center, Baltimore). A
schematic diagram of the constructs generated for this study is shown
in Fig. 1.
Stable Transfection--
NIH3T3 cells were plated at 1.5 × 105 cells per 100-mm dish. After 24 h cells were
transfected with 1 µg of each plasmid DNA, by the calcium phosphate
coprecipitation method (25) as described (26). Cultures were incubated
with the DNA precipitates for 18-20 h and then washed in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% calf serum (Life
Technologies, Inc.). To ensure selection of cells coexpressing WNT-2
and FRP, different selectable markers were utilized in each construct.
Cotransfectants were doubly selected by addition of 750 µg/ml
geneticin (Life Technologies, Inc.) and 2 µg/ml puromycin
(Calbiochem) to the medium. Cells expressing WNT-2 or
FRP individually were cotransfected with control vectors
containing the other selectable marker.
Transient Transfection--
For transient transfection 70-80%
confluent cultures of 293T cells were transfected by the calcium
phosphate method using 3 µg of each plasmid DNA. When necessary the
amounts of DNAs used for cotransfection were adjusted to yield similar
levels of protein expression. Cells were exposed for 5-6 h to the DNA
precipitates and then washed in growth medium. After 48 h, cells
were harvested and cell lysates prepared for analysis as indicated below.
GST-E-cadherin Binding Assay--
GST-E-cadherin was expressed
in bacteria and purified from the bacterial lysates by binding to
glutathione-Sepharose beads as described previously (12). Cells were
lysed in immunoprecipitation buffer (10 mM sodium
phosphate, pH 7, 0.15 M NaCl, 1% Nonidet P-40, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 2 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride), and cell extracts were
clarified by centrifugation. In order to perform a quantitative
analysis, two different amounts of total protein (0.1 and 1 mg) were
analyzed for each sample by incubation with Sepharose beads (Amersham
Pharmacia Biotech) bound to the GST-E-cadherin. After 1 h of
incubation with rotation at 4 °C, the beads were collected by
centrifugation, washed, and dissolved in Laemmli buffer. Samples were
subjected to SDS-PAGE followed by immunoblotting with 0.5 µg/ml
anti-
-catenin antibody (Transduction Laboratories). Detection was
per- performed with 125I-labeled protein A (Amersham
Pharmacia Biotech).
Paracrine Assay for Wnt Signaling--
The paracrine assay was
performed as described.2
Briefly, 293T cells transiently transfected with 1 µg of
pLNC/Wnt-1-HA or with an empty vector were cocultured with
293T cells transiently transfected with 1 µg of
pcDNA/FRP-HA, pGL3-OT, or pGL3-OF and 0.1 µg of
-galactosidase expression vector. After 48 h, the relative luciferase units (RLU) were measured using the Promega luciferase assay
system according to the manufacturer's protocol. The activities were
normalized for transfection efficiency using
-galactosidase activity. TCF/LEF-dependent luciferase activity was
calculated by subtracting RLU levels obtained with the pGL3-OF reporter
from those obtained by pGL3-OT (27).
Western Blot and Coimmunoprecipitation--
Cultures were
solubilized in lysing buffer (0.01 M phosphate buffer, 1%
Triton, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 M NaCl, 5 µM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM sodium vanadate, 2 mM phenylmethylsulfonyl
fluoride). Around 100 µg of total cell lysates were resolved by
SDS-PAGE, and proteins were transferred to Immobilon-P membranes. After
blocking with 5% bovine serum albumin in PBS, filters were incubated
with the specific primary antibody (2.6 µg/ml horseradish
peroxidase-conjugated rabbit anti-mouse HFc, Dako; 10 µg/ml anti-FLAG
M2, Kodak; 1.5 µg/ml anti-HA obtained from the Hybridoma Center,
Mount Sinai School of Medicine, New York). When the anti-FLAG or the
anti-HA primary antibodies were used, membranes were washed in
PBS/Tween 20 and incubated for 1 h with a secondary horseradish
peroxidase-conjugated rabbit anti-mouse antibody. After washing 6 times
in PBS/Tween 20, membranes were subjected to ECL analysis (Amersham
Pharmacia Biotech). For coimmunoprecipitation analysis, cells were
solubilized in lysing buffer (see above), and extracts were clarified
by centrifugation at 10,000 × g for 20 min at 4 °C.
Around 1.5-3 mg of total cell lysates were incubated with the specific
antibody (24 µg of goat anti-mouse HFc, Pierce; 10 µg of anti-FLAG
M2; 15 µg of anti-HA) for 1 h on ice and then incubated with
rotation with protein A beads (Pierce) for 1 h at 4 °C. The
beads were collected by centrifugation, washed three times in
immunoprecipitation buffer, dissolved in Laemmli buffer, and analyzed
by SDS-PAGE, followed by detection with each specific antibody. The
extracellular matrix fraction was prepared as follows. Cultures were
washed twice with PBS containing 1 mg/ml aminocaproic acid and 1 mM phenylmethylsulfonyl fluoride and then incubated with
the same solution containing 5 mM EGTA for 15 min at
37 °C. Cells were removed, and the plates were washed 2-3 times
with the PBS solution and twice with distilled H2O. The
extracellular matrix was then solubilized in 200 µl of Laemmli buffer.
Metabolic Labeling--
Subconfluent 293T cultures were
transiently transfected with 3 µg of each plasmid DNA. Forty hours
later, cells were washed and incubated for 30 min in methionine- or
methionine- and cysteine-free DMEM in the absence of serum. Cells were
then labeled for 3 h with 3.5 ml of the same medium containing 200 µCi of [35S]methionine (~1,175 Ci/mmol, NEN Life
Science Products) or 200 µCi of [35S]methionine and 200 µCi of [35S]cysteine (~1,075 Ci/mmol, NEN Life
Science Products). Labeled cells were rinsed with cold PBS, lysed in
0.5 ml of lysing buffer, and clarified by centrifugation. Cell extracts
were incubated with 24 µg of goat anti-mouse HFc (Pierce), 15 µg of
anti-HA, 10 µg of anti-PDGF, or 10 µg of anti-FLAG.
Immunoprecipitates were recovered with protein A (Pierce) beads,
dissolved in Laemmli buffer, and analyzed by SDS-PAGE. After
electrophoresis, the gels were treated with En3hance
(DuPont), dried, and autoradiographed.
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RESULTS |
FRP Inhibits Wnt Function in Both Autocrine and Paracrine
Modes--
Studies to date showing that FRPs antagonize Wnt action
have generally utilized in vivo models, with the exception
of one recent report in which cotransfection of 293 cells with
Frzb-1 and WNT-1 resulted in the inhibition of
Wnt-induced accumulation of cytosolic
-catenin (23). To confirm and
extend these findings with respect to FRP, we cotransfected 293T cells
with WNT-1 and FRP. This transient expression
system has the advantage of inducing high levels of protein expression.
However, we observed that cotransfection led to variable levels of
expression of the exogenous proteins. To overcome this difficulty,
WNT-1-HFc (12) was cotransfected with the vector control or
HA-tagged FRP (Fig. 1) at
different DNA ratios in order to ensure that similar WNT-1 protein
levels could be obtained in the presence or absence of FRP
expression.

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Fig. 1.
Schematic diagram of the constructs generated
for use in this study (see "Materials and Methods").
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Under conditions in which 293T cells transfected with WNT-1,
in the presence or absence of FRP, expressed similar WNT-1 levels (Fig.
2A), FRP was also specifically
detected as a 36-kDa species in WNT-1/FRP-cotransfected
cells (Fig. 2B). Total and uncomplexed
-catenin levels
(12) were quantitated in the same cells using a GST-E-cadherin binding
assay as described under "Materials and Methods." Although total
-catenin levels were not appreciably altered (data not shown), Fig.
2C shows that WNT-1 induced an increase of around 10-fold in
the amount of uncomplexed
-catenin over that of the vector control.
Of note, expression of FRP dramatically inhibited this increase (Fig.
2C). Similar results were obtained with WNT-2 (data not
shown). These findings established, under carefully controlled
conditions, that FRP acts to inhibit Wnt signaling functions
responsible for increased free
-catenin levels.

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Fig. 2.
FRP inhibition of WNT-1-induced increase in
uncomplexed -catenin levels. 293T cells
were transiently transfected with either vector control,
pCEV/WNT-1-HFc, or pCEV/WNT-1-HFc and
pcDNA/FRP-HA. Around 48 h following transfection,
cultures were solubilized, and cell extracts were analyzed by SDS-PAGE,
followed by immunoblotting with anti-HFc (A) or anti-HA
(B). Analysis of free -catenin levels was performed using
either 0.1 (lanes a) or 1 mg (lanes b) of total
cell lysates (C), as described under "Materials and
Methods."
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We next investigated the ability of FRP to inhibit Wnt functions in a
stable transformation model. To do so, WNT-2 and
FRP genes were cotransfected using different selectable
markers into NIH3T3 cells, in which both WNT-1 and
-2 induce morphologic alterations that include increased
saturation density as well as higher steady state levels of uncomplexed
-catenin (12). Whereas WNT-2 transfectants exhibited
abnormal growth and achieved higher cell density, double marker
selected cultures expressing both FRP and WNT-2
showed little if any morphological differences from vector control or FRP transfectants (Fig. 3A).
Similar results were obtained in multiple experiments. Moreover,
inhibition of the transformed phenotype by FRP was correlated with
inhibition of Wnt function as indicated by the substantially lower
levels of WNT-2-induced free
-catenin in the stable
cotranfectants (Fig. 3B).

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Fig. 3.
FRP antagonizes Wnt functions in stable
transfectants. NIH3T3 cells were cotransfected with pBabe
puro and pMMT neo vector controls, pBabe
puro and pMMT/FRP neo, pBabe/WNT-2-HFc
puro and pMMT neo, or pMMT/FRP neo and
pBabe/WNT-2-HFc puro. Double marker selected cultures were
obtained by addition of 750 µg of geneticin and 2 µg/ml puromycin.
A, marker-selected cultures were grown post-confluence for 6 days and then photographed. Scale bar, 150 µm.
B, confluent cultures were solubilized in lysing buffer and
0.1 or 1 mg of total cell extracts analyzed for levels of free
-catenin as described under "Materials and Methods."
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Both Wnt and FRP proteins are processed through the secretory pathway
but remain associated with cells that have been analyzed to date (21,
28, 29). The sites of interactions and mechanisms by which FRP exerts
its inhibitory functions on Wnt signaling remain to be elucidated. To
address this question, we first analyzed the localization of FRP in
transfected 293T cells. As shown in Fig.
4, FRP was found to be present in cell
lysates and the extracellular matrix but was not detected in the medium
using conditions known to optimize FRP release into the medium of
cultured fibroblasts (21). When the results were normalized for the
amount of cell lysate, culture fluid, or extracellular matrix in the
same Petri dish, FRP was found to be predominantly cell-associated
(90%) with the remainder present in the extracellular matrix (Fig. 4). These results indicate that FRP remains tightly cell-associated when
expressed by 293T cells.

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Fig. 4.
Cellular localization of FRP in transfected
293T cultures. 24 h after transfection cells were incubated
in serum-free medium containing 10 µg/ml heparin. Twenty-four hours
later cell lysates (C.L.), conditioned medium
(C.M.), and extracellular matrix (E.C.M.) were
collected. Aliquots reflecting 1/50 of the cell lysates and culture
fluids, respectively, as well as 1/7 of the extracellular matrix from
the same culture dish were analyzed by SDS-PAGE followed by
immunoblotting with anti-HA as described under "Materials and
Methods." Similar results were obtained with heparin at higher
concentrations (50 µg/ml).
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Experiments were next performed to analyze the ability of FRP to
inhibit Wnt actions in a paracrine mode. To do so we utilized a
recently developed transient assay involving a luciferase reporter for
TCF transcriptional activation (27) as an indirect marker of Wnt-1
function. 293T cells have previously been shown to respond to autocrine
Wnt-1 by stimulating TCF-dependent transcription (13). We
established a cocultivation assay in which cells transiently transfected with Wnt-1 are able to stimulate the activation
of the TCF reporter in target cells in a paracrine mode.2
293T cells were transfected with Wnt-1 or vector control and then cocultivated with 293T cells, which were cotransfected with the
TCF reporter and either the vector control or FRP. As shown in Fig. 5, TCF reporter activity was
increased over 20-fold in 293T cells in response to cocultivation with
Wnt-1-expressing cells. However, the responsiveness of FRP
expressing 293T cells to paracrine-acting Wnt-1 was not
significantly different from those expressing vector alone. These
results indicate that Wnt-1 function can be inhibited by FRP in a
paracrine mode.

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Fig. 5.
FRP inhibits Wnt-1-induced
TCF/LEF-dependent transcription in a paracrine mode.
293T cells in 6-well plates were cotransfected with FRP or empty
vector, as well as either pGL3-OT or pGL3-OF TCF luciferase reporters
and a -galactosidase expression vector. The transfected cultures
were then cocultivated with 293T cells transfected with either
Wnt-1 or vector as described under "Materials and
Methods." After 48 h luciferase activity was measured and levels
of Wnt-1-induced RLU presented relative to the RLU obtained
by coculturing with the empty vector transfectants. Results of one
representative experiment of three independently performed assays are
shown. Determinations were made in duplicate for each experimental
point, and the standard deviation was less than 4%.
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FRP Interacts with Wnts--
Previous studies have shown that
Frzb-1 coimmunoprecipitates several members of the Wnt family (20, 23).
To confirm and extend these findings with respect to FRP, we performed
immunoprecipitation experiments with lysates of 293T cells
cotransfected with WNT-1-HFc or WNT-2-HFc in
combination with either FLAG-tagged FRP or FLAG-tagged C-terminally truncated FRP encoding only its CRD (Fig. 1).
Antiserum against the HFc tag was used for immunoprecipitation followed by immunoblotting with anti-FLAG to detect FRP or CRD-FRP. As shown in
Fig. 6A, FRP and CRD-FRP could
be immunoprecipitated in complexes with either WNT-1 or -2. The
specificity of these interactions was demonstrated by the fact that
anti-HFc failed to immunoprecipitate FRP or CRD-FRP in the absence of
Wnt-1 coexpression and by the fact that Wnt with a different tag, HA,
was similarly able to immunoprecipitate FRP-FLAG (data not shown).

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Fig. 6.
A, coimmunoprecipitation of FRP or
CRD-FRP with WNT-1 or WNT-2. 293T cells were cotransfected with
FLAG-tagged pcDNA/FRP or pcDNA/CRD-FRP
together with HFc-tagged pCEV/WNT-1 or
pCEV/WNT-2. The lysates were subjected to
immunoprecipitation (IP) with anti-HFc, followed by
immunoblotting with anti-FLAG to detect FRP and CRD-FRP. B,
analysis of FRP-WNT-2 interactions in metabolically labeled cells. 293T
cells were transfected with either vector control HFc-tagged
pcDNA/WNT-2 alone or in the presence of
pcDNA/FRP. After labeling, cell lysates were
immunoprecipitated with anti-HFc, followed by SDS-PAGE analysis.
C, PDGF does not form complexes with Wnt or FRP. Lysates
from labeled 293T cells transfected with vector,
pcDNA/PDGFB, or pcDNA/PDGFB cotransfected
with HA-tagged WNT-2 or FRP were subjected to immunoprecipitation with
either anti-PDGF or anti-HA and analyzed by SDS-PAGE.
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To investigate further the nature of Wnt-FRP interactions as well as to
examine the stoichiometry of the complex, we performed biosynthetic
experiments. Thus, 293T cells were transfected with WNT-2-HFc in the presence or absence of FRP.
Around 40 h later cultures were labeled for 3 h with
[35S]methionine followed by immunoprecipitation analysis
of cell lysates with anti-HFc. Fig. 6B shows that anti-HFc
specifically immunoprecipitated the radiolabeled 66-kDa WNT-2-HFc from
lysates of WNT-2-transfected 293T cells. In contrast, both
WNT-2-HFc and a 36-kDa species corresponding to FRP were
coimmunoprecipitated from cotransfected cultures. No other major bands
were observed, arguing against the possibility that other protein(s)
participated in FRP-WNT-2 binding interactions. Of note, the intensity
of the band corresponding to FRP was significantly greater than the
signal associated with WNT-2-HFc in these complexes (Fig.
6B). Similar results were obtained in several independent
experiments. The ratio between the intensity of the FRP and the WNT
bands was around 1.7 as calculated by densitometry. As a similar number
of methionines are present in both proteins, these results raised
the possibility that Wnt-2 might be present in a complex containing
more than one FRP molecule.
In order to establish the specificity of the Wnt-FRP interactions, we
performed biosynthetic experiments using PDGFB as a control for binding
interactions. PDGFB, like Wnt, contains multiple cysteine residues and
is also processed through the secretory pathway (30, 31).
FRP-HA and WNT-2-HA were cotransfected with PDGFB, and cells were labeled with
[35S]methionine and [35S]cysteine. Cell
lysates were subjected to immunoprecipitation with either a PDGFB
monoclonal antibody (32) or anti-HA. Fig. 6C shows that
neither WNT-2 nor FRP interacted with PDGFB. These results strongly
support the specificity of the Wnt-FRP complexes observed (Fig. 6,
A and B).
Analysis of FRP Structure--
We took advantage of the
availability of both HA- and FLAG-tagged FRP and
CRD-FRP constructs (Fig. 1) to investigate FRP structure. Thus, 293T cells were transfected individually or cotransfected with
the FLAG- and HA-tagged versions of FRP or
CRD-FRP, respectively. Fig.
7A shows the analysis of
transfected cell lysates by immunoblotting with anti-FLAG
(top) or following immunoprecipitation with the same
antibody (bottom). The results revealed that each of the FLAG-tagged proteins was expressed well and was immunoprecipitable by
anti-FLAG. Fig. 7B further shows that anti-HA detected
FRP-HA and CRD-FRP-HA in cells transfected with these expression
vectors (Fig. 7B, top). Of note, FRP-HA was specifically
immunoprecipitated by anti-FLAG from cells coexpressing
FRP-FLAG. Similarly, CRD-FRP-HA was specifically
immunoprecipitated from lysates of cells cotransfected with
CRD-FRP-FLAG (Fig. 7B, bottom). Furthermore,
antibody specificity controls indicated that FRP-HA and CRD-FRP-HA were
immunoprecipitated by anti-FLAG only in the presence of their
FLAG-tagged versions. These findings indicate that FRP, itself, can
form complexes and that its CRD is sufficient to mediate these
interactions.

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Fig. 7.
FRP complex formation in transfected 293T
cells. A, following transfection or cotransfection with
the HA and FLAG-tagged versions of pcDNA/FRP and
pcDNA/CRD-FRP, cell lysates were analyzed by
immunoblotting with anti-FLAG directly (top) and after
immunoprecipitation (IP) with the same antibody
(bottom). B, the same cell lysates were
immunoblotted with anti-HA directly (top) or after
immunoprecipitation with anti-FLAG (bottom).
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We next investigated whether FRP was capable of forming such complexes
following secretion. Because FRP remains tightly cell-associated (Fig.
4), experiments were performed by independently transfecting HA or
FLAG-tagged FRPs in 293T cells, followed by cocultivation of
the transfectants for 48 h. Immunoprecipitation of cell lysates revealed FRP-HA-FRP-FLAG complexes indicating that these interactions can occur following secretion from cells (Fig.
8).

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Fig. 8.
FRP complex formation in autocrine and
paracrine modes. In the autocrine assay, 293T cells were
cotransfected with either the HA- and FLAG-tagged versions of
pcDNA/FRP or with the FLAG-tagged
pcDNA/FRP and the vector. In the paracrine assay, the
pcDNA/FRP-FLAG transfectants were cocultivated with the
293T cells transfected with either the vector or the
pcDNA-FRP-HA. Cell lysates were analyzed by
immunoblotting with anti-FLAG directly (top) or after
immunoprecipitation (IP) with anti-HA antibody
(bottom).
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FRP Forms Complexes with the Hfz6 Receptor--
Evidence that the
CRD of FRP has a predicted structure similar to that of fz (21)
suggested the possibility that fz itself might interact with FRP. We
have cloned several members of the fz family including Hfz6 from a
human cDNA library. For the present studies, we generated an
expression vector encoding a FLAG-tagged C-terminal truncated version
of Hfz6 containing its CRD region but lacking the entire transmembrane
and cytoplasmic domains (Fig. 1). This construct, designated
CRD-Hfz6-FLAG, was cotransfected into 293T cells with either
vector control, FRP-HA, or CRD-FRP-HA. Immunoblotting analysis with anti-FLAG revealed expression (Fig. 9A, top) and specific
immunoprecipitation (Fig. 9A, bottom) of FLAG-tagged CRD-Hfz6. The expression of HA-tagged FRP and CRD-FRP in
transfectants can be seen in Fig. 9B (top). When
anti-HA immunoblotting analysis of anti-FLAG immunoprecipitates was
performed, the results showed the specific interaction of CRD-Hfz6 with
both FRP and CRD-FRP (Fig. 9B, bottom). Immunoprecipitation
experiments performed following cocultivation of cells independently
transfected with CRD-Hfz6 or FRP confirmed these
interactions (data not shown). These findings establish the ability of
FRP to form complexes with a prototype fz and provide evidence that
these interactions are mediated by their homologous CRD regions.

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Fig. 9.
FRP interaction with Hfz6. 293T cells
were transfected with pcDNA/CRD-Hfz6 FLAG-tagged,
pcDNA/FRP-HA, or pcDNA/ CRD-FRP-HA or
cotransfected with CRD-Hfz6-FLAG together with FRP-HA or
CRD-FRP-HA. A, transfected cell lysates were then
subjected to anti-FLAG immunoblotting directly (top) or
following immunoprecipitation (IP) with the same antibody.
B, the same cell lysates were immunoblotted with anti-HA
directly (top) or after immunoprecipitation with anti-FLAG
(bottom).
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To assess the stoichiometry of the FRP-CRD-HFz6 complexes, we performed
biosynthetic experiments. 293T transfectants expressing FRP-HA and
CRD-Hfz6-FLAG individually or together were labeled with
[35S]methionine and [35S]cysteine. Cell
lysates were then immunoprecipitated with anti-HA or anti-FLAG. As
shown in Fig. 10, each protein was
readily detected in singly transfected cultures, with FRP expressed at
higher levels than CRD-Hfz6. When cotransfectants were subjected to
coimmunoprecipitation with anti-HA or anti-FLAG, FRP-CRD-Hfz6 complexes
were detectable in both cases. When the lysates were immunoprecipitated
with anti-HA, the band corresponding to FRP was stronger than that
corresponding to CRD-Hfz6, consistent with the higher level of FRP
expression. However, when anti-FLAG was used to immunoprecipitate
CRD-Hfz6, present in limiting amounts, the coprecipitated FRP band
showed a very similar intensity. The ratio between the intensity of the signal for FRP and the CRD-Hfz6 band was found to be 1.0 when analyzed
by densitometry. Since the number of cysteines and methionines in the
two molecules is similar, these observations support the heterodimeric
interaction between the two proteins. No other species was consistently
detectable in both anti-HA and anti-FLAG coimmunoprecipitates. However,
we cannot rigorously exclude the presence of other molecules in the
complex. To confirm the specificity of the FRP-CRD-Hfz6 interactions,
cotransfection of CRD-Hfz6 with PDGFB revealed the absence of any
detectable complex.

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Fig. 10.
Hfz6 forms complexes with FRP but does not
interact with PDGF. 293T cells were transfected with vector
control, PDGFB, FRP-HA, or CRD-Hfz6-FLAG alone or in
combination. Metabolically labeled cultures were lysed and
immunoprecipitated (IP) with anti-PDGF, anti-HA, or
anti-FLAG and subjected to SDS-PAGE. The additional band of around 32 kDa observed in lanes 5 and 7 could either
represent a protein associated with FRP or a breakdown product of the
FRP protein itself.
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DISCUSSION |
Our present studies provide evidence concerning mechanisms
involved in antagonism of Wnt signaling by members of the FRP family. Several previous studies have reported that Wnts can bind to fz receptors or FRPs presented at the cell surface (17, 22). There are
also reports of Wnt binding to Frzb as measured by in vitro
or in vivo coexpression and coimmunoprecipitation (20, 23,
33). We confirmed these observations with respect to FRP, further
demonstrating complexes containing WNT-1 or WNT-2 and FRP in
coexpressing cells. Metabolic labeling analysis suggested a direct
interaction between FRP and Wnt, which does not require the
participation of any additional major protein. Moreover, this binding
appeared to involve more than one FRP interacting with each Wnt molecule.
By use of different immunologic tags, FRP complexes containing both
tags were specifically detectable. Such complexes were further shown to
involve the CRD region of the molecule. FRP and fz contain analogous
CRD domains (21), and it was possible to demonstrate that FRP also
possessed the ability to form complexes with the CRD of a prototype fz,
Hfz6. Biosynthetic experiments further revealed that these complexes
were consistent with FRP-CRD-Hfz6 heterodimers. Thus, FRP has the
ability to independently interact with Wnts and with their receptors.
It might be argued that cysteine-rich proteins such as Wnt, FRP, and
Hfz6, which are processed through the same secretory pathway, could
form illegitimate complexes by improper disulfide bond formation under
conditions of exogenous coexpression. However, cotransfection with
another cysteine-rich, secreted molecule, PDGFB (30, 31), yielded no
evidence of complexes with any of the same molecules. Moreover, FRP
complexes were detected when cells were independently transfected with
FRPs containing different tags and cocultivated. All of these findings
strongly support the specificity of the complexes involving FRP and
either Wnts or Hfz6. The strength of the binding interactions for each
of these complexes was sufficient to survive exposure to 0.1% SDS. Such conditions do not impair antigen-antibody binding but do disrupt
interactions between heterodimers of tyrosine kinase growth factor
receptors (34, 35). Continued investigation of the physical and
biochemical characteristics of the complexes involving FRP with Wnts
and the fz receptors should provide further insights into the nature of
these interactions.
Our present studies are consistent with at least two models by which
FRP may function as a naturally occurring antagonist of Wnt signaling.
According to the first, FRP binds Wnt and exerts its inhibitory
function by competing for the ability of the Wnt ligand to interact
with the fz receptor. However, a second model emerges from our results
showing that FRP forms complexes with the fz receptor and that these
interactions are mediated by their homologous CRDs. Such findings
support the possibility that fz, itself, may form complexes which
function as the signaling and/or binding receptor for Wnt. According to
this model, FRP could act by a dominant negative mechanism, interacting
with the fz receptor and forming nonfunctional complexes that are
incapable of transmitting the Wnt signal.
In several assay systems, FRP acted to inhibit Wnt-induced alterations
of
-catenin regulation and TCF transcription. Moreover, we showed
that FRP inhibited Wnt function in both autocrine or paracrine modes.
Similar conclusions were derived from microinjection of
Xenopus embryos (20). Although as shown here and in previous studies (21), secreted FRP remains predominantly associated with the
cell or extracellular matrix, it could be postulated to inhibit
paracrine-acting Wnts by either of the above models. Lin et
al. (23) reported that certain Frzb deletion mutants that retained
the ability to bind WNT-1 failed to block WNT-1-induced axis
duplication. This lack of a strict correlation between the ability of
Frzb to interact physically with Wnts and its ability to inhibit Wnt
signaling suggests that the mechanism of antagonism is more complex
than can be explained solely by Wnt binding. Genetic analysis aimed at
localizing FRP domains required for Wnt binding, as well as for
formation of complexes with the fz receptor, should be helpful in
assessing the proposed models for FRP inhibition of Wnt signaling.
These models are not mutually exclusive, and cooperation of the two
mechanisms might be necessary to ensure more complete functional
inhibition of Wnt signaling.
The FRP family is not the only example of naturally occurring Wnt
antagonists. Recently, dickkopf-1 (dkk-1), which
encodes Dkk-1, a secreted inducer of Spemman's organizer in
Xenopus was isolated by an expression cloning strategy (36).
This 40-kDa secreted protein contains two cysteine-rich domains and
represents a new multigene family. Dkk-1 and Frzb have overlapping
patterns of expression during development. Genetic analysis indicates
further that Dkk-1 inhibits Wnt signaling upstream of dishevelled (36). It will be of interest to determine whether Dkk-1 acts like FRP/Frzb to
bind Wnt ligands and/or their fz receptors or, indirectly, by
activating an independent Wnt inhibitory pathway.