Cancer Biology and Genetics Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
Author for correspondence (e-mail:
feng.cong{at}pharma.novartis.com)
Accepted 21 June 2004
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SUMMARY |
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Key words: Wnt, LDL-receptor-related proteins 5 and 6, Frizzled, Drosophila
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Introduction |
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Frizzled receptors are a family of seven-transmembrane proteins, which
interact with Wnt proteins through their N-terminal extracellular
cysteine-rich domain (CRD) (Bhanot et al.,
1996; Hsieh et al.,
1999
). There are three major pathways downstream of Frizzled: the
canonical Wnt/ß-catenin pathway, the planar polarity pathway and the
Wnt-Ca2+ pathway. It is unclear whether Frizzled receptors are
directly coupled to the same set of downstream signaling proteins in these
distinct signaling pathways. Like Wnt proteins, Frizzled receptors have been
grouped into two classes: one class is thought to stimulate the ß-catenin
pathway and the other class to stimulate the Ca2+ pathway
(Miller et al., 1999a
). The
mechanism by which Frizzled activates the Wnt/ß-catenin pathway is still
uncertain, although a link with heterotrimeric G proteins has been proposed
(Liu et al., 2001
). Dvl is, at
present, the only known component closely downstream of Frizzled and it is
involved in all three pathways downstream of Frizzled.
LRP co-receptors (LRP5/6, Arrow) are single transmembrane proteins that
comprise a subfamily of LDL-receptor related proteins and play an essential
role in the canonical ß-catenin pathway
(Pinson et al., 2000;
Tamai et al., 2000
;
Wehrli et al., 2000
). The
intracellular domains of LRP5 and Arrow have been shown to bind to Axin
(Mao et al., 2001b
;
Tolwinski et al., 2003
).
Recent studies have demonstrated that Wnt induces membrane translocation and
destabilization of Axin (Cliffe et al.,
2003
; Mao et al.,
2001a
; Tolwinski et al.,
2003
). Although it is widely accepted that Wnt binds to Frizzled,
whether Wnt directly interacts with LRP is less certain
(Tamai et al., 2000
;
Wu and Nusse, 2002
). LRP has a
large extracellular domain that contains four EGF repeats and three LDLR
repeats. The role of the extracellular domain of LRP and the functional
significance of the potential physical interaction between this domain and Wnt
are unclear.
Both Frizzled and LRP receptors are essential for the signaling activity of
Wnt. There are three potential mechanisms that could explain the
interdependence between endogenous Frizzled and LRP in transmitting the Wnt
signal. First, Wnt, Frizzled and LRP could form a single signaling complex,
leading to the transmission of one signal. Second, Wnt could form two separate
complexes with Frizzled and LRP, and generate signals that merge in downstream
signaling cascades. Third, Wnt could primarily interact with one receptor,
inducing activation of the other receptor through intracellular signaling. In
the last two scenarios, proximity between Frizzled and LRP might be
unnecessary and insufficient for signaling. Based on the finding that the
extracellular domain of LRP6 binds to Wnt1 and associates with the
extracellular domain of Frizzled in a Wnt-dependent manner, it has been
proposed that Wnt triggers ß-catenin signaling by bridging between
Frizzled and LRP (Tamai et al.,
2000). However, this model has not been rigorously tested.
In this study, we have analyzed the mechanisms by which Frizzled and LRP transmit a Wnt signal in cell culture systems. We have identified key amino acid residues of Frizzled essential for transducing the Wnt signal, and demonstrated that LRP signaling activity can be stimulated by oligomerization. We have provided several lines of evidence suggesting that Wnt activates ß-catenin signaling by bringing Frizzled and LRP into proximity.
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Materials and methods |
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HFz5, LRP6, LRP6EGF, LRP6
EGF-FKBP and LRP6
N were
cloned into a mammalian expression vector under the control of the
cytomegalovirus (CMV) promoter. GFP was fused to the C terminus of human FZ5
and YFP was fused to the C termini of LRP6 and LRP6
N. MESD
(Hsieh et al., 2003
),
Myc-tagged Dsh and mFz8-CRD-IgG (Hsieh et
al., 1999
) were provided by Jen-Chih Hsieh. LRP6N-IgG and LDLR-IgG
were provided by Xi He (Tamai et al.,
2000
). The LEF-1 reporter constructs were provided by Rudolf
Grosschedl. TOP-FLASH reporter was provided by Hans Clevers.
Mammalian cell culture, transfection and luciferase assays
Human 293 cells and monkey COS7 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum at 37°C in 5%
CO2. Cells were transfected with Fugene 6 (Roche) according to the
manufacturer's instructions. Luciferase assays were performed with the Dual
Luciferase Assay kit (Promega) according to the manufacturer's
instructions.
Drosophila S2 cell transfection and dsRNA experiments
Drosophila S2 cells were grown at room temperature in Schneider's
Drosophila medium supplemented with 10% fetal bovine serum.
Transfection of S2 cells and dsRNA experiments were performed as described
(Cong et al., 2004). The
following genes were targeted with dsRNA: Drosophila Arrow,
DDBJ/EMBL/GenBank Accession Number NM_079998; Dsh, Accession Number L26974;
Drosophila stimulatory G protein
subunit G
s, Accession
Number M23233. G
s-dsRNA was used as a control in the RNA interference
experiments.
Co-immunoprecipitation and immunoblotting assay
Co-immunoprecipitation of Frizzled and Dvl was performed as described
(Chen et al., 2003).
Co-immunoprecipitation of LRP6 and Axin was performed as described
(Liu et al., 2003
). Commercial
antibodies used in this study include anti-GFP polyclonal antibodies
(Clontech), anti-
-tubulin monoclonal antibodies (Sigma), anti-HA
(HA.11) monoclonal antibodies (Covance), anti-ß-catenin monoclonal
antibodies (Transduction Laboratories) and anti-Myc (9E10) monoclonal
antibodies (Santa Cruz).
Membrane biotinylation assay
Cells were washed twice with ice-cold PBS (pH 8.0), and suspended at a
concentration of 1x107 cells/ml. Cells were incubated with
0.5 mg/ml Sulfo-NHS-LC-Biotin (Pierce) at room temperature for 30 minutes.
Cells were washed once with 50 mM Tris (pH 8.0) and twice with ice-cold PBS to
remove any remaining biotinylation reagents. Cells were then lysed with RIPA
buffer and cell lysates were incubated with immobilized monomeric Avidin beads
(Pierce) for 3 hours. Beads were washed four times with RIPA buffer, and the
bound proteins were eluted with Laemmli sample buffer.
Cell fractionation assay
Cells were washed and scraped on ice into TBS (10 mM Tris-HCl, pH 7.5, 140
mM NaCl, 2 mM DTT, protease inhibitors). Cells were homogenized with 30
strokes in a dounce homogenizer, and the nuclei were removed by low speed
centrifugation. The post-nuclear supernatants were spun at 100,000
g for 90 minutes at 4°C to generate a supernatant, or
cytosolic, fraction and membrane-rich pellet fraction. Samples normalized for
protein content were analyzed by SDS-PAGE.
Fluorescence microscopy
COS cells were transfected with Myc-tagged Dsh and GFP-tagged HFz5. The
immunofluorescence experiment was performed as described
(Cong et al., 2003). 293 cells
were transfected with LRP6-YFP and LRP6
N-YFP expression constructs.
Thirty-six hours after transfection, cells were fixed with 2%
paraformaldehyde. Cells were washed with PBS, mounted using Vetashield
mounting medium and examined by laser scanning microscopy (Zeiss).
Liquid binding assay
Liquid binding assay was performed as previously described
(Semenov et al., 2001;
Tamai et al., 2000
). All
recombinant proteins were used as conditioned medium (CM). LRP6N-IgG, LDLR-IgG
and mFz8CRD-IgG were produced in 293 cells via transient transfection. MESD
was co-expressed with LRP6N-IgG to facilitate the secretion of LRP6N-IgG.
Wnt3A and Wnt3A C77S-HA CM were collected from Rat-2 cells stably transfected
with Wnt3A or Wnt3A C77S.
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Results |
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Identification of amino acid residues in the intracellular domains of Frizzled receptors essential for Wnt/ß-catenin signaling
We systematically mutagenized the intracellular domains of rat Fz1 to
identify critical amino acid residues for Wnt/ß-catenin signaling using
the S2 cell culture assay. Thirty rat Fz1 mutants were generated to make
changes in all three intracellular loops and in the proximal membrane region
of the C-terminal tail, a region known to be essential for Wnt signaling
(Umbhauer et al., 2000), and
two mutants were generated with changes in the extracellular loops. In each
mutant, two neighboring amino acid residues were simultaneously changed to
Ala. As expected, mutations in the extracellular loops did not affect the
signaling activity of rat Fz1, while mutations in the intracellular domains
had various effects (Fig. 2A).
All rat Fz1 mutants were tagged with a GFP epitope at their C termini;
immunoblotting demonstrated that the levels of mutant proteins were
approximately equal (data not shown).
|
We then mutated Arg263, Leu443 and Lys525 of human FZ5, residues analogous to Arg 340, Leu524 and Lys619 of rat Fz1, and found that mutating these residues abolished the signaling activity of human FZ5 without affecting its membrane localization (Fig. 2C). All three residues are well conserved in all Frizzled receptors (data not shown).
The Trp residue in the essential Lys-Thr-x-x-x-Trp motif is conserved in
most Frizzled receptors, but substituted by a Tyr residue in
Drosophila Fz3 and mom-5 of C. elegans. As
Drosophila Fz3 and mom-5 appear to be weak or defective Frizzled
receptors (Rocheleau et al.,
1997; Sato et al.,
1999
; Thorpe et al.,
1997
), it has been speculated that the Trp to Tyr substitution
might be responsible for the defective signaling activities of
Drosophila Fz3 and mom-5
(Umbhauer et al., 2000
). We
have found that mutating the corresponding Trp residue in rat Fz1 and human
FZ5 to Ala significantly reduced the signaling activity of Frizzled receptors,
but mutating it to Tyr had no significant effect
(Fig. 2D). Therefore,
replacement of Trp with Tyr at this position in Drosophila Fz3 and
mom-5 is unlikely to be responsible for their deficiencies in Wnt
signaling.
Membrane relocation of Dvl by wild-type and mutant Frizzled receptors
Overexpression of Frizzled induces membrane translocation of Dvl. We tested
whether the residues of Frizzled identified as crucial for ß-catenin
signaling were also important for Frizzled-induced Dvl membrane translocation.
Dsh was localized in a punctated pattern in the cytoplasm when expressed alone
in COS cells, and co-expression of human FZ5 recruited Dsh to the plasma
membrane (Fig. 3A). By
contrast, the inactive human FZ5 mutants R253A, L443A and K525A were unable to
translocate Dsh to the plasma membrane
(Fig. 3A), although they were
all present on the plasma membrane at levels similar to that of wild-type
protein as determined by membrane biotinylation (data not shown).
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Activation of LRP6 signaling activity by inducible oligomerization
Overexpression of LRP without Wnt and Frizzled stimulates ß-catenin
signaling in various systems (Mao et al.,
2001b; Schweizer and Varmus,
2003
; Tamai et al.,
2000
), possibly by titrating endogenous Axin. As many receptors
are activated by ligand-induced oligomerization
(Weiss and Schlessinger,
1998
), it is conceivable that ligand-induced oligomerization of
LRP increases its signaling activity.
We tested this hypothesis by generating various LRP chimeric proteins. The
FK506-binding protein, FKBP, has been engineered to serve as an inducible
intracellular dimerization domain (Klemm
et al., 1998). LRP6
EGF-FKBP was generated by fusing two
copies of a modified FKBP domain (Ariad) to the C terminus of LRP6
EGF,
an LRP6 N-terminal deletion mutant with all four EGF repeats removed
(Fig. 4A) and its activity was
tested in 293 cells using a LEF-luciferase reporter as readout. AP20187, a
synthetic dimerizer (Clackson et al.,
1998
), significantly increased the signaling activity of
LRP6
EGF-FKBP, but not that of LRP6
EGF
(Fig. 4B). Furthermore, AP20187
increased LRP6
EGF-FKBP-induced, but not LRP6
EGF-induced,
stabilization of ß-catenin in 293 cells
(Fig. 4C). These results
suggest that oligomerization of LRP increases the signaling activity of LRP in
the ß-catenin pathway. This conclusion directly conflicts with a recent
report by Liu et al. (Liu et al.,
2003
), as discussed later.
|
Next, we tested the effect of oligomerization on the signaling activity of
LRP6 in Drosophila S2 cells using a different strategy, involving an
extracellular oligomerization signal. The extracellular domain of TrkC, a
neurotrophin receptor, was fused N-terminal to the transmembrane domain of
LRP6 to form TrkN-LRP6C (Fig.
4A); neurotrophin 3 (NT3), a specific ligand of TrkC, was used to
induce oligomerization of TrkN-LRP6C. Treatment of Drosophila S2
cells with NT3 increased the signaling activity of TrkN-LRP6C by almost
tenfold (Fig. 4E), but had no
effect on the signaling activity of LRP6, LRP6N and HFz5N-LRP6C, a
chimeric protein with the N-terminal extracellular domain of human FZ5 fused
upstream of the transmembrane domain of LRP6
(Fig. 4A). These data further
strengthen the claim that the signaling activity of LRP can be increased by
oligomerization.
We next tested whether Dvl is required for the signaling activity of LRP in
these experiments. Although Dsh-dsRNA significantly decreased the signaling
activity of Wg and Fz5 (Fig.
4F), it had no effect on the signaling activity of LRP6N,
consistent with previous findings (Li et
al., 2002
; Schweizer and
Varmus, 2003
). Importantly, Dsh-dsRNA also had no effect on the
activity of TrkN-LRP6C, even in the presence of NT3. These results demonstrate
that activation of ß-catenin signaling by overexpression of LRP, with or
without inducible oligomerization, does not involve Dvl. Presumably,
overexpressed LRP stimulates ß-catenin signaling by recruiting Axin to
the plasma membrane and inactivating it, a process that does not require Dvl;
oligomerized LRP has a higher affinity for Axin.
The extracellular domain of LRP6 plays a positive role in Wnt signaling, and can be replaced by the N-terminal extracellular domain of human FZ5
Both LRP and Frizzled are required to transduce a Wnt signal. Could this
occur by formation of hetero-oligomers and, if so, how? It is also uncertain
whether all Wnt proteins bind to the extracellular domain of LRP and, if so,
whether such a binding is functionally significant. It has been suggested that
the extracellular domain, specifically the region containing the first two EGF
repeats, plays an important role in transducing the Wnt signal
(Mao et al., 2001a).
The extracellular domain of LRP might play a positive role and actively
transmit the Wnt signal. Alternatively, as it has recently been suggested
(Liu et al., 2003), the
extracellular domain could play a negative role by restraining the signaling
activity of LRP in the absence of Wnt; binding to Wnt might then release the
inhibitory effect. Consistent with the second possibility, an LRP mutant
lacking the whole extracellular domain appears to be hyperactive and does not
synergize with Wnt (Liu et al.,
2003
; Mao et al.,
2001a
; Mao et al.,
2001b
; Schweizer and Varmus,
2003
).
We tested the signaling activity of LRP6 and LRP6N in 293 cells
using the TOP-FLASH reporter. Consistent with previous observations,
LRP6
N appeared to be much more active than LRP6
(Fig. 5B). However, a close
examination of the subcellular localizations of these two proteins tagged at
their C termini with YFP revealed that LRP6
N was mostly localized on
the plasma membrane, while LRP6 was mostly localized in the perinuclear
region, presumably in the endoplasmic reticulum
(Fig. 5A). This observation is
consistent with recent findings that correct folding and membrane
translocation of LRP proteins require MESD (Boca in Drosophila), a
chaperone protein found in the endoplasmic reticulum
(Culi and Mann, 2003
;
Hsieh et al., 2003
).
Co-expression of MESD increased the membrane localization of LRP6
(Fig. 5A). Importantly, the
signaling activities of LRP6 and LRP6
N became comparable upon
co-expression of MESD (Fig.
5B). Therefore, the apparent lower activity of the full-length
LRP6 is most likely due to its inefficient transport to the plasma membrane,
and the extracellular domain of LRP6 does not play a negative role.
|
HFz5N-LRP6C was generated by fusing the extracellular domain of human FZ5
N-terminal to the transmembrane domain of LRP6
(Fig. 4A), and tested for its
ability to complement endogenous Arrow in S2 cells. Consistent with our
previous results (Schweizer and Varmus,
2003), full-length LRP, but not the N-terminally truncated LRP6,
complemented endogenous Arrow to transduce the Wnt signal
(Fig. 6). Significantly,
HFz5N-LRP6C, like full-length LRP6, also complemented endogenous Arrow in this
assay (Fig. 6). No synergy
between HFz5N-LRP6C and human FZ5 was observed when Wg was not co-expressed
(data not shown). The inability of LRP6
N to complement endogenous Arrow
is not due to its lower signaling activity, as LRP6
N has a
significantly higher activity than LRP6 and HFz5N-LRP6C when expressed alone
(Fig. 4). Taken together, these
data suggest that the extracellular domain of LRP plays a positive role in
transducing the Wnt signal, possibly by binding to Wnt.
|
A recent study by Tolwinski et al. supports the first model by showing that
a Frizzled-LRP chimeric protein with the intracellular domain of Arrow fused
to the C terminus of Drosophila Fz2 is more active than Arrow or
Drosophila Fz2 and does not require endogenous Wg
(Tolwinski et al., 2003).
However, it is not clear from their study whether Drosophila Fz2
plays an active signaling role in the chimera. As overexpression of LRP
induces ligand-independent signaling in Xenopus embryos and cultured
cells (Mao et al., 2001a
;
Mao et al., 2001b
;
Schweizer and Varmus, 2003
;
Tamai et al., 2000
), the
ligand-independent signaling of the Drosophila Fz2-Arrow chimera
could result from enhanced stability of Arrow or increased membrane levels of
Arrow. Furthermore, fusing Drosophila Fz2 to Arrow will increase
oligomerization and therefore increase the signaling activity of Arrow, as
Frizzled receptors are known to form constitutive homo-oligomers
(Kaykas et al., 2004
).
To explore these possibilities, we fused the intracellular domain of LRP6
to the C terminus of human FZ5, forming HFz5-LRP6C, then tested the signaling
activity of this chimera in S2 cells. HFz5-LRP6C was much more active than
human FZ5 or LRP6N (Fig.
7), consistent with the previous findings
(Tolwinski et al., 2003
).
Furthermore, mutating three residues essential for human FZ5 function, Arg263,
Leu443 and Lys525 of HFz5-LRP6C, decreased the signaling activity of the
chimera to a level similar to that of LRP6 alone, suggesting that human FZ5 is
essential for signaling by the chimera. Mutating these residues in HFz5-LRP6C
did not affect the membrane concentration of these proteins, as demonstrated
by membrane biotinylation (Fig.
7). Importantly, the signaling activity of HFz5-LRP6C was
Dsh-dependent, while the residual activity of mutated HFz5-LRP6C was Dsh
independent (Fig. 7). Since the
signaling activity of LRP6 is strictly Dsh-independent
(Fig. 4), the residual activity
of mutated HFz5-LRP6C is most probably contributed by only the LRP6 part of
the chimera, and the higher signaling activity of HFz5-LRP6C could not result
from enhanced oligomerization of LRP6. Taken together, these data suggest that
Frizzled and LRP synergize when the two proteins are in proximity.
|
As NT3 is the ligand for TrkC, we fused NT3 to the N terminus of human FZ5
via a Gly-rich sequence linker to form NT3-HFz5
(Fig. 8A). Like human FZ5,
NT3-HFz5 had no signaling activity by itself, but strongly synergized with Wg
(Fig. 8B). Importantly,
NT3-HFz5 strongly synergized with TrkN-LRP6C, which contains the extracellular
domain of TrkC, but not with LRP6N
(Fig. 8B). Likewise, TrkN-LRP6C
did not synergize with human FZ5 or with NT3-HFz5 K535A
(Fig. 8B). Furthermore, the
synergistic activity of TrkN-LRP6C and NT3-Fz5 was Dsh dependent (data not
shown), implying that the human FZ5 part of the NT3-HFz5 chimera played an
active signaling role. These experiments further strengthen the argument that
bringing LRP and human FZ5 into proximity significantly increases their
signaling activity via the ß-catenin signaling pathway.
|
It has previously been shown that fusing XWnt8 to the N terminus of human
FZ5 results in a hyperactive molecule
(Holmen et al., 2002). We
generated a similar chimera by fusing Wg to the N terminus of human FZ5
(Fig. 9A); as anticipated, this
chimera is hyperactive in S2 cells (Fig.
9B). The signaling activity of Wg-Fz5, like that of co-expression
of Wg and human FZ5, was found to be dependent on both Arrow and Dsh
(Fig. 9B).
|
Taken together, the above experiments demonstrate that the requirement for native Wnt protein for ß-catenin-mediated signaling can be by-passed by fusing Wnt to either Frizzled or LRP. This is consistent with the Wnt-induced dimerization model.
Potential roles for Wnt palmitoylation revealed by Wnt-Frizzled and Wnt-LRP chimeras
Wnt proteins are palmitoylated at the conserved Cys residue closest to the
N terminus in the Wnt family of proteins, and this modification is required
for the function of Wnt proteins (Willert
et al., 2003). It is not known, however, why palmitoylation is
required for Wnt function. Although palmitoylation does not appear to be
required for secretion of at least some Wnt proteins, it might facilitate the
ability of Wnt to interact with Frizzled, LRP, or some unknown molecules;
alternatively, the palmitate group may promote membrane localization of
Wnt.
A model of Wnt-dependent dimerization of receptors predicts that the main function of the Wg region of the Wg-HFz5 chimera is to interact with LRP, while the primary role of the Wg region of the Wg-TrkN-LRP6C chimera is to interact with Frizzled. Thus, these chimeric receptors provided an opportunity to test the role of palmitoylation in the interplay of Wnt with its two putative receptors.
We first generated a Wg mutant (WgCS), with the conserved potential palmitoylation site Cys93 mutated to Ser, and tested its signaling activity in S2 cells. As shown in Fig. 9C, WgCS was expressed at a similar level as Wg (data not shown), but completely lacked signaling activity, confirming the importance of palmitoylation. Mutating Cys93 to Ser in Wg-Fz5 (WgCS-HFz5) also severely affected the signaling activity of Wg-Fz5 without altering its membrane localization (Fig. 9C). Assuming that the primary role of Wg in the Wg-HFz5 chimera is to interact with Arrow, these results imply that the palmitate modification of Wnt proteins is important for the interaction between Wnt and LRP.
We next mutated Cys93 to Ser in Wg-TrkN-LRP6C to make WgCS-TrkN-LRP6C, and
tested its signaling activity in S2 cells. Although WgCS-TrkN-LRP6C and
Wg-TrkN-LRP6C were present at similar levels in the membrane, the signaling
activity of WgCS-TrkN-LRP6C is significantly lower
(Fig. 9D), suggesting that
palmitoylation of Wnt is important for its binding to Frizzled. Consistent
with this result, the WgCS-HFz5 mutant was significantly more active than
human FZ5 plus WgCS, although the expression level of WgCS-HFz5 was lower than
that of human FZ5 (Fig. 9C), so
the signaling activity of WgCS can be partially rescued by fusing WgCS to
human FZ5. This is consistent with the hypothesis that palmitoylation of Wnt
might be important for the binding of Wnt to Frizzled. However, it is also
possible that fusion of human FZ5 to WgCS enhances secretion of WgCS
(Nusse, 2003) or tethers WgCS
to the plasma membrane.
Physical interactions between Wnt and Frizzled or LRP
We have provided evidence to suggest that the major function of the
extracellular domain of LRP is to bind to Wnt, and that Wnt most probably
triggers the ß-catenin pathway by forming Frizzled-LRP oligomers.
However, the binding of Wnt to LRP is less well established than the binding
of Wnt to Frizzled (Tamai et al.,
2000; Wu and Nusse,
2002
). We therefore examined the binding between Wnt and LRP using
a liquid binding assay in which conditioned medium (CM) from Rat2 cells
overexpressing HA-tagged Wnt3A was mixed with CM from 293 cells overexpressing
the extracellular domain of LRP6 tagged with the immunoglobulin-
Fc
epitope (LRP6-IgG) or the extracellular domain of LDL receptor tagged with the
immunoglobulin-
Fc epitope (LDLR-IgG). Wnt3A bound to LRP6N-IgG, but
not to LDLR-IgG (Fig. 10A),
consistent with a previous report (Tamai
et al., 2000
). Notably, the Wnt3A palmitoylation mutant (C77S)
bound less well to LRP6-IgG. In addition, we examined the ability of
mFz8CRD-IgG to interact with Wnt3A and Wnt 3A C77S, and found that Wnt3A C77S
also bound less well to mouse Fz8CRD (Fig.
10B).
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Discussion |
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What is the mechanism by which Frizzled transduces a Wnt signal? We have
found that the mutations that disrupt the signaling activity of Frizzled also
affect the ability of Frizzled to induce membrane translocation of Dvl and
reduce physical interaction between Frizzled and Dvl
(Fig. 3), suggesting that a
physical interaction between Frizzled and Dvl is required for the signaling
activity of Frizzled. We propose that Frizzled might function as a docking
site for Dvl in ß-catenin signaling. Our results are consistent with a
previous finding that the Lys-Thr-x-x-x-Trp motif at the C-terminal tail of
Frizzled is not only required for activating ß-catenin signaling, but
also for inducing Dvl membrane translocation
(Umbhauer et al., 2000).
Interestingly, a recent study has demonstrated that the PDZ domain of Dvl
directly binds to a peptide of C-terminal region of Frizzled containing the
Lys-Thr-x-x-x-Trp motif, and this peptide can inhibit Wnt/ß-catenin
signaling in Xenopus (Wong et
al., 2003
). However, the binding is relatively weak
(Kd
10 µM). Our results suggest that multiple regions of
Frizzled might be involved in the binding with Dvl and could increase the
binding affinity.
The same structural elements may be required for Frizzled to function in
both the planar polarity and the ß-catenin pathways, as membrane
translocation of Dvl has been implicated in planar polarity signaling
(Axelrod, 2001;
Rothbacher et al., 2000
), and
residues essential for the activity of Frizzled in ß-catenin signaling
are also important for Frizzled-induced translocation of Dvl to the plasma
membrane (Fig. 3). It is
possible that other proteins in the Frizzled-Dvl complex, such as LRP in
ß-catenin signaling and Flamingo in planar polarity signaling, determine
the signaling consequences of interaction between Frizzled and Dvl.
What is the role of LRP in transmitting the Wnt signal and what is the
function of its extracellular domain of LRP for receiving the Wnt signal? An
in vitro binding assay has suggested that Wnt1 is able to bind to the
extracellular domain of LRP (Tamai et al.,
2000), but analogous binding was not observed in studies with Wg
protein (Wu and Nusse, 2002
).
Results from in vitro binding assays need to be treated cautiously, as the
concentrations of ligands and receptors in these assays could be significantly
higher than in physiological situations, and certain components normally
involved in formation of the receptor complex could be missing in these
assays. Therefore, functional data are necessary to address the significance
of potential binding between Wnt and LRP. We have shown that the extracellular
domain of LRP can be functionally replaced by the extracellular domain of
Frizzled (Fig. 6), suggesting a
physiological role for a direct, or indirect, interaction of Wnt with the
extracellular domain of LRP.
LRP can also transmit a signal via ß-catenin without a requirement for
Wnt (Mao et al., 2001a;
Mao et al., 2001b
;
Schweizer and Varmus, 2003
).
We have taken advantage of two commonly used inducible oligomerization
strategies to demonstrate that oligomerization of LRP6 increases its signaling
activity and its interaction with Axin
(Fig. 4). Interestingly, it has
been shown that the second cysteine-rich domain of DKK2 stimulates
ß-catenin signaling via LRP independently of Dvl
(Li et al., 2002
). Further
experiments are needed to determine whether this DKK2 fragment activates LRP
by altering the oligomerization status of LRP.
A recent study has suggested that the extracellular domain of LRP might
negatively regulate the signaling activity of LRP through dimerization, which
can be relieved by Wnt proteins (Liu et
al., 2003). By contrast, we have shown that the signaling activity
of LRP was markedly increased by oligomerization
(Fig. 4). The source of this
discrepancy is currently unclear. However, we have found that, when
overexpressed in 293 cells, full-length LRP6 was less efficiently transferred
to the plasma membrane than was LRP6
N
(Fig. 5), an observation that
correlated with the lower signaling activity of LRP6. In addition, upon
co-expression of MESD, a chaperone of LRP, the signaling activities of LRP6
and LRP6
N became equivalent (Fig.
5). These data suggest that the low signaling activity of
full-length LRP6 is most likely due to its poor membrane localization, and
strongly argue against a negative role of the extracellular domain of LRP in
Wnt signaling. It should be also noted that only dimers of LRP6 could be
induced in the previous study (Liu et al.,
2003
), whereas we have used LRP6 fusion proteins able to form
oligomers. We have found that AP20187 increased the signaling activity of
LRP6
EGF fused to two copies of FKBP
(Fig. 4), but did not affect
the signaling activity of LRP6
EGF fused to one copy of FKBP (data not
shown). It is possible that in different conditions of dimer and oligomer
formation, the intracellular domains of LRP6 are placed at different positions
relative to each other, affecting the ability to bind to Axin.
Although Wnt can bind to both Frizzled and LRP, both receptors are essential for transducing the Wnt signal. It is possible that Wnt, Frizzled and LRP form one signaling complex. Alternatively, Wnt proteins might form separate complexes with Frizzled and LRP, which turn on separate signaling pathways that converge downstream.
We have provided several lines of evidence that support the first model, and our data suggest that bringing Frizzled and LRP into proximity is sufficient to trigger signaling through ß-catenin signaling. We have shown that the Wnt signaling pathway can be fully stimulated by oligomerizing Frizzled and LRP either through the intracellular region, by directly fusing the intracellular domain of LRP6 to the C terminus of human FZ5 (Fig. 7), or through the extracellular region, by co-expressing TrkN-LRP6C and NT3-HFz5 (Fig. 8). Furthermore, we have shown that the requirement for free Wnt proteins can be bypassed by fusing Wnt to either Frizzled or LRP (Fig. 9). These results suggest that Wnt, Frizzled and LRP form a single signaling complex, and the function of Wnt is to form a bridge between Frizzled and LRP. We recognize, however, that Wnt-induced oligomerization of endogenous Frizzled and LRP in living cells has not been demonstrated, nor have we characterized the physical properties of the proposed Wnt-induced oligomers.
Why is it necessary and sufficient to bring LRP and Frizzled into proximity
for transducing the Wnt signal? Our RNA interference studies have indicated
that signaling by overexpressed LRP is strictly Dvl independent, and Dvl
becomes important once Wnt and Frizzled are involved. Axin is known to
interact with the C terminus of LRP (Mao
et al., 2001b; Tolwinski et
al., 2003
), and Dvl can interact with Frizzled
(Fig. 3)
(Wong et al., 2003
).
Presumably, once overexpressed, a high concentration of membrane LRP is able
to bring endogenous Axin to the plasma membrane, based solely on its affinity
with Axin, so that Axin might be inactivated or degraded
(Mao et al., 2001b
;
Tolwinski et al., 2003
;
Willert et al., 1999
). This
would explain why the signaling activity of ectopically expressed LRP is Dvl
independent. Under normal physiological conditions, Frizzled and Dvl might be
required to translocate Axin to the membrane LRP upon Wnt signaling. Dvl might
function as a molecular chaperone to deliver Axin to the Frizzled-LRP complex,
based on its affinity with both Frizzled and Axin. In addition, Frizzled and
Dvl might also enhance the binding affinity between LRP and Axin through
promoting phosphorylation of LRP (Tamai et
al., 2004
). Therefore, the Wnt-Frizzled-LRP complex might serve as
a high-affinity docking site for Axin
(Fig. 11). This model is also
in agreement with the recent finding that Wnt induces translocation of Axin to
the membrane in a Dvl-dependent manner
(Cliffe et al., 2003
).
Consistent with its proposed role as a shuttle, Dvl is associated with
intracellular vesicles, and interacts with both actin stress fibers and
microtubules (Capelluto et al.,
2002
; Miller et al.,
1999b
).
|
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ACKNOWLEDGMENTS |
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Footnotes |
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