Laboratoire de Biologie du Développement, Groupe de Biologie Expérimentale, UMR CNRS 7622, Université Paris VI, 9 quai Saint-Bernard, 75005, Paris, France
* Author for correspondence (e-mail: umbhauer{at}ccr.jussieu.fr)
Accepted 4 March 2003
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Summary |
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Key words: Frizzled, Wnt, Dimerization, Xenopus
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Introduction |
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A crucial function of the Wnt pathway is to activate
ß-catenin-dependent transcription. In the absence of Wnt receptor
activation, the modular protein Axin provides a scaffold for the binding of
glycogen synthase kinase-3ß (GSK3), adenomatous polyposis coli protein
(APC) and ß-catenin. This, in turn, facilitates ß-catenin
phosphorylation by GSK3 (Ikeda et al.,
1998; Itoh et al.,
1998
) and leads to the rapid degradation of ß-catenin via the
ubiquitin pathway (Aberle et al.,
1997
). Upon Wnt's binding of the frizzled receptor, the
AxinGSK3APCß-catenin complex is disrupted by a
process involving the cytoplasmic protein Dishevelled (Dsh), and
dephosphorylation and recruitment of Axin to LRP5, which is associated with
Axin destabilization (Mao et al.,
2001
; Smalley et al.,
1999
; Yamamoto et al.,
1999
). Subsequently, the ß-catenin is no longer targeted for
ubiquitin degradation and has been shown to accumulate in the nuclei
(Fagotto et al., 1998
;
Yost et al., 1996
), where it
may interact with members of the lymphoid enhancer factor (LEF)/T-cell factor
(TCF) classes of transcription factors to regulate the expression of target
genes such as siamois (Brannon et
al., 1997
; Carnac et al.,
1996
; Lemaire et al.,
1995
; Molenaar et al.,
1996
). In Xenopus, Wnt/ß-catenin signaling plays a
crucial role in dorsoventral axis specification. Ventral overexpression of
certain Wnts or frizzleds, Dsh or ß-catenin ectopically activates
siamois transcription and leads to the generation of a complete
secondary axis (Moon and Kimelman,
1998
; Sokol,
1999
). In normal embryos, ß-catenin protein accumulates in
the cytoplasm and nuclei of dorsal blastomeres during early cleavage stages
(Larabell et al., 1997
).
Moreover, overepression of GSK3 and Axin or depletion of maternal
ß-catenin RNA causes deficiencies in dorsal structures
(He et al., 1995
;
Heasman et al., 1994
;
Yost et al., 1998
;
Zeng et al., 1997
).
The biochemical mechanisms by which the binding of the Wnt ligand to its
frizzled receptor elicits signal transduction within the cell are poorly
characterized. Numerous studies have suggested that several G-protein-coupled
receptor (GPCR) families exist as dimers or even higher structures
(Devi, 2001;
Gouldson et al., 2000
;
Hebert and Bouvier, 1998
;
Milligan, 2001
). Recently,
biophysical methods based on luminescence and fluorescence energy transfer
have confirmed the existence of such oligomeric complexes in living cells
(Angers et al., 2000
;
Angers et al., 2001
;
Kroeger et al., 2001
;
Overton and Blumer, 2000
).
However, whether dimerization is a general property of this class of receptors
and whether this is functionally relevant for signal transduction remains
controversial (Cvejic and Devi,
1997
; George et al.,
1998
; Gouldson et al.,
1998
; Hebert et al.,
1998
; Marshall et al.,
1999
). In several cases, receptors appear to fold as constitutive
dimers shortly after biosynthesis, whereas ligand-promoted dimerization at the
cell surface has been proposed for others
(Jones et al., 1998
;
Kaupmann et al., 1998
;
Kuner et al., 1999
;
White et al., 1998
).
Dimerization is required for normal functioning of ß-adrenergic receptors
and has been shown to rescue the function of mutant forms of ß-adrenergic
and angiotensin type I receptors (Hebert
et al., 1998
).
In this report, we address the question of the potential dimerization
property of two Xenopus frizzled receptors and the potential role of
this dimerization in Wnt/ß-catenin signal transduction. Taking advantage
of the differential ability of Xfz3 and Xfz7 to activate the
Wnt/ß-catenin pathway when overexpressed in the Xenopus embryo
(Umbhauer et al., 2000a), we
carried out a comparative study of Xfz3 and Xfz7, which revealed an unexpected
correlation between the presence of frizzled dimers and activation of the
Wnt/ß-catenin pathway. We show that Xfz3 but not Xfz7 exists as a
homodimer when expressed in the Xenopus embryo and activates
transcription of the Wnt/ß-catenin target gene siamois.
Moreover, forced homodimerization of Xfz7 is sufficient for activation of the
Wnt/ß-catenin pathway, even in the absence of the CRD ligand-binding
domain. We therefore propose that dimerization is a crucial step in the
transduction of the Wnt/ß-catenin signal by frizzled receptors.
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Materials and Methods |
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To obtain Xfz3-HA, the coding sequence of Xfz3 was PCR amplified with an
EcoRV site added at the C-terminus and cloned in frame with two HA
epitopes. To obtain Xfz7C-flag, a BamHI/ClaI digested
fragment corresponding to the 526 first amino acids of Xfz7 was inserted in
frame in the pCS2-extraXfz7-flag (Djiane
et al., 2000
) digested by BamHI and ClaI. To
make a myc version of the N-terminal extracellular region of Xfz3
(extra3-myc), a nontagged construct corresponding to the first 196 amino acids
of Xfz3 was first cloned in pSP64T (Krieg
and Melton, 1987
). Then, a XhoI/XbaI fragment
was inserted in pCS2-Xfz3-myc digested with XhoI and XbaI.
To obtain extra3-flag, pCS2-extraXfz7-flag
(Djiane et al., 2000
) was
digested by BamHI and ClaI to replace extraXfz7 with a PCR
fragment corresponding to the first 196 amino acids of Xfz3. The antisense
primer was 5'-TGAATCGATAGGCAAAGGAGAG-3' and the sense was the SP6
primer.
The Xfz7-BD-myc construct was obtained by EcoRI/BglII
digestion of pCS2-Xfz7-myc and insertion of an
EcoRI/BglII-digested PCR fragment amplified from TELmod
vector (Lopez et al., 1999).
The upstream primer was: 5'-CGGGAATTCGCGCTTGCAGCCAA-TT-3'; the
downstream primer was: 5'-GCAAGATCTGCTGAAGG-AGTTCATAGAG-3'.
The fibroblast growth factor receptor-1 (FGFR-1) fusion proteins were
obtained as follows: for extra3-R1, the coding sequence for the transmembrane
and intracellular domains of FGFR-1
(Friesel and Dawid, 1991) was
PCR amplified (upstream primer, 5'-CGGAGCT-CCAACTGGAAATT-3';
downstream primer, 5'-ATTATCTAGATC-AGCGTTTTTTAAG-3'), digested by
SacI and XbaI and inserted into pCS2-Xfz3-myc digested by
the same enzymes. For Xfz3
C-R1, the intracellular region of FGFR-1 was
PCR amplified (upstream primer, 5'-CGCTTCGAACACCCGTCGAAG-3';
downstream primer, 5'-ATTATCTAGATCAGCGTTTTTTAAG-3'), digested by
BstBI and XbaI and inserted into pCS2-Xfz3-myc digested by
the same enzymes. Torso-FGFR-1 has been previously described in
(Umbhauer et al., 2000b
). The
wild-type FGFR-1 is as in Amaya et al.
(Amaya et al., 1991
).
All constructs were checked by sequencing. Plasma membrane subcellular localization in blastula animal cap cells was verified by immunodetection and confocal microscopy for all myc and HA tagged constructs bearing a transmembrane domain.
Xenopus embryos and mRNA microinjections
Xenopus eggs were obtained from females injected with 500 IU of
human chorionic gonadotropin (Sigma), and artificially fertilized. Eggs were
dejellied with 2% cysteine hydrochloride (pH 7.8) and embryos were staged
according to Nieuwkoop and Faber
(Nieuwkoop and Faber,
1967).
Capped mRNAs were synthesized from linearized plasmids using SP6 RNA polymerase (Boehringer Mannheim) in the presence of 500 µM 5'-mGpppG-3' cap analog, 500 µM each rUTP, rATP, rCTP and 50 µM rGTP. Synthetic mRNA was purified using a Sephadex G-50 column (Pharmacia, Les Ulis, France) and recovered by ethanol precipitation. Microinjection of embryos was performed in 0.1x MBS (Modified Barth's Solution) containing 3% Ficoll 400 using a PLI-100 reproducible pico-injector (Medical Systems, Greenvale, NY). Unless specified, a volume of 10 nl corresponding to 250 pg of synthetic mRNA was injected into each blastomere at the animal pole region of two-cell-stage embryos.
RT-PCR
Animal cap explants from control and injected embryos were dissected at
mid-blastula stage (stage 8) and cultured to early gastrula stage (stage
10.5). Extraction of total RNA was as previously described
(Umbhauer et al., 2000a).
About 5 µg of total RNA were reverse-transcribed in the presence of 100 IU
of MLV (Murine Leukemia Virus) reverse transcriptase (Life Technologies,
Invitrogen, Cergy-Pontoise, France). In each experiment, 5 µg of total RNA
from whole embryos treated the same way but without reverse transcriptase were
used as a control for PCR specificity. One tenth of the reverse-transcribed
cDNA was used for PCR amplification in a 25 µl reaction mixture consisting
of 1x PCR buffer (Perkin Elmer Cetus, Life Science, Courtaboeuf,
France), dNTP at 0.2 mM each, 25 pmol of sense and antisense primers and 0.3
IU of Taq DNA polymerase (Perkin Elmer Cetus). PCR primers were the following:
Siamois (Lemaire et al.,
1995
) (5'-AAGGAACCCCACCAGGATAA-3' and
5'-TACTGGTGGCTGGAGAAATA-3'; 30 cycles); Xbra as in Henry
et al. (Henry et al., 1996
),
30 cycles; and ornithine decarboxylase (ODC)
(Bassez et al., 1990
)
(5'-GTCAATGATGGAGTGTATGGATC-3' and
5'-TCCATTCCGCTCTCCTGAGCAC-3'; 26 cycles). PCR products were
resolved on a 2% agarose gel containing 1 µg/ml ethidium bromide.
Western blotting and immunoprecipitation
Embryos at the early gastrula stage were extracted in lysis buffer [10 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% aprotinin (Sigma), 2
mM phenylmethylsulfonyl fluoride (PMSF) and 0.1% leupeptin (Sigma)]. After 10
minutes centrifugation at 13,200 g at 4°C, supernatants
were diluted in 2x Laemmli buffer without ß-mercaptoethanol for
direct western blotting analysis. For these analyses, samples of protein
extracts from the equivalent of 0.5 embryos were fractionated by
electrophoresis on SDS-polyacrylamide gels according to Laemmli
(Laemmli, 1970). For
immunoprecipitation experiments, extracted proteins from an equivalent of ten
embryos were separated in two equal parts and processed for
immunoprecipitation using an anti-c-myc (9E10, Santa Cruz, Santa Cruz, CA), an
anti-influenza-HA (12CA5, Boehringer Mannheim) or an anti-flag (M2, Sigma)
monoclonal antibody. Immunoblotting analyses were performed after gel
electrophoresis in reducing conditions and electrotransfer of separated
proteins on nitrocellulose sheets (HYBON-C, Amersham, Les Ulis, France). The
membranes were incubated in the presence of a 1/1000 dilution of monoclonal
antibodies 9E10 or 12CA5 or in the presence of a 1/500 dilution of the M2
antibody. After incubation with horseradish peroxidase-conjugated secondary
antibody (Immunotech Beckman Coulter Company, Maseille, London, 1/5000
dilution), bands were visualized using the ECL chemiluminescence kit
(Amersham).
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Results |
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To confirm that the species of higher molecular weight observed in direct
western blot corresponded to an Xfz3 homodimer, we devised a differential
co-immunoprecipitation strategy. Xfz3-HA, Xfz3C-myc and Xfz3-myc were
expressed either separately or in combination in Xenopus embryos. The
receptors were then immunoprecipitated with an anti-HA or an anti-myc
antibody, subjected to SDS-PAGE and blotted with one or the other antibody.
The anti-myc antibody specifically precipitated Xfz3
C-myc, whereas the
anti-HA antibody did not (Fig.
3, lanes 1 and 2). Immunoprecipitation of protein extracts from
embryos co-expressing Xfz3
C-myc and Xfz3-HA with the anti-HA antibody
resulted in the co-precipitation of Xfz3
C-myc
(Fig. 3, lane 3). Similar
results were obtained for the wild-type Xfz3-HA and Xfz3-myc
(Fig. 3, lanes 5 and 6). In
both cases, two bands corresponding in size to the monomeric and the dimeric
forms of the receptors were co-immunoprecipitated, suggesting that Xfz3 was
also present as an oligomer. Overexpression of the same tagged proteins in COS
cells yielded identical results (not shown). Taken together, these results
show that Xfz3 protein is able to form homodimers when expressed in
Xenopus embryos.
|
Xfz3 dimerization is disrupted by reducing agents such as
ß-mercaptoethanol
To determine whether disulfide bonds are important for Xfz3 dimer
formation, treatment with 2% or 20% ß-mercaptoethanol was performed
before SDS-PAGE on protein extracts from embryos expressing Xfz3C-myc.
In parallel, to prevent artificial disulfide bonds, protein extracts were also
treated with 10 mM iodoacetamide (IAA). As shown in
Fig. 4, ß-mercaptoethanol
treatment led to a significant diminution of the dimer/monomer ratio. In the
IAA-treated extracts, the dimeric form remained, excluding the possibility
that the band with high molecular mass was an artefact due to disulfide bond
exchange reactions during the preparation of samples. Thus, Xfz3 is able to
form a SDS resistant dimer in Xenopus embryo and its dimerization
relies, at least partly, on disulfide linkages.
|
The N-terminal extracellular region of Xfz3 is sufficient for
dimerization
The N-terminal extracellular region of frizzled receptors contains a
conserved CRD interacting with Wnt ligand. We have tested whether this region
alone is capable of dimerization when expressed in Xenopus embryos.
Embryos were injected with mRNA encoding the first 196 N-terminal amino acids
of Xfz3 (corresponding to the N-terminal extracellular region including the
CRD) bearing either a flag (extra3-flag) or a myc (extra3-myc) tag.
Co-immunoprecipitation experiments were performed using an anti-myc or an
anti-flag antibody. In protein extracts derived from embryos expressing both
constructs, anti-flag co-immunoprecipitated the extra3-myc protein
(Fig. 5A, lane 5), revealing
the presence of extra3-myc/extra3-flag dimers. In the embryo, the N-terminal
extracellular region of Xfz3 has therefore an intrinsic capacity to form a
homodimer. We asked next whether this region was sufficient to induce
dimerization when added to a membrane-bound heterologous context. Two chimeric
constructs between Xfz3 and the tyrosine kinase FGFR-1 receptor were devised.
For one construct (extra3-R1), the extracellular domain of FGFR-1, which
contains the FGF ligand binding site, has been replaced by the N-terminal
extracellular region of Xfz3 (including the CRD)
(Fig. 1). The other construct,
Xfz3C-R1, was made by replacing the C-terminal cytoplasmic region of
Xfz3 by the intracellular portion of the FGFR-1 receptor. The experimental
concept is based on the fact that tyrosine kinase receptors are known to
transduce intracellular signaling via dimerization on ligand binding. If
dimerization of the chimeric receptors occurs, the Xfz3 part of the chimeric
protein should be responsible for this dimerization, as no FGF ligand-binding
site is present in these receptors.
|
The chimeric receptors extra3-R1 and Xfz3C-R1 were overexpressed in
Xenopus blastula animal cap cells and transcriptional activation of
Xbra was tested by RT-PCR. Xbra is a target gene of the
transduction pathway activated by the FGFR-1 in response to FGF ligand in
ectoderm animal cap cells. Activation of its transcription will indicate the
presence of receptor dimers. As a positive control, we overexpressed a
constitutive form of FGFR-1 receptor, torso-R1
(Umbhauer et al., 2000b
) and
the wild-type FGFR-1 as a negative control. The same quantity of mRNA (50 pg)
was injected for each construct. As shown in
Fig. 5B, extra3-R1 and
Xfz3
C-R1 overexpression leads to the transcriptional activation of
Xbra, as does the torso-R1 positive control. This ability is
correlated with phosphorylation on tyrosine residues of these hybrid proteins
(Fig. 5C). In the same
experiment, Xbra was not expressed in response to overexpressing the
wild-type FGFR-1 (Fig. 5C).
These experiments show that the N-terminal extracellular portion of Xfz3 is
sufficient to induce dimerization either in a frizzled receptor or in an
unrelated context.
Functional correlation between frizzled dimerization and activation
of the Wnt/ß-catenin pathway
In previous work, we have shown that overexpression of Xfz3 in
Xenopus blastula animal cap cells is sufficient to activate
transcription of the Wnt/ß-catenin target gene siamois.
Overexpression of the receptor Xfz7, however, does not lead to
siamois expression (Umbhauer et
al., 2000a). Using the differential ability of these two receptors
to activate the Wnt/ß-catenin pathway after overexpression in
Xenopus embryo, we have addressed the functional significance of
frizzled dimerization.
We first analyzed the ability of the receptor Xfz7 to form dimers when
overexpressed in Xenopus embryos. Western blot analysis of protein
extracts derived from Xfz7-myc-injected embryos revealed only one group of
three bands corresponding to the expected molecular weight of the monomeric
Xfz7-myc (Fig. 6A). In most
cases, no other detectable signal was observed, although in some cases (two of
ten), a faint band corresponding to the size of a putative Xfz7-myc dimer was
visible. Co-immunoprecipitation experiments confirmed these western blot
analyses. The anti-flag antibody did immunoprecipitate Xfz7C-flag
(Fig. 6B, lane 4) but it did
not co-precipitate Xfz7-myc in protein extracts derived from embryos injected
with Xfz7-myc and Xfz7
C-flag, (Fig.
6B, lane 3). These results show that, unlike Xfz3, Xfz7 protein is
almost solely present in a monomeric form when expressed in the embryo.
|
Activation of the Wnt/ß-catenin pathway in blastula animal cap cells in response to Xfz3 but not Xfz7 may be due to the endogenous expression of a specific ligand for Xfz3. If this is true, co-expression of the N-terminal extracellular region of Xfz3 should block activation of the Wnt/ß-catenin pathway in response to Xfz3 by sequestering the ligand in the extracellular compartment. Embryos were injected at the two-cell stage with Xfz7-myc RNA, Xfz3-HA RNA or a combination of Xfz3-HA and extra3-myc RNA. Animal caps were dissected at the blastula stage, cultured to early gastrula and analysed by RT-PCR for siamois expression (Fig. 7A). As expected, Xfz3-HA but not Xfz7-myc activated siamois expression. Notably, the level of siamois expression in caps co-expressing at the same time Xfz3-HA and extra3-myc was very similar to the level obtained in response to Xfz3-HA alone (Fig. 7A). Moreover, co-injection of extra3-myc did not inhibit the formation of Xfz3 dimers, as shown by immunoprecipitation and western blot analysis (Fig. 7B). These results suggest that Xfz3 dimerizes and activates the Wnt/ß-catenin pathway in a ligand-independent manner, at least in ectoderm animal cap cells.
|
These results described above show a correlation between the presence of
dimers and the activation of the Wnt/ß-catenin pathway. To determine
whether activation of the Wnt/ß-catenin pathway was indeed directly
related to frizzled receptor dimerization, we designed a construct to
artificially force the dimerization of the Xfz7 receptor. We interchanged the
CRD of Xfz7 with an unrelated oligomerization domain (Xfz7-BD), the B domain
of ETS transcription factor TEL (translocated Ets leukemia)
(Lopez et al., 1999). Western
blot and immunodetection analyses revealed that this protein indeed migrates
as monomeric and dimeric forms, and even higher molecular forms
(Fig. 8A), and is correctly
expressed at the plasma membrane (Fig.
8B). Expression of Xfz7-BD in animal cap cells induces
siamois expression (Fig.
8C) at a similar level to that induced by Xfz3. Induced
dimerization of Xfz7 is therefore sufficient to activate the
Wnt/ß-catenin pathway.
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Discussion |
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The sensitivity of the dimeric Xfz3 receptor to reducing agents indicates
that disulfide bonds are important for Xfz3 dimer formation and/or
stabilization. The ß-mercaptoethanol treatment can either disrupt
intramolecular disulfide bonds and consequently affect conformation of the
receptor in a manner that impedes dimerization, or it can disrupt
intermolecular disulfide bonds directly responsible for dimer formation. If
intermolecular disulfide linkages are implicated in Xfz3 dimerization, none of
the ten cysteines located in the CRD is likely to be involved in these
interactions as these conserved cysteines have been shown to be engaged in
pairwise intramolecular bonds in the secreted frizzled-related proteins sFRP-1
and sFRP-3, as well as in the frizzled module recently determined in rat Ror1
receptor tyrosine kinase (Chong et al.,
2002; Roszmusz et al.,
2001
). However, cysteines outside the CRD could be involved in
intermolecular disulfide bonds implicated in Xfz3 dimerization. Several
cysteines located in the first and second extracellular loops are conserved
among the frizzled proteins and, interestingly, two conserved cysteines,
located on these same loops in the m3 muscarinic receptors have been
identified as key residues for covalent dimer formation by site-directed
mutagenesis (Zeng and Wess,
1999
).
Although dimerization has been shown for several GPCRs, the functional
roles of such a process are currently unclear. The GABA-B receptors associate
in the endoplasmic reticulum as heterodimers and are targeted to the plasma
membrane as preformed dimers, independent of agonist regulation
(Jones et al., 1998;
White et al., 1998
).
Dimerization of these receptors provides a mechanism to control the efficient
delivery of active GPCRs to the cell surface. The ß-adrenergic receptor,
however, undergoes ligand-dependent dimerization and activation, suggesting
that dimerization favors receptor/G-protein coupling efficiency
(Angers et al., 2000
;
Hebert and Bouvier, 1998
;
Hebert et al., 1996
). In the
case of frizzled receptors, our results strongly suggest that dimerization
plays a role in transducing the signal through the Wnt/ß-catenin pathway.
When overexpressed in blastula animal cap cells, Xfz3 exists as a dimer and
leads to activation of the Wnt/ß-catenin pathway, whereas Xfz7 remains
monomeric and does not activate the Wnt/ß-catenin pathway. Moreover, the
addition of a heterologous dimerization domain to Xfz7 is sufficient to
activate the Wnt/ß-catenin pathway in the animal cap assay. Activation of
this pathway can also be obtained when Xfz7 is co-expressed with an
appropriate Wnt ligand, suggesting that the lack of activity of Xfz7 in
Wnt/ß-catenin signaling in the animal cap cells may be due to the absence
of a specific ligand (Umbhauer et al.,
2000a
).
Although it is tempting to speculate that frizzled dimerization is induced
by the Wnt ligand, we have failed to show any reproducible effect of Wnt
expression on the frizzled dimer/monomer ratio using western blot or
co-immunoprecipitation studies (data not shown). In blastula animal cap cells,
the N-terminal extracellular domain of Xfz3 was not sufficient to inhibit Xfz3
dimerization and activation of the Wnt/ß-catenin pathway in response to
the full-length receptor Xfz3. These results suggest that activation of the
Wnt/ß-catenin pathway and formation of Xfz3 dimers are both ligand
independent, but they do not exclude a different mechanism for the receptor
Xfz7. The question of whether ligands affect GPCR oligomerization is currently
largely debated. Agonist treatment of several GPCRs has variably been reported
to increase, decrease or have no effect on the oligomeric complexes. This may
reflect the specific behavior of each receptor or it may be a consequence of
using different experimental approaches. For example, immunoprecipitation
experiments have suggested that agonists favor monomer formation of the
-opioid receptor (Cvejic and Devi,
1997
), whereas the use of both bioluminescence resonance energy
transfer (BRET) and time-resolved fluorescence resonance energy transfer
(FRET) approaches does not reveal any effect of agonist or antagonist on this
receptor at the cell surface (McVey et
al., 2001
).
In addition to the canonical Wnt/ß-catenin pathway, Wnt and frizzled
have been shown to transduce signals through at least two other pathways
(Kuhl et al., 2000b;
Mlodzik, 1999
). Wnt5A and rat
frizzled 2 trigger intracellular calcium release
(Slusarski et al., 1997
) and
activate protein kinase C and calmodulin kinase II (CamKII)
(Kuhl et al., 2000a
;
Sheldahl et al., 1999
) in a
heterotrimeric G-protein-dependent manner. In Drosophila, the planar
cell polarity pathway (PCP) identified downstream of DFz1 involves the
cytoplasmic protein Dsh, the small GTPase RhoA and a Jun N-terminal kinase
cascade. A similar PCP pathway might be implicated in the control of
gastrulation movements in Xenopus
(Wallingford et al., 2000
;
Yamanaka et al., 2002
). Dorsal
overexpression of Xfz7 or Xwnt11 perturbs convergence extension movements,
which can be rescued by co-injection of a dominant-negative form of the small
GTPase (Djiane et al., 2000
;
Medina et al., 2000
;
Tada and Smith, 2000
). Our
results suggest that dimerization of frizzled receptors might not be required
for the activation of the PCP pathway, as overexpression of Xfz7 as a monomer
is sufficient to alter gastrulation movements. Because several frizzleds seem
to be bifunctional receptors capable of transducing signals via two
biochemically distinct pathways, dimerization could enable the discrimination
between the alternate pathways. Therefore, dimerization of frizzled is
emerging as a possible mechanism for transduction specificity.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aberle, H., Bauer, A., Stappert, J., Kispert, A. and Kemler,
R. (1997). Beta-catenin is a target for the
ubiquitin-proteasome pathway. EMBO J.
16,3797
-3804.
Amaya, E., Musci, T. J. and Kirschner, M. W. (1991). Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 66,257 -270.[Medline]
Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D.,
Dennis, M. and Bouvier, M. (2000). Detection of beta
2-adrenergic receptor dimerization in living cells using bioluminescence
resonance energy transfer (BRET). Proc. Natl. Acad. Sci.
USA 97,3684
-3689.
Angers, S., Salahpour, A. and Bouvier, M. (2001). Biochemical and biophysical demonstration of GPCR oligomerization in mammalian cells. Life Sci. 68,2243 -2250.[CrossRef][Medline]
Bai, M., Trivedi, S. and Brown, E. M. (1998).
Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell
surface of CaR-transfected HEK293 cells. J. Biol.
Chem. 273,23605
-23610.
Bassez, T., Paris, J., Omilli, F., Dorel, C. and Osborne, H. B. (1990). Post-transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes. Development 110,955 -962.[Abstract]
Bhanot, P., Brink, M., Samos, C. H., Hsieh, J. C., Wang, Y., Macke, J. P., Andrew, D., Nathans, J. and Nusse, R. (1996). A new member of the frizzled family from Drosophila functions as a Wingless receptor. Nature 382,225 -230.[CrossRef][Medline]
Bhanot, P., Fish, M., Jemison, J. A., Nusse, R., Nathans, J. and
Cadigan, K. M. (1999). Frizzled and Dfrizzled-2
function as redundant receptors for Wingless during Drosophila
embryonic development. Development
126,4175
-4186.
Bhat, K. M. (1998). frizzled and frizzled 2 play a partially redundant role in wingless signaling and have similar requirements to wingless in neurogenesis. Cell 95,1027 -1036.[Medline]
Brannon, M., Gomperts, M., Sumoy, L., Moon, R. T. and Kimelman,
D. (1997). A beta-catenin/XTcf-3 complex binds to the siamois
promoter to regulate dorsal axis specification in Xenopus.
Genes Dev. 11,2359
-2370.
Cadigan, K. M. and Nusse, R. (1997). Wnt
signaling: a common theme in animal development. Genes
Dev. 11,3286
-3305.
Cadigan, K. M., Fish, M. P., Rulifson, E. J. and Nusse, R. (1998). Wingless repression of Drosophila frizzled 2 expression shapes the Wingless morphogen gradient in the wing. Cell 93,767 -777.[Medline]
Carnac, G., Kodjabachian, L., Gurdon, J. B. and Lemaire, P.
(1996). The homeobox gene Siamois is a target of the Wnt
dorsalisation pathway and triggers organiser activity in the absence of
mesoderm. Development
122,3055
-3065.
Chen, C. M. and Struhl, G. (1999). Wingless
transduction by the Frizzled and Frizzled2 proteins of Drosophila.
Development 126,5441
-5452.
Chong, J. M., Uren, A., Rubin, J. S. and Speicher, D. W.
(2002). Disulfide bond assignments of secreted Frizzled-related
protein-1 provide insights about Frizzled homology and netrin modules.
J. Biol. Chem. 277,5134
-5144.
Cvejic, S. and Devi, L. A. (1997). Dimerization
of the delta opioid receptor: implication for a role in receptor
internalization. J. Biol. Chem.
272,26959
-26964.
Dann, C. E., Hsieh, J. C., Rattner, A., Sharma, D., Nathans, J. and Leahy, D. J. (2001). Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains. Nature 412,86 -90.[CrossRef][Medline]
Devi, L. A. (2001). Heterodimerization of G-protein-coupled receptors: pharmacology, signaling and trafficking. Trends Pharmacol. Sci. 22,532 -537.[CrossRef][Medline]
Djiane, A., Riou, J., Umbhauer, M., Boucaut, J. and Shi, D.
(2000). Role of frizzled 7 in the regulation of convergent
extension movements during gastrulation in Xenopus laevis.
Development 127,3091
-3100.
Fagotto, F., Gluck, U. and Gumbiner, B. M. (1998). Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of beta-catenin. Curr. Biol. 8,181 -190.[Medline]
Friesel, R. and Dawid, I. B. (1991). cDNA cloning and developmental expression of fibroblast growth factor receptors from Xenopus laevis. Mol. Cell. Biol. 11,2481 -2488.[Medline]
George, S. R., Lee, S. P., Varghese, G., Zeman, P. R., Seeman,
P., Ng, G. Y. and O'Dowd, B. F. (1998). A
transmembrane domain-derived peptide inhibits D1 dopamine receptor function
without affecting receptor oligomerization. J. Biol.
Chem. 273,30244
-30248.
Gouldson, P. R., Snell, C. R., Bywater, R. P., Higgs, C. and Reynolds, C. A. (1998). Domain swapping in G-protein coupled receptor dimers. Protein Eng. 11,1181 -1193.[Abstract]
Gouldson, P. R., Higgs, C., Smith, R. E., Dean, M. K., Gkoutos, G. V. and Reynolds, C. A. (2000). Dimerization and domain swapping in G-protein-coupled receptors: a computational study. Neuropsychopharmacology 23,S60 -S77.[CrossRef][Medline]
He, X., Saint-Jeannet, J. P., Woodgett, J. R., Varmus, H. E. and Dawid, I. B. (1995). Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos. Nature 374,617 -622.[CrossRef][Medline]
Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., McCrea, P., Kintner, C., Noro, C. Y. and Wylie, C. (1994). Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell 79,791 -803.[Medline]
Hebert, T. E. and Bouvier, M. (1998). Structural and functional aspects of G protein-coupled receptor oligomerization. Biochem. Cell Biol. 76, 1-11.[CrossRef][Medline]
Hebert, T. E., Moffett, S., Morello, J. P., Loisel, T. P.,
Bichet, D. G., Barret, C. and Bouvier, M. (1996). A
peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits
both receptor dimerization and activation. J. Biol.
Chem. 271,16384
-16392.
Hebert, T. E., Loisel, T. P., Adam, L., Ethier, N., Onge, S. S. and Bouvier, M. (1998). Functional rescue of a constitutively desensitized beta2AR through receptor dimerization. Biochem. J. 330,287 -293.[Medline]
Henry, G. L., Brivanlou, I. H., Kessler, D. S.,
Hemmati-Brivanlou, A. and Melton, D. A. (1996).
TGF-beta signals and a pattern in Xenopus laevis endodermal
development. Development
122,1007
-1015.
Hsieh, J. C., Kodjabachian, L., Rebbert, M. L., Rattner, A., Smallwood, P. M., Samos, C. H., Nusse, R., Dawid, I. B. and Nathans, J. (1999a). A new secreted protein that binds to Wnt proteins and inhibits their activities. Nature 398,431 -436.[CrossRef][Medline]
Hsieh, J. C., Rattner, A., Smallwood, P. M. and Nathans, J.
(1999b). Biochemical characterization of Wnt-frizzled
interactions using a soluble, biologically active vertebrate Wnt protein.
Proc. Natl. Acad. Sci. USA
96,3546
-3551.
Ikeda, S., Kishida, S., Yamamoto, H., Murai, H., Koyama, S.
and Kikuchi, A. (1998). Axin, a negative regulator of
the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and
promotes GSK-3beta-dependent phosphorylation of beta-catenin. EMBO
J. 17,1371
-1384.
Itoh, K., Krupnik, V. E. and Sokol, S. Y. (1998). Axis determination in Xenopus involves biochemical interactions of axin, glycogen synthase kinase 3 and beta-catenin. Curr. Biol. 8,591 -594.[Medline]
Jones, K. A., Borowsky, B., Tamm, J. A., Craig, D. A., Durkin, M. M., Dai, M., Yao, W. J., Johnson, M., Gunwaldsen, C., Huang, L. Y. et al. (1998). GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2. Nature 396,674 -679.[CrossRef][Medline]
Kaupmann, K., Malitschek, B., Schuler, V., Heid, J., Froestl, W., Beck, P., Mosbacher, J., Bischoff, S., Kulik, A., Shigemoto, R. et al. (1998). GABA(B)-receptor subtypes assemble into functional heteromeric complexes. Nature 396,683 -687.[CrossRef][Medline]
Kennerdell, J. R. and Carthew, R. W. (1998). Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95,1017 -1026.[Medline]
Krieg, P. A. and Melton, D. A. (1987). In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 155,397 -415.[Medline]
Kroeger, K. M., Hanyaloglu, A. C., Seeber, R. M., Miles, L. E.
and Eidne, K. A. (2001). Constitutive and
agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone
receptor. Detection in living cells using bioluminescence resonance energy
transfer. J. Biol. Chem.
276,12736
-12743.
Kuhl, M., Sheldahl, L. C., Malbon, C. C. and Moon, R. T.
(2000a). Ca(2+)/calmodulin-dependent protein kinase II is
stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in
Xenopus. J. Biol. Chem.
275,12701
-12711.
Kuhl, M., Sheldahl, L. C., Park, M., Miller, J. R. and Moon, R. T. (2000b). The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet. 16,279 -283.[CrossRef][Medline]
Kuner, R., Kohr, G., Grunewald, S., Eisenhardt, G., Bach, A. and
Kornau, H. C. (1999). Role of heteromer formation in
GABAB receptor function. Science
283, 74-77.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Larabell, C. A., Torres, M., Rowning, B. A., Yost, C., Miller,
J. R., Wu, M., Kimelman, D. and Moon, R. T. (1997).
Establishment of the dorsoventral axis in Xenopus embryos is presaged
by early asymmetries in beta-catenin that are modulated by the Wnt signaling
pathway. J. Cell Biol.
136,1123
-1136.
Lemaire, P., Garrett, N. and Gurdon, J. B. (1995). Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell 81, 85-94.[Medline]
Lopez, R. G., Carron, C., Oury, C., Gardellin, P., Bernard, O.
and Ghysdael, J. (1999). TEL is a sequence-specific
transcriptional repressor. J. Biol. Chem.
274,30132
-30138.
Mao, J., Wang, J., Liu, B., Pan, W., Farr, G. H., III, Flynn, C., Yuan, H., Takada, S., Kimelman, D., Li, L. et al. (2001). Low-density lipoprotein receptor-related protein-5 binds to Axin and regulates the canonical Wnt signaling pathway. Mol. Cell 7,801 -809.[CrossRef][Medline]
Marshall, F. H., White, J., Main, M., Green, A. and Wise, A. (1999). GABA(B) receptors function as heterodimers. Biochem. Soc. Trans. 27,530 -535.[Medline]
McVey, M., Ramsay, D., Kellett, E., Rees, S., Wilson, S., Pope,
A. J. and Milligan, G. (2001). Monitoring receptor
oligomerization using time-resolved fluorescence resonance energy transfer and
bioluminescence resonance energy transfer. The human delta-opioid receptor
displays constitutive oligomerization at the cell surface, which is not
regulated by receptor occupancy. J. Biol. Chem.
276,14092
-14099.
Medina, A., Reintsch, W. and Steinbeisser, H. (2000). Xenopus frizzled 7 can act in canonical and non-canonical Wnt signaling pathways: implications on early patterning and morphogenesis. Mech. Dev. 92,227 -237.[CrossRef][Medline]
Miller, J. R., Hocking, A. M., Brown, J. D. and Moon, R. T. (1999). Mechanism and function of signal transduction by the Wnt/beta-catenin and Wnt/Ca2+ pathways. Oncogene 18,7860 -7872.[CrossRef][Medline]
Milligan, G. (2001). Oligomerisation of
G-protein-coupled receptors. J. Cell Sci.
114,1265
-1271.
Mlodzik, M. (1999). Planar polarity in the
Drosophila eye: a multifaceted view of signaling specificity and
cross-talk. EMBO J. 18,6873
-6879.
Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destree, O. and Clevers, H. (1996). XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86,391 -399.[Medline]
Moon, R. T. and Kimelman, D. (1998). From cortical rotation to organizer gene expression: toward a molecular explanation of axis specification in Xenopus. Bioessays 20,536 -545.[CrossRef][Medline]
Musci, T. J., Amaya, E. and Kirschner, M. W. (1990). Regulation of the fibroblast growth factor receptor in early Xenopus embryos. Proc. Natl. Acad. Sci. USA 87,8365 -8369.[Abstract]
Nieuwkoop, P. D. and Faber, J. (1967). Normal Table of Xenopus laevis (Daudin). Amsterdam: Amsterdam/North-Holland Publishing Company.
Overton, M. C. and Blumer, K. J. (2000). G-protein-coupled receptors function as oligomers in vivo. Curr. Biol. 10,341 -344.[CrossRef][Medline]
Pinson, K. I., Brennan, J., Monkley, S., Avery, B. J. and Skarnes, W. C. (2000). An LDL-receptor-related protein mediates Wnt signalling in mice. Nature 407,535 -538.[CrossRef][Medline]
Romano, C., Yang, W. L. and O'Malley, K. L.
(1996). Metabotropic glutamate receptor 5 is a disulfide-linked
dimer. J. Biol. Chem.
271,28612
-28616.
Romano, C., Miller, J. K., Hyrc, K., Dikranian, S., Mennerick,
S., Takeuchi, Y., Goldberg, M. P. and O'Malley, K. L.
(2001). Covalent and noncovalent interactions mediate
metabotropic glutamate receptor mGlu5 dimerization. Mol.
Pharmacol. 59,46
-53.
Roszmusz, E., Patthy, A., Trexler, M. and Patthy, L.
(2001). Localization of disulfide bonds in the frizzled module of
ror1 receptor tyrosine kinase. J. Biol. Chem.
276,18485
-18490.
Rulifson, E. J., Wu, C. H. and Nusse, R. (2000). Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless. Mol. Cell 6, 117-126.[Medline]
Sheldahl, L. C., Park, M., Malbon, C. C. and Moon, R. T. (1999). Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner. Curr. Biol. 9,695 -698.[CrossRef][Medline]
Shi, D. L., Goisset, C. and Boucaut, J. C. (1998). Expression of Xfz3, a Xenopus frizzled family member, is restricted to the early nervous system. Mech. Dev. 70,35 -47.[CrossRef][Medline]
Slusarski, D. C., Corces, V. G. and Moon, R. T. (1997). Interaction of Wnt and a Frizzled homologue triggers G-protein-linked phosphatidylinositol signalling. Nature 390,410 -413.[CrossRef][Medline]
Smalley, M. J., Sara, E., Paterson, H., Naylor, S., Cook, D.,
Jayatilake, H., Fryer, L. G., Hutchinson, L., Fry, M. J. and Dale, T.
C. (1999). Interaction of axin and Dvl-2 proteins regulates
Dvl-2-stimulated TCF-dependent transcription. EMBO J.
18,2823
-2835.
Sokol, S. Y. (1999). Wnt signaling and dorsoventral axis specification in vertebrates. Curr. Opin. Genet. Dev. 9,405 -410.[CrossRef][Medline]
Tada, M. and Smith, J. C. (2000). Xwnt11 is a
target of Xenopus Brachyury: regulation of gastrulation movements via
Dishevelled, but not through the canonical Wnt pathway.
Development 127,2227
-2238.
Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., Hess, F., Saint-Jeannet, J. P. and He, X. (2000). LDL-receptor-related proteins in Wnt signal transduction. Nature 407,530 -535.[CrossRef][Medline]
Turner, D. L. and Weintraub, H. (1994). Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8,1434 -1447.[Abstract]
Umbhauer, M., Djiane, A., Goisset, C., Penzo-Mendez, A., Riou,
J. F., Boucaut, J. C. and Shi, D. L. (2000a). The
C-terminal cytoplasmic Lysthr-X-X-X-Trp motif in frizzled receptors mediates
Wnt/beta-catenin signalling. EMBO J.
19,4944
-4954.
Umbhauer, M., Penzo-Mendez, A., Clavilier, L., Boucaut, J. and
Riou, J. (2000b). Signaling specificities of fibroblast
growth factor receptors in early Xenopus embryo. J. Cell
Sci. 113,2865
-2875.
Wallingford, J. B., Rowning, B. A., Vogeli, K. M., Rothbacher, U., Fraser, S. E. and Harland, R. M. (2000). Dishevelled controls cell polarity during Xenopus gastrulation. Nature 405,81 -85.[CrossRef][Medline]
Wang, S., Krinks, M., Lin, K., Luyten, F. P. and Moos, M., Jr (1997). Frzb, a secreted protein expressed in the Spemann organizer, binds and inhibits Wnt-8. Cell 88,757 -766.[Medline]
Wehrli, M., Dougan, S. T., Caldwell, K., O'Keefe, L., Schwartz, S., Vaizel- Ohayon, D., Schejter, E., Tomlinson, A. and DiNardo, S. (2000). Arrow encodes an LDL-receptor-related protein essential for Wingless signalling. Nature 407,527 -530.[CrossRef][Medline]
White, J. H., Wise, A., Main, M. J., Green, A., Fraser, N. J., Disney, G. H., Barnes, A. A., Emson, P., Foord, S. M. and Marshall, F. H. (1998). Heterodimerization is required for the formation of a functional GABA(B) receptor. Nature 396,679 -682.[CrossRef][Medline]
Yamamoto, H., Kishida, S., Kishida, M., Ikeda, S., Takada, S.
and Kikuchi, A. (1999). Phosphorylation of axin, a Wnt
signal negative regulator, by glycogen synthase kinase-3beta regulates its
stability. J. Biol. Chem.
274,10681
-10684.
Yamanaka, H., Moriguchi, T., Masuyama, N., Kusakabe, M.,
Hanafusa, H., Takada, R., Takada, S. and Nishida, E.
(2002). JNK functions in the non-canonical Wnt pathway to
regulate convergent extension movements in vertebrates. EMBO
Rep. 3,69
-75.
Yost, C., Torres, M., Miller, J. R., Huang, E., Kimelman, D. and Moon, R. T. (1996). The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev. 10,1443 -1454.[Abstract]
Yost, C., Farr, G. H., III, Pierce, S. B., Ferkey, D. M., Chen, M. M. and Kimelman, D. (1998). GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis. Cell 93,1031 -1041.[Medline]
Zeng, F. Y. and Wess, J. (1999). Identification
and molecular characterization of m3 muscarinic receptor dimers. J.
Biol. Chem. 274,19487
-19497.
Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J., Perry, W. L., III, Lee, J. J., Tilghman, S. M., Gumbiner, B. M. and Costantini, F. (1997). The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell 90,181 -192.[Medline]
Zhang, J. and Carthew, R. W. (1998).
Interactions between Wingless and DFz2 during Drosophila wing
development. Development
125,3075
-3085.