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First published online 30 November 2006
doi: 10.1242/dev.02699
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Review |
Hubrecht Laboratory and Center for Biomedical Genetics, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.
* Author for correspondence (e-mail: rkors{at}niob.knaw.nl)
In this review, we look at the processes that occur `behind the scenes' in Wnt signalling, within the Wnt-producing cells. The Wnt community has long been focused upon events that occur downstream of Wnt binding to its receptors, but the recent discovery that the maturation of the Wnt protein may have a profound effect on its signalling properties has excited great interest. In the last 2 years, several key regulators of Wnt production have been discovered, but our global understanding of this process remains relatively poor. Several models that reconcile former and recent observations of Wnt modification, sorting and secretion, and which highlight the potential of this emerging field, are presented here.
Introduction
Wnt glycoproteins are extracellular ligands that can be found in many
species, ranging from the sea anemone Nematostella vectensis to human
(Kusserow et al., 2005
). Wnts
act as morphogens and control the patterning of the developing embryo by
triggering concentration-dependent responses in cells located at a distance
from the signal-sending domain (Neumann
and Cohen, 1997; Strigini and
Cohen, 2000; Zecca et al.,
1996). During animal development, Wnt ligands regulate several key
processes, such as cell proliferation, cell migration and cell differentiation
(Cadigan and Nusse, 1997;
Moon et al., 2002;
Wodarz and Nusse, 1998).
Furthermore, deregulation of the Wnt signalling pathway has been implicated in
many pathological disorders, including colon cancer
(Bienz and Clevers, 2000;
Logan and Nusse, 2004). The
intracellular mechanisms that transduce the Wnt signal downstream of its
receptors and modulate the expression of its target genes have been
extensively studied, and will not be discussed here (for review, see
Cadigan and Liu, 2006;
Cadigan and Nusse, 1997;
He et al., 2004;
Moon et al., 2002;
van Es et al., 2003) (also see
the Wnt Homepage
http://www.stanford.edu/~rnusse/wntwindow.html).
However, little is known about the different steps that control the synthesis
and secretion of a functional Wnt protein, and which ensure its delivery to
the responding cells along the morphogenetic field. In this review, we focus
on Wnt-producing cells and discuss the mechanisms that lead to the production
of a fully active Wnt. All known aspects of Wnt maturation will be addressed:
from its post-translational modification in the endoplasmic reticulum (ER) to
its sorting within the expressing cells and, finally, to its secretion as a
long-range-acting protein. Although the influence of Wnt properties on the
formation of an extracellular concentration gradient will be mentioned, the
multiple mechanisms that regulate the spreading of Wnt will not be presented
here. For overviews of these processes, the reader is referred to other
reviews (Cadigan, 2002;
Eaton, 2006;
Strigini and Cohen, 2000;
Vincent and Dubois, 2002).
Structure of Wnts
Except for Drosophila Wingless (Wg), Wnt3/5 and Wnt4, which are
all larger proteins, the different Wnts generally have similar structural
characteristics (Miller, 2002)
(Table 1). Thus, most Wnts are
approximately 350 amino acids long and have a molecular weight of about 40
kDa. Wnts contain several charged residues and an average of 23 to 25
cysteines, some of which are highly conserved between different species
(Fig. 1). Several of these
cysteines are involved in inter- and intra-molecular disulfide bonds,
participating in Wnt folding and multimerization
(Tanaka et al., 2002).
Surprisingly, despite the presence of charged residues in their primary
sequences, mouse Wnt3A and Drosophila Wg were found to be hydrophobic
molecules (Willert et al.,
2003; Zhai et al.,
2004). This hydrophobicity confers a strong affinity for cell
membranes, which raises questions as to how these proteins spread in the
extracellular space. Wnts do not contain any defined domains, but do have an
N-terminal hydrophobic signal sequence that targets them to the ER.
Finally, further analysis of the Wnt sequences reveals the presence of a
number of potential N-glycosylation sites, the functional relevance
of which will be discussed below (Fig.
1).
|
Post-translational modifications of Wnt in the ER
When they reach the ER, immature Wnts undergo important posttranslational
modifications. First, the oligosaccharyl transferase complex (OST) rapidly and
efficiently attaches N-linked oligosaccharide chains to the
appropriate residues on the peptide backbone (Asn108 and
Asn414 in Wg) (Tanaka et al.,
2002). However, the biological function of Wnt
N-glycosylation remains unclear. Indeed, mutation of part or all of
the potential glycosylation sites of mouse Wnt1 does not affect its function
in a cell culture-based assay (Mason et
al., 1992). In cell culture, the secretion of many Wnts by their
producing cells is inefficient and most newly synthesized Wnt is retained in
the ER (Burrus and McMahon,
1995; McMahon and Moon,
1989; St-Arnaud et al.,
1989). Within this compartment, Wnt has been found to associate
with the immunoglobulin heavy-chainbinding protein BiP, a member of the Hsp70
family of heat-shock proteins (Burrus and
McMahon, 1995; Kitajewski et
al., 1992). BiP acts as a chaperone for numerous proteins, and
allows the further sorting and secretion of mature and functional proteins
only (Gething and Sambrook,
1990; Hurtley et al.,
1989; Kassenbrock et al.,
1988). Thus, BiP can sequester certain misfolded or unassembled
proteins within the ER. Inhibition of Wnt N-glycosylation does not
increase the relative amount of Wnt bound to BiP, suggesting that this does
not severely affect Wnt folding and structure
(Burrus and McMahon, 1995).
Finally, similarly to other secreted proteins, Wnt N-glycosylation
may be one of the determinant signals for its apical secretion, which may
explain the absence of effect when glycosylation is inhibited in a
non-polarized cell assay (Mason et al.,
1992). However, this hypothesis has not been experimentally
proven, and the exact role of N-glycosylation in the production
and/or activity of Wnt remains to be elucidated.
The recent purification of mouse Wnt3A from cell culture medium identified
an additional post-translational modification
(Willert et al., 2003).
Specifically, a palmitate group is attached to a cysteine located in the
N-terminal part of the protein (C77) (see
Fig. 1). Wg was also found to
be lipid-modified (Zhai et al.,
2004), and the high conservation of the cysteine involved suggests
that this palmitoylation is a common feature of all Wnts. The function of this
second modification is also unclear. The hydrophobic lipid group may promote
Wnt targeting to the ER membrane and subsequently allow efficient
N-glycosylation by the membrane-associated OST complex. However, this
cannot be the sole function of the palmitate moiety because, in contrast to
loss of N-glycosylation of Wnt1, the absence of palmitoylation
abrogates Wnt3A function in cell culture
(Willert et al., 2003).
Alternatively, Wnt palmitoylation may protect the modified cysteine from
forming a disulfide bond, thereby preventing the aberrant folding of the
protein and its retention in the ER by BiP. Palmitate groups are also known to
act as intracellular sorting signals
(Bijlmakers and Marsh, 2003).
Thus, Wnt lipid-modification may allow the proper routing of the protein
through different compartments of the Wnt-producing cells, where Wnts acquire
their signalling potential. Finally, Wnt3A hydrophobicity has been found to
depend largely upon the presence of the palmitate moiety
(Willert et al., 2003).
Therefore, this lipid-modification may play a role after secretion by
targeting Wnt to the membranes of cells along the morphogenetic field. Such a
mechanism may control the spatial distribution of Wnt and increase its local
concentration at its receptors, allowing efficient signalling. This model is
indeed supported by the capacity of a mutant form of Wnt3A that lacks the
palmitoylated cysteine (C77A) to activate a Tcf reporter gene when the
potential drop in local concentration is compensated for by overexpression
(Willert et al., 2003).
However, such a model implies that another mechanism exists that allows Wnt to
spread away from its production site by partially balancing the hydrophobicity
that is provided by the lipid group.
There is some evidence that the protein Porcupine, which resides in the ER
and is a member of the membrane-bound O-acyltransferase family
(MBOAT), has an influence on both of these post-translational modifications.
With respect to the N-glycosylation, in the absence of Porcupine, the
attachment of the oligosaccharide chains is impaired. Conversely, Porcupine
overexpression results in ectopic glycosylation of Wg
(Tanaka et al., 2002). The
precise role of Porcupine in this process is unknown. Interestingly, the
Porcupine C-terminal region binds an N-terminal domain of Wg
that contains conserved cysteines. Furthermore, Wg N-glycosylation
competes with the formation of intracellular disulfide bonds, as suggested by
the enhanced glycosylation observed after treatment by a reducing agent such
as Dithiotreitol (DTT) (Tanaka et al.,
2002). Porcupine may therefore protect these residues from forming
disulfide bonds. This theory was initially supported by the absence of
additional effects of Porcupine overexpression under fully reducing
conditions. However, Porcupine does not affect the formation of disulfide
bonds (Tanaka et al., 2002),
although a transient effect delaying this process, which would promote Wg
glycosylation, cannot be ruled out. Another model can be envisaged where
Porcupine targets Wnt to the ER membrane, facilitating its modification by the
OST complex. This may be directly achieved by the physical binding of Wnt to
membrane-bound Porcupine, or indirectly by the Porcupine-dependent acylation
of Wnt (see below). Consistent with this model, a membrane-tethered form of Wg
is more efficiently glycosylated (Tanaka
et al., 2002). In addition to targeting Wnt to the ER membrane,
the binding of Porcupine may have an effect on Wnt conformation, allowing
optimal access of the OST complex to the different N-glycosylation
sites.
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). Second,
the Porcupine-binding domain of Wg spans the region where the modified
cysteine is located (Fig. 1)
(Tanaka et al., 2002).
Furthermore, similarly to loss of palmitoylation, loss of Porcupine abrogates
Wg signalling (Riggleman et al.,
1990; van den Heuvel et al.,
1993). However, in Drosophila embryos, Wg secretion is
inhibited in the absence of Porcupine, whereas Wnt3A(C77A), lacking the
palmitoylated cysteine, is secreted normally in mammalian cell culture
(Kadowaki et al., 1996;
van den Heuvel et al., 1993;
Willert et al., 2003),
indicating that catalyzing the palmitoylation of this conserved cysteine is
not the only function that Porcupine may play in the Wnt-maturation process.
Recent studies may, nevertheless, confirm Porcupine as the Wnt-acylating
enzyme. In addition to being palmitoylated at this N-terminally
located cysteine, Burrus and colleagues found that Wnt is lipid-modified at
other positions in a Porcupine-dependent manner as well. Indeed, Wnt3A(C77A)
was shown to still harbour one or more lipid modifications despite lacking the
palmitoylated cysteine. Additionally, overexpression of Porcupine further
increased the fraction of Wnt3A(C77A) found in the detergent phase in phase
separation experiments (L. Burrus, personal communication). This suggests that
residues other than those previously described can be modified by Porcupine
and can confer hydrophobicity. Interestingly, the group of Shinji Takada has
shown that mutation of another specific amino acid in Wnt3A, besides C77,
prevents all lipid modification and results in the retention of the protein in
the ER (S. Takada, personal communication). The different lipid groups may
therefore have separate functions. The previously identified palmitoylation
may affect Wnt function itself, either by allowing its sorting to specialized
compartments where Wnt acquires crucial features, or by enhancing its
signalling potential at the level of the responding cells. Additional
acylation(s) may be required for the proper sorting of Wnt out of the ER and
may therefore affect its secretion. All those modifications appear to be
dependent on Porcupine, explaining the retention of Wnt within its producing
cells when Porcupine function is inhibited.
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Before being released into the extracellular space, Wnts within the
producing cells move from the ER to the trans-Golgi network (TGN), and from
there through different subcellular compartments
(Gonzalez et al., 1991). It is
tempting to speculate that fully modified and functional Wnt can leave the ER
and enter the bulk flow secretory pathway, thereby exiting its expressing
cells via unregulated secretion vesicles. However, some results suggest that
Wnt may use an alternative secretion route. First, in Wg-expressing cells, a
fraction of Wg has been found to exist in the endosomal compartment (notably
in multi-vesicular bodies), as well as in recycling vesicles that potentially
arise from endosomes and are directed to the plasma membrane
(Gonzalez et al., 1991;
Pfeiffer et al., 2002;
van den Heuvel et al., 1989).
This localization may result from the direct endocytosis of secreted Wg by the
producing cells themselves. Alternatively, Wg may be actively targeted to this
subcellular compartment within the expressing cells, before secretion. Second,
the comparison of Wnt with Hedgehog also supports the hypothesis of a
specialized secretion pathway. Wnt and Hedgehog are morphogens that share
several properties at the structural (both are lipid-modified) and functional
levels (Nusse, 2003). A recent
study showed that Hedgehog does not enter the default secretory pathway
(Gallet et al., 2003): by
monitoring Hedgehog localization in its producing cells, the authors found
that it segregates away from a secreted form of GFP. Wnt may follow a similar
route. Finally, Wg has also been found to be associated with lipid rafts in
cell culture (Zhai et al.,
2004). Lipid rafts are separate microdomains of ordered lipid
phases within the bilayer of the membrane and are known to be involved in
multiple aspects of membrane transport
(Ikonen, 2001). It is
especially interesting to notice that rafts can act as platforms that
segregate subgroups of proteins from other secreted proteins, partitioning
them into specialized sorting and secreting routes
(Schmidt et al., 2001).
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).
Furthermore, a putative Wnt cargo receptor at the membrane of the TGN may bind
Wnt and direct its transport to the endosomal compartment. Again, lipid rafts
have been shown to mediate the inclusion of sorting receptors and cargo
molecules into the forming vesicle
(Dhanvantari and Loh, 2000).
The existence of such a cargo receptor has not yet been reported. However, the
transmembrane protein Wntless/Evi, which is required within Wnt-producing
cells, may be a good candidate for this mechanism (see below). Although a
requirement for additional intracellular sorting events cannot be ruled out,
Wnt is likely to leave the endosomal compartment via recycling vesicles,
resulting in its release outside of its producing cells.
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Wnt is associated with lipoprotein particles
As has been thoroughly demonstrated for Wg in Drosophila
(Neumann and Cohen, 1997;
Strigini and Cohen, 2000;
Zecca et al., 1996), in other
species, Wnts also act as morphogens and can reach responding cells that are
located several cell diameters away from the Wnt source
(Coudreuse et al., 2006;
Kiecker and Niehrs, 2001). The
migration of Wnt along the morphogenetic field, and whether this occurs via
extracellular diffusion or transcytosis, has long been a subject of debate
(Vincent and Dubois, 2002).
Although some discrepancies remain, the majority of recent studies favour a
model in which Wnt can spread in a controlled, but extracellular, manner
(Baeg et al., 2001;
Han et al., 2005;
Lander et al., 2002;
Strigini and Cohen, 2000).
Wg was recently found to associate with lipoprotein particles
(Panakova et al., 2005).
Lipoprotein particles consist of phospholipid monolayers that surround a core
of esterified cholesterol and triglycerides, scaffolded by members of the
apolipoprotein family. In Drosophila, the formation of such a
Wg-lipoprotein-particle complex is required for long-range signalling in the
wing imaginal disc, but it does not seem to dramatically influence short-range
effects of Wg. The function of this association in long-range signalling of
Wnt is unclear, and it has so far not been formally proven that this complex
has signalling activity. Several models can, however, be envisaged. First,
lipoprotein particles may displace hydrophobic Wnt from the membranes of its
producing cells and from cells located in their direct vicinity, thereby
allowing its further spreading. Second, it may tightly regulate the extension
and steepness of the Wnt gradient by mediating Wnt interactions with members
of the heparan sulfate proteoglycan family (HSPG)
(Baeg et al., 2001;
Giraldez et al., 2002;
Han et al., 2005;
Kirkpatrick et al., 2004;
Kreuger et al., 2004). Third,
although possibly counteracting its membrane targeting at the site of
production, the transport of Wnt on lipoprotein particles may prevent its
dilution in the extracellular space and therefore increase its local
concentration at its receptors. This may be achieved directly or indirectly by
interaction with HSPGs. Finally, lipoprotein particles may stabilize
extracellular Wnt and allow the clustering of Wnt molecules, facilitating the
presentation of efficient amounts of Wnt to the receptors. This may be
especially important for signalling at a distance from the source, where the
concentration of Wnt becomes limiting.
The manner and location in which Wnt may associate with these lipoprotein
particles is particularly interesting in the context of this review.
Consistent with the displacement hypothesis, one model suggests that Wnt binds
lipoprotein particles outside of its producing cells, after secretion
(Fig. 2A). However, the
potential sorting of Wnt to endosomes suggests an alternative process. Thus,
lipoprotein particles may be endocytosed by low density lipoprotein (LDL)- or
high density lipoprotein (HDL)-receptors at the surface of the Wnt-producing
cells. Internalized lipoprotein particles may then reach the endosomes where
the mature Wnt has been sorted to. The association of Wnt with these particles
may therefore occur within the Wnt-producing cells, before Wnt secretion
(Fig. 2B). The requirement for
a mechanism that extracts Wnt out of the default secretory pathway may
therefore be related to the binding of Wnt to lipoprotein particles. However,
Wnt is likely to use both the constitutive and a specialized secretion route.
Indeed, only a low fraction of total Wg was found to be bound to lipoprotein
particles (Panakova et al.,
2005). Most Wnt may therefore use the default secretory pathway
and signal at a short distance from the producing cells, whereas a small
fraction of Wnt, dedicated to long-range signalling, may be sorted to the
endosomes to be associated with lipoprotein particles
(Fig. 2B). Endosomal budding
and formation of recycling vesicles may subsequently release the
Wnt-lipoprotein-particle complex outside of the producing cells, allowing
optimal signalling along the morphogenetic field.
Wntless/Evi and Wnt secretion
Irrespective of the pathway, Wnt secretion appears to be tightly regulated
by the highly conserved seven-pass transmembrane protein Wntless/Evi (Wls),
which physically interacts with Wnt
(Banziger et al., 2006;
Bartscherer et al., 2006). In
the absence of Wls, Wnt is retained within its producing cells, resulting in a
Wnt loss-of-function phenotype. The function of Wls in Wnt signalling is
conserved in C. elegans, where the Wls orthologue MOM-3 (also known
as MIG-14) is required for signalling by all Wnts tested
(Eisenmann and Kim, 2000;
Harris et al., 1996;
Thorpe et al., 1997). Previous
results from genetic studies on MIG-14, and especially the recent analysis of
Wls function in Drosophila and mammalian cells
(Banziger et al., 2006;
Bartscherer et al., 2006), have
demonstrated that it is specifically required within the Wnt-producing cells
for Wnt secretion.
Conflicting results on Wls subcellular localization have been obtained by
different groups. The Boutros laboratory identified Wls mainly at the plasma
membrane of Drosophila imaginal disc cells, whereas Bänziger and
colleagues suggest that Wls is located in the Golgi, and in vesicles between
the Golgi and the cell surface in Drosophila, as well as in mammalian
cells (Banziger et al., 2006;
Bartscherer et al., 2006).
However, such differences may result from variations in the expression levels
of the Wls transgenes. The potential localization of Wls in vesicles suggests
that it may regulate the intracellular trafficking of Wnt between different
compartments of the producing cells. Mis-sorting of Wnt to certain
compartments may preclude its secretion. Consistent with this hypothesis, in
the absence of Wls, Wnt3A does not reach the surface of its producing cells.
In the study by the Basler laboratory, the subcellular localization of Wnt3A
was not noticeably modified by the loss of Wls function in cell culture
(Banziger et al., 2006).
However, it cannot be ruled out that the sorting defect is relatively subtle
and only detectable by a detailed compartmental analysis, or by
cofractionation experiments. Interestingly, Bartscherer and colleagues show
that the apical localization of Wg in the wing disc epithelium is lost in the
absence of Wls (Bartscherer et al.,
2006). Instead, Wg appears to be located throughout the expressing
cells. Thus, in a Wls-mutant background, Wnt may not reach the appropriate
apical compartment or the compartment where it acquires its capacity to be
apically sorted and secreted. However, the interpretation of this defect in
apical localization remains difficult, as Wls was shown to be required for Wnt
secretion in non-polarized assays (Banziger
et al., 2006).
The aberrant sorting of Wnt during its maturation may also indirectly
disrupt its secretion. Similarly to the loss of Porcupine or mutation of a
specific residue in Wnt3A (S. Takada, personal communication), the mis-sorting
of Wnt may prevent post-translational modifications that are essential for its
secretion. However, Porcupine-mediated acylation itself is unlikely to be
affected by Wls, because Wls was not detected in the ER and was found to act
later in the secretion process. Furthermore, inhibition of Wnt3A acylation
results in its retention in the ER, whereas Wg is localized throughout the
expressing cells in the absence of Wls function. Finally,
N-glycosylation of Wnt3A, which may serve as a signal for its
polarized secretion, was found to be intact in the absence of Wls
(Banziger et al., 2006).
Wls-dependent trafficking of Wnt may, therefore, regulate other, as yet
unknown, modifications. Alternatively, Wls may allow the optimal
post-translational modification of Wnt by means other than the control of its
subcellular localization. Thus, Wls may directly act as a chaperone and
notably control the modification of residues that are crucial for Wnt
secretion. This might be achieved by presenting Wnt to the appropriate enzyme
or by facilitating the access of the enzyme to the correct residues.
Our currently limited understanding of Wnt maturation allows only for speculative models, and it cannot be ruled out that Wls controls unknown steps of Wnt production that are required for its secretion. A detailed description of the biochemical properties of mature Wnt, as well as a complete study of the route followed by Wnt during its production by the Wnt-expressing cells, will be necessary to understand the functions of proteins such as Wls.
The role of the retromer complex
An additional level of complexity has recently been added to this picture
with the identification of the retromer complex as a key player in the
production of a functional Wnt (Coudreuse
et al., 2006). The retromer is a highly conserved multiprotein
complex, the core of which consists of the subunits Vps35, Vps29 and Vps26
(Seaman et al., 1998). It was
initially discovered in yeast for its role in the intracellular sorting of the
carboxypeptidase Y (CPY) cargo receptor Vps10p
(Paravicini et al., 1992;
Seaman et al., 1997). Vps10p
binds CPY (which are lysosomal enzymes) in the TGN and ensures their transit
to late endosomes. After the pH-dependent release of CPY in endosomes, Vps10p
is recycled back to the TGN. This retrograde transport is mediated by the
interaction of the cytoplasmic tail of Vps10p with Vps35p
(Nothwehr et al., 2000). In
the absence of retromer function, Vps10p accumulates in the endosomes and is
eventually targeted to the lysosomes. Vps10p subsequently becomes limiting in
the TGN, resulting in the secretion of CPY via the constitutive secretion
pathway. Similarly, the mammalian mannose 6-phosphate receptor, which allows
the transport of lysosomal hydrolase precursors, is recycled to the TGN by the
retromer complex (Arighi et al.,
2004). In mammalian cells, the retromer complex was also shown to
mediate basal-to-apical transcytosis of the
immunoglobulin-A-immunoglobulin-receptor complex
(Verges et al., 2004).
A recent study using both C. elegans and vertebrate systems
suggests that the retromer is also required within the Wnt-producing cells for
proper Wnt signalling (Coudreuse et al.,
2006). Thus, loss of the core protein VPS-35 abrogates EGL-20/Wnt
signalling in C. elegans (see
Prasad and Clark, 2006).
Similarly, the knockdown of Vps35 in mammalian cells and Xenopus
tropicalis inhibits Wnt target gene expression
(Coudreuse et al., 2006). In
C. elegans, retromer function appears to be specific to Wnt
signalling, as no other major signalling pathway is significantly affected in
mutants of retromer components. Interestingly, the retromer was found to be
required within the EGL-20/Wnt-producing cells for the formation of an
anteroposterior gradient of EGL-20. Consistent with these findings, loss of
the retromer was shown to fully impair long-range, but to only mildly affect
short-range, EGL-20 signalling. Finally, phenotypic studies in C.
elegans and biochemical analyses in mammalian cells have shown that Wnt
secretion itself is not inhibited in the absence of retromer function
(Coudreuse et al., 2006).
Several models can be proposed for the function of the retromer in Wnt-gradient formation. As discussed earlier, Wnt harbours post-translational modifications, such as N-glycosylations and palmitoylations. Additional, as yet unidentified, modifications may also occur during Wnt maturation. Some of these modifications, acquired in various specialized subcellular compartments of the expressing cells, may be required for the formation of a Wnt gradient, and therefore for the long-range signalling activity of Wnt. Thus, the retromer may promote Wnt maturation by keeping the different Wnt-modifying enzymes in their assigned compartments. This may be achieved through the direct binding of the retromer complex to the cytoplasmic tail of membrane-bound enzymes, or by the interaction of this complex with associated cargo receptors. As a member of the MBOAT family, Porcupine localization may be directly or indirectly regulated by the retromer complex. However, Porcupine is required for Wg secretion, whereas loss of retromer function does not affect Wnt secretion, indicating that the retromer is probably not required for Porcupine function.
Interestingly, the effect of loss of retromer function is strikingly
similar to the effect observed for Wg and Hedgehog when their association with
lipoprotein particles is impaired
(Panakova et al., 2005). Thus,
in the absence of the Drosophila lipoprotein lipophorin, which is an
essential component of the lipoprotein particles in Drosophila,
long-range Wg signalling is more strongly affected than is short-range Wg
signalling. If the binding of Wnt to lipoprotein particles occurs within the
endosomal compartment after sorting of Wnt by a cargo receptor, at least two
functions can be envisaged for the retromer complex. First, the retromer may
directly bind the cytoplasmic tail of the Wnt cargo receptor at the TGN and
allow the continuous transport of a fraction of newly synthesized Wnt to the
endosomes (Fig. 3A).
Alternatively, it may promote the binding of a cytoplasmic transporter to the
Wnt cargo receptor. In the absence of retromer function, the
Wnt-Wnt-cargo-receptor complex may accumulate in the TGN. Wnt may subsequently
be released and enter the bulk flow secretion. This dissociation may occur
within the TGN, or at later stages, because of a change in chemical
environment along the default secretory pathway. Consequently, the endosomal
association of Wnt with lipoprotein particles is prevented, resulting in
constitutive secretion of a non-spreading, short-range-signalling form of Wnt.
A second model emerges from the known recycling function of the retromer in
yeast. This model implies that the amount of Wnt cargo receptor at the TGN is
limiting. Thus, similar to its function in Vps10p recycling, the retromer may
retrieve Wnt cargo receptors from the endosomes to the TGN, allowing
additional rounds of Wnt cycling (Fig.
3B). In the absence of retromer function, the Wnt cargo receptors
may accumulate in the endosomes and may eventually be degraded in the
lysosomes. The subsequent shortage in cargo receptors would prevent Wnt from
exiting the TGN. Again, this would result in constitutive secretion of a
short-range signalling form of Wnt. In addition to requiring retromer
function, Wnt association with lipoprotein particles may also depend on the
N-terminal palmitate group. Thus, when the palmitoylated cysteine is
mutated into a serine, EGL-20 is similarly unable to form an anteroposterior
gradient in C. elegans (our unpublished data).
These two models, however, rely on the actual sorting of Wnt to the
endosomes before secretion as well as on the hypothetical existence of a Wnt
cargo receptor. Furthermore, they imply that the association of Wnt with
lipoprotein particles, which is necessary for the spreading of Wnt along the
morphogenetic field, is not the means by which hydrophobic Wnt avoids
sequestration at the membrane of cells directly adjacent to its producing
cells. Indeed, Wnt3A was found in normal amounts in the medium of vertebrate
cells after the depletion of Vps35 by RNAi, suggesting that it does not stick
to the nearby membranes despite the potential absence of lipoprotein-particle
association (Coudreuse et al.,
2006). Further studies are obviously required to understand the
exact role of the lipoprotein particles and the consequences of the loss of
retromer function on the association of these particles with Wnt.
Wls, the retromer complex and the association of Wnt with lipoprotein particles
Both Wls and the retromer complex may be involved in intracellular trafficking of Wnt within Wnt-expressing cells. It is therefore attractive to speculate on models in which Wls and the retromer functionally interact to allow the efficient maturation of Wnt and its association with lipoprotein particles. The molecular nature of Wls, a transmembrane protein that binds Wnt, suggests that it may act as the previously discussed Wnt cargo receptor, allowing the transport of Wnt from the TGN to the endosomes. In this context, the retromer may directly or indirectly bind Wls and mediate the anterograde transport of the Wls-Wnt complex from the TGN. Alternatively, it may regulate the retrieval of Wls from the endosomes, thereby ensuring the further cycling of Wnt molecules. However, such models seem inconsistent with the inhibition of Wnt secretion observed in the absence of Wls, whereas normal amounts of Wnt appear to exit the cells when retromer function is impaired. Further investigation is therefore needed to reconcile these results and to demonstrate if Wls, the retromer and the association of Wnt with lipoprotein particles are different steps of the same mechanism (Fig. 4).
Conclusions
The question of the maturation, sorting and secretion of a functional, long-range-acting Wnt protein has long been left aside in favour of the study of Wnt signal transduction downstream of its receptors. However, it has recently become clear that the function of Wnt in vivo and its capacity to spread and precisely pattern the developing embryo are dependent on key processes that occur within Wnt-producing cells before secretion. Specific post-translational modifications, the controlled sorting of Wnt to specialized subcellular compartments and its association with extracellular vehicles could be at the tip of the iceberg of mechanisms that contribute to the release of an optimal Wnt protein. A concerted effort to investigate these processes needs to be made in order to propose new models and to elucidate some of the many questions and contradictions that we have highlighted in this review. The better characterization of the properties that Wnt acquires during its maturation, as well as an in-depth description of Wnt biochemistry, are inevitable steps towards a better understanding of its function in animal development.
ACKNOWLEDGMENTS
We would like to thank Laura Burrus and Shinji Takada for sharing unpublished results; and Claire McNulty for critically reading the manuscript. This work was funded by grants (H.C.K.) from the Dutch Cancer Foundation and the EU Sixth Framework Program `Cells into Organs'.
REFERENCES
Arighi, C. N., Hartnell, L. M., Aguilar, R. C., Haft, C. R. and
Bonifacino, J. S. (2004). Role of the mammalian retromer in
sorting of the cation-independent mannose 6-phosphate receptor. J.
Cell Biol. 165,123
-133.
Baeg, G. H., Lin, X., Khare, N., Baumgartner, S. and Perrimon, N. (2001). Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. Development 128,87 -94.[Abstract]
Banziger, C., Soldini, D., Schutt, C., Zipperlen, P., Hausmann, G. and Basler, K. (2006). Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125,509 -522.[CrossRef][Medline]
Bartscherer, K., Pelte, N., Ingelfinger, D. and Boutros, M. (2006). Secretion of Wnt ligands requires Evi, a conserved transmembrane protein. Cell 125,523 -533.[CrossRef][Medline]
Bienz, M. and Clevers, H. (2000). Linking colorectal cancer to Wnt signaling. Cell 103,311 -320.[CrossRef][Medline]
Bijlmakers, M. J. and Marsh, M. (2003). The on-off story of protein palmitoylation. Trends Cell. Biol. 13,32 -42.[CrossRef][Medline]
Burrus, L. W. and McMahon, A. P. (1995). Biochemical analysis of murine Wnt proteins reveals both shared and distinct properties. Exp. Cell Res. 220,363 -373.[CrossRef][Medline]
Cadigan, K. M. (2002). Regulating morphogen gradients in the Drosophila wing. Semin. Cell Dev. Biol. 13,83 -90.[CrossRef][Medline]
Cadigan, K. M. and Nusse, R. (1997). Wnt
signaling: a common theme in animal development. Genes
Dev. 11,3286
-3305.
Cadigan, K. M. and Liu, Y. I. (2006). Wnt
signaling: complexity at the surface. J. Cell Sci.
119,395
-402.
Coudreuse, D. Y., Roel, G., Betist, M. C., Destree, O. and
Korswagen, H. C. (2006). Wnt gradient formation requires
retromer function in Wnt-producing cells. Science
312,921
-924.
Dhanvantari, S. and Loh, Y. P. (2000). Lipid
raft association of carboxypeptidase E is necessary for its function as a
regulated secretory pathway sorting receptor. J. Biol.
Chem. 275,29887
-29893.
Eaton, S. (2006). Release and trafficking of lipid-linked morphogens. Curr. Opin. Genet. Dev. 16, 17-22.[CrossRef][Medline]
Eisenmann, D. M. and Kim, S. K. (2000).
Protruding vulva mutants identify novel loci and Wnt signaling factors that
function during Caenorhabditis elegans vulva development.
Genetics 156,1097
-1116.
Gallet, A., Rodriguez, R., Ruel, L. and Therond, P. P. (2003). Cholesterol modification of hedgehog is required for trafficking and movement, revealing an asymmetric cellular response to hedgehog. Dev. Cell 4,191 -204.[CrossRef][Medline]
Gething, M. J. and Sambrook, J. (1990). Transport and assembly processes in the endoplasmic reticulum. Semin. Cell Biol. 1,65 -72.[Medline]
Giraldez, A. J., Copley, R. R. and Cohen, S. M. (2002). HSPG modification by the secreted enzyme Notum shapes the Wingless morphogen gradient. Dev. Cell 2, 667-676.[CrossRef][Medline]
Gonzalez, F., Swales, L., Bejsovec, A., Skaer, H. and Martinez Arias, A. (1991). Secretion and movement of wingless protein in the epidermis of the Drosophila embryo. Mech. Dev. 35,43 -54.[CrossRef][Medline]
Han, C., Yan, D., Belenkaya, T. Y. and Lin, X.
(2005). Drosophila glypicans Dally and Dally-like shape
the extracellular Wingless morphogen gradient in the wing disc.
Development 132,667
-679.
Harris, J., Honigberg, L., Robinson, N. and Kenyon, C. (1996). Neuronal cell migration in C. elegans: regulation of Hox gene expression and cell position. Development 122,3117 -3131.[Abstract]
He, X., Semenov, M., Tamai, K. and Zeng, X.
(2004). LDL receptor-related proteins 5 and 6 in
Wnt/ß-catenin signaling: arrows point the way.
Development 131,1663
-1677.
Hurtley, S. M., Bole, D. G., Hoover-Litty, H., Helenius, A. and
Copeland, C. S. (1989). Interactions of misfolded influenza
virus hemagglutinin with binding protein (BiP). J. Cell
Biol. 108,2117
-2126.
Ikonen, E. (2001). Roles of lipid rafts in membrane transport. Curr. Opin. Cell Biol. 13,470 -477.[CrossRef][Medline]
Kadowaki, T., Wilder, E., Klingensmith, J., Zachary, K. and
Perrimon, N. (1996). The segment polarity gene porcupine
encodes a putative multitransmembrane protein involved in Wingless processing.
Genes Dev. 10,3116
-3128.
Kassenbrock, C. K., Garcia, P. D., Walter, P. and Kelly, R. B. (1988). Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro. Nature 333, 90-93.[CrossRef][Medline]
Kiecker, C. and Niehrs, C. (2001). A morphogen
gradient of Wnt/ß-catenin signalling regulates anteroposterior neural
patterning in Xenopus. Development
128,4189
-4201.
Kirkpatrick, C. A., Dimitroff, B. D., Rawson, J. M. and Selleck, S. B. (2004). Spatial regulation of Wingless morphogen distribution and signaling by Dally-like protein. Dev. Cell 7,513 -523.[CrossRef][Medline]
Kitajewski, J., Mason, J. O. and Varmus, H. E.
(1992). Interaction of Wnt-1 proteins with the binding protein
BiP. Mol. Cell. Biol.
12,784
-790.
Kreuger, J., Perez, L., Giraldez, A. J. and Cohen, S. M. (2004). Opposing activities of Dally-like glypican at high and low levels of Wingless morphogen activity. Dev. Cell 7, 503-512.[CrossRef][Medline]
Kusserow, A., Pang, K., Sturm, C., Hrouda, M., Lentfer, J., Schmidt, H. A., Technau, U., von Haeseler, A., Hobmayer, B., Martindale, M. Q. et al. (2005). Unexpected complexity of the Wnt gene family in a sea anemone. Nature 433,156 -160.[CrossRef][Medline]
Lander, A. D., Nie, Q. and Wan, F. Y. (2002). Do morphogen gradients arise by diffusion? Dev. Cell 2, 785-796.[CrossRef][Medline]
Logan, C. Y. and Nusse, R. (2004). The Wnt signaling pathway in development and disease. Annu. Rev Cell Dev. Biol. 20,781 -810.[CrossRef][Medline]
Mason, J. O., Kitajewski, J. and Varmus, H. E. (1992). Mutational analysis of mouse Wnt-1 identifies two temperature-sensitive alleles and attributes of Wnt-1 protein essential for transformation of a mammary cell line. Mol. Biol. Cell 3, 521-533.[Abstract]
McMahon, A. P. and Moon, R. T. (1989). int-1, a proto-oncogene involved in cell signalling. Development 107 Suppl,161 -167.
Miller, J. R. (2002). The Wnts. Genome Biol. 3, REVIEWS3001.
Moon, R. T., Bowerman, B., Boutros, M. and Perrimon, N.
(2002). The promise and perils of Wnt signaling through
ß-catenin. Science
296,1644
-1646.
Neumann, C. J. and Cohen, S. M. (1997). Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development 124,871 -880.[Abstract]
Nothwehr, S. F., Ha, S. A. and Bruinsma, P.
(2000). Sorting of yeast membrane proteins into an
endosome-to-Golgi pathway involves direct interaction of their cytosolic
domains with Vps35p. J. Cell Biol.
151,297
-310.
Nusse, R. (2003). Wnts and Hedgehogs:
lipid-modified proteins and similarities in signaling mechanisms at the cell
surface. Development
130,5297
-5305.
Panakova, D., Sprong, H., Marois, E., Thiele, C. and Eaton, S. (2005). Lipoprotein particles are required for Hedgehog and Wingless signalling. Nature 435, 58-65.[CrossRef][Medline]
Paravicini, G., Horazdovsky, B. F. and Emr, S. D. (1992). Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. Mol. Biol. Cell 3, 415-427.[Abstract]
Pfeiffer, S., Ricardo, S., Manneville, J. B., Alexandre, C. and Vincent, J. P. (2002). Producing cells retain and recycle Wingless in Drosophila embryos. Curr. Biol. 12,957 -962.[CrossRef][Medline]
Prasad, B. C. and Clark, S. G. (2006). Wnt
signaling establishes anteroposterior neuronal polarity and requires retromer
in C. elegans. Development
133,1757
-1766.
Riggleman, B., Schedl, P. and Wieschaus, E. (1990). Spatial expression of the Drosophila segment polarity gene armadillo is posttranscriptionally regulated by wingless. Cell 63,549 -560.[CrossRef][Medline]
Schmidt, K., Schrader, M., Kern, H. F. and Kleene, R.
(2001). Regulated apical secretion of zymogens in rat pancreas.
Involvement of the glycosylphosphatidylinositol-anchored glycoprotein GP-2,
the lectin ZG16p, and cholesterol-glycosphingolipid-enriched microdomains.
J. Biol. Chem. 276,14315
-14323.
Seaman, M. N., Marcusson, E. G., Cereghino, J. L. and Emr, S.
D. (1997). Endosome to Golgi retrieval of the vacuolar
protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30,
and VPS35 gene products. J. Cell Biol.
137, 79-92.
Seaman, M. N., McCaffery, J. M. and Emr, S. D.
(1998). A membrane coat complex essential for endosome-to-Golgi
retrograde transport in yeast. J. Cell Biol.
142,665
-681.
St-Arnaud, R., Craig, J., McBurney, M. W. and Papkoff, J. (1989). The int-1 proto-oncogene is transcriptionally activated during neuroectodermal differentiation of P19 mouse embryonal carcinoma cells. Oncogene 4,1077 -1080.[Medline]
Strigini, M. and Cohen, S. M. (2000). Wingless gradient formation in the Drosophila wing. Curr. Biol. 10,293 -300.[CrossRef][Medline]
Tanaka, K., Kitagawa, Y. and Kadowaki, T.
(2002). Drosophila segment polarity gene product
porcupine stimulates the posttranslational N-glycosylation of wingless in the
endoplasmic reticulum. J. Biol. Chem.
277,12816
-12823.
Thorpe, C. J., Schlesinger, A., Carter, J. C. and Bowerman, B. (1997). Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm. Cell 90,695 -705.[CrossRef][Medline]
van den Heuvel, M., Nusse, R., Johnston, P. and Lawrence, P. A. (1989). Distribution of the wingless gene product in Drosophila embryos: a protein involved in cell-cell communication. Cell 59,739 -749.[CrossRef][Medline]
van den Heuvel, M., Harryman-Samos, C., Klingensmith, J., Perrimon, N. and Nusse, R. (1993). Mutations in the segment polarity genes wingless and porcupine impair secretion of the wingless protein. EMBO J. 12,5293 -5302.[Medline]
van Es, J. H., Barker, N. and Clevers, H. (2003). You Wnt some, you lose some: oncogenes in the Wnt signaling pathway. Curr. Opin. Genet. Dev. 13, 28-33.[CrossRef][Medline]
Verges, M., Luton, F., Gruber, C., Tiemann, F., Reinders, L. G., Huang, L., Burlingame, A. L., Haft, C. R. and Mostov, K. E. (2004). The mammalian retromer regulates transcytosis of the polymeric immunoglobulin receptor. Nat. Cell. Biol. 6, 763-769.[CrossRef][Medline]
Vincent, J. P. and Dubois, L. (2002). Morphogen transport along epithelia, an integrated trafficking problem. Dev. Cell 3,615 -623.[CrossRef][Medline]
Willert, K., Brown, J. D., Danenberg, E., Duncan, A. W., Weissman, I. L., Reya, T., Yates, J. R., 3rd and Nusse, R. (2003). Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423,448 -452.[CrossRef][Medline]
Wodarz, A. and Nusse, R. (1998). Mechanisms of Wnt signaling in development. Annu. Rev. Cell Dev. Biol. 14,59 -88.[CrossRef][Medline]
Zecca, M., Basler, K. and Struhl, G. (1996). Direct and long-range action of a wingless morphogen gradient. Cell 87,833 -844.[CrossRef][Medline]
Zhai, L., Chaturvedi, D. and Cumberledge, S.
(2004). Drosophila wnt-1 undergoes a hydrophobic
modification and is targeted to lipid rafts, a process that requires
porcupine. J. Biol. Chem.
279,33220
-33227.
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