Division of Neuroscience, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
* Author for correspondence (e-mail: xi.he{at}childrens.harvard.edu)
SUMMARY
Wnt signaling through the canonical ß-catenin pathway plays essential roles in development and disease. Low-density-lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6) in vertebrates, and their Drosophila ortholog Arrow, are single-span transmembrane proteins that are indispensable for Wnt/ß-catenin signaling, and are likely to act as Wnt co-receptors. This review highlights recent progress and unresolved issues in understanding the function and regulation of Arrow/Lrp5/Lrp6 in Wnt signaling. We discuss Arrow/Lrp5/Lrp6 interactions with Wnt and the Frizzled family of Wnt receptors, and with the intracellular ß-catenin degradation apparatus. We also discuss the regulation of Lrp5/Lrp6 by other extracellular ligands, and LRP5 mutations associated with familial osteoporosis and other disorders.
Introduction
Signaling by the Wnt family of secreted growth factors has key roles in
development and disease (Wodarz and
Nusse, 1998; Veeman et al.,
2003
). Since the discovery of Wnt1 as an oncogene that
causes mouse mammary tumorigenesis (Nusse
and Varmus, 1982
), the Wnt gene family, which includes 19 members
in the human genome, has been found in all animal species examined. Wnt
proteins regulate many stages of development, from patterning of the embryo
and generation of tissues and cell types, to regulation of cell movements,
polarity, axon guidance and synapse formation
(Nusse, 2003
;
Packard et al., 2003
;
Strutt, 2003
;
Veeman et al., 2003
).
Defective Wnt signaling plays major roles in diseases such as cancer
(Bienz and Clevers, 2000
;
Polakis, 2000
) and
osteoporosis (Patel and Karsenty,
2002
). Therefore, the investigation of Wnt signal transduction is
crucial for understanding development and disease.
The interaction of Wnt proteins with their receptors on the cell surface is
the first step in transducing the extracellular signal into intracellular
responses. The first identified Wnt receptors were members of the Frizzled
(Fz) family of seven-pass transmembrane receptors
(Wodarz and Nusse, 1998), 10
of which are encoded in the human genome. In addition to Fz proteins, the
canonical Wnt/ß-catenin signaling pathway requires single-span
transmembrane proteins that belong to a subfamily of low-density-lipoprotein
(LDL) receptor related proteins (LRPs): vertebrate Lrp5 and Lrp6, and their
Drosophila ortholog Arrow (Pinson
et al., 2000
; Tamai et al.,
2000
; Wehrli et al.,
2000
), which are the focus of this review.
We will discuss the structure and function of Arrow/Lrp5/Lrp6, their
interactions with Wnt, Fz and the intracellular ß-catenin signaling
apparatus, their biogenesis and modulation by extracellular antagonists, and,
finally, the roles of LRP5 mutations in human diseases. Because of their
related biochemical properties in Wnt signaling, we will often, unless
otherwise specified, use Lrp5/Lrp6 to refer Lrp5 and Lrp6 together in the
discussion. For an overview of Wnt signaling, including accounts of various
Wnt transduction pathways and components, readers may refer to many excellent
reviews (Wodarz and Nusse,
1998; Adler, 2002
;
Huelsken and Behrens, 2002
;
Nusse, 2003
;
Strutt, 2003
;
Veeman et al., 2003
).
Canonical Wnt/ß-catenin signaling
ß-catenin phosphorylation and degradation
The outcome of the most intensively studied Wnt pathway the
canonical Wnt/ß-catenin signaling pathway relies to a large
extent on the regulation of the stability/abundance of the ß-catenin
protein (Fig. 1). It is widely
accepted that in this Wnt pathway ß-catenin associates with, and acts as
an obligatory nuclear co-activator for, the TCF/LEF (T cell factor/Lymphoid
enhancer factor) family of transcription factors
(Bienz and Clevers, 2003;
Cong et al., 2003
;
Tolwinski and Wieschaus,
2004
). In the absence of a Wnt ligand, the level of cytosolic
ß-catenin is kept low as a result of its amino-terminal
phosphorylation-dependent ubiquitination/proteosome degradation. When
ß-catenin is low, TCF/LEF is associated with transcriptional
co-repressors and suppresses Wnt-responsive gene expression
(Fig. 1A). Upon Wnt
stimulation, ß-catenin phosphorylation and degradation is inhibited, and
the accumulation of ß-catenin promotes its association with TCF/LEF,
leading to the activation of Wnt-responsive transcription
(Fig. 1B).
|
How Wnt inhibits ß-catenin phosphorylation is not well defined, but
may be achieved by any of, or combinations of, the following
(Fig. 1B): (1) degradation of
the Axin protein (Willert et al.,
1999; Yamamoto et al.,
1999
; Mao et al.,
2001b
; Tolwinski et al.,
2003
); (2) alteration of the composition of the Axin complex (such
as by dissociation of Gsk3 or ß-catenin from Axin)
(Kishida et al., 1999a
;
Willert et al., 1999
;
Li et al., 1999
;
Itoh et al., 2000
); (3)
binding of the Gsk3 binding protein (GBP) to Gsk3
(Farr et al., 2000
;
Salic et al., 2000
); and (4)
inhibition of Gsk3 (or Ck1) kinase activity
(Cook et al., 1996
). A recent
genetic study in Drosophila
(Tolwinski et al., 2003
),
however, suggested that Wnt/ß-catenin signaling can occur in a
Gsk3-independent manner (Box
1). Another cytoplasmic protein Dishevelled has a crucial but
poorly understood role in the inhibition of the Axin/Gsk3 complex, and is
discussed in more detail in later sections.
Fz proteins are Wnt receptors
The Wnt-Fz ligand-receptor relationship is best characterized in
Drosophila. Studies using cultured Drosophila cells indicate
that two members of the Fz family, Dfz1 and Dfz2 (Fz and Fz2 FlyBase),
are Wingless (Wg, Drosophila Wnt1) receptors, which bind Wg with high
affinities (Kd=108 M and 109 M
for Dfz1 and Dfz2, respectively) (Bhanot et
al., 1996; Rulifson et al.,
2000
). Fly mutants lacking both Dfz1 and Dfz2,
but not mutants lacking either, have severely defective Wg signaling
(Bhat, 1998
;
Kennerdell and Carthew, 1998
;
Bhanot et al., 1999
;
Chen and Struhl, 1999
;
Muller et al., 1999
),
providing unambiguous evidence that, in many contexts, Dfz1 and Dfz2 are
redundant Wg receptors.
Studies in nematodes, Xenopus and mammalian cells support the
Wnt-Fz ligand-receptor relationship (Sawa
et al., 1996; Yang-Snyder et
al., 1996
; He et al.,
1997
; Rocheleau et al.,
1997
; Thorpe et al.,
1997
; Hsieh et al.,
1999
). However, the specificity of Wnt-Fz interactions remains
largely unresolved, particularly in vertebrates, because of difficulties in
producing soluble Wnt proteins, the large numbers of Wnt and Fz genes, and the
potential multitudes of Wnt-Fz interactions and functional redundancies.
Nevertheless, recent success in purification of the first Wnt protein, mouse
Wnt3a, may help to pave the way for comprehensive biochemical studies of
Wnt-Fz interactions in vitro (Willert et
al., 2003
).
Arrow/Lrp5/Lrp6: primary structure and function
Arrow/Lrp5/Lrp6 and Wnt/ß-catenin signaling
The roles of Arrow and Lrp6 in Wnt signaling were discovered via genetic
studies. Drosophila mutants lacking arrow phenotypically
resemble the wg mutant (Wehrli
et al., 2000), and mutant mice lacking Lrp6 exhibit
composite phenotypes similar to mutations of several individual Wnt genes
(Pinson et al., 2000
). In
Xenopus embryos, dominant-negative Lrp6 blocks signaling by several
Wnt proteins, whereas overexpression of Lrp6 cooperates with Wnt and Fz to
activate Wnt/ß-catenin signaling
(Tamai et al., 2000
).
Furthermore, Arrow and Lrp6 are required for cells to respond to Wnt, and act
upstream of known intracellular Wnt signaling components in
Drosophila and Xenopus
(Tamai et al., 2000
;
Wehrli et al., 2000
),
pinpointing a role for Arrow/Lrp6 in Wg/Wnt signal reception. A recent genetic
study in mice indicates that Lrp5 also has a role in Wnt signaling
(Kelly et al., 2004
).
Unlike Fz, which is required for multiple Wnt pathways
(Strutt, 2003;
Veeman et al., 2003
), Arrow
and Lrp6 appear to be specifically required for Wnt/ß-catenin signaling.
In Drosophila, Dfz1, but not Dfz2, plays a central role in planar
cell polarity (PCP) determination (reviewed by
Adler, 2002
;
Strutt, 2003
), whereas
arrow mutants exhibit normal PCP
(Wehrli et al., 2000
). This
indicates that Arrow is not required for Dfz1 PCP function. Similarly, in
Xenopus, blocking Lrp6 function has little effect on gastrulation
movements (Semenov et al.,
2001
), which are regulated by a Wnt11/Fz pathway analogous to
Dfz1/PCP signaling (Heisenberg et al.,
2000
; Tada and Smith,
2000
; Wallingford et al.,
2000
).
Lrp5/Lrp6: redundancy and Wnt specificity
Lrp5 and Lrp6 are highly homologous, and are widely co-expressed during
embryogenesis and in adult tissues (Dong
et al., 1998; Hey et al.,
1998
; Kim et al.,
1998
; Pinson et al.,
2000
; Houston and Wylie,
2002
; Kato et al.,
2002
; Fujino et al.,
2003
; Kelly et al.,
2004
). Lrp6/ mice are perinatal
lethal and exhibit mid/hindbrain defects, posterior truncation and abnormal
limb patterning, which resemble the defects of mice mutant for Wnt1,
Wnt3a and Wnt7a (Pinson et
al., 2000
). Lrp5/ mice have
normal embryogenesis, grow to adulthood and are fertile, but show osteoporosis
(Kato et al., 2002
) and some
metabolic abnormalities (Magoori et al., 2002;
Fujino et al., 2003
). Thus,
the Lrp6 loss-of-function phenotype is much more severe than the Lrp5
loss-of-function phenotype, indicating that Lrp6 has a more influential role
than Lrp5 during embryogenesis. There is a hint of functional redundancy
between Lrp5 and Lrp6, as defects in Lrp6/
embryos are less severe than those observed in individual Wnt mutants
(Pinson et al., 2000
). Indeed,
Lrp5/;
Lrp6/ double mutant mice die during gastrulation
(much earlier than Lrp6/ mice); they lack
the primitive streak and nascent mesoderm
(Kelly et al., 2004
), and thus
phenotypically resemble the Wnt3 mutant
(Liu et al., 1999
). Thus,
genetic studies in mice indicate that Wnt3 requires either Lrp5 or Lrp6 in
order to function, whereas Wnt1, Wnt3a and Wnt7a rely on Lrp6 primarily and
Lrp5 to some extent. Whether signaling by other Wnt proteins requires Lrp5
and/or Lrp6 cannot be inferred at the moment due to the early lethality of
double Lrp5/;
Lrp6/ mutants and the degree of redundancy
between the two proteins. It seems unlikely that Lrp5 and Lrp6 are involved in
signaling by distinct Wnt proteins during development (note that
Lrp5/ mice have normal embryogenesis),
although more genetic studies are needed to clarify this issue. It is also
unclear whether, in Drosophila, Arrow is required for signaling by
other Wnt proteins besides Wg.
An allelic series of compound mutants reveal the following order of
severity of developmental abnormalities: Lrp5+/
(normal)<Lrp6+/<Lrp5/<Lrp5+/;
Lrp6+/<Lrp5/;
Lrp6+/<Lrp6/<Lrp5+/;
Lrp6/<Lrp5/;
Lrp6/ (Kelly
et al., 2004). This is likely to reflect the severity of loss of
Wnt/ß-catenin signaling, and supports the view that Lrp5 and Lrp6 share
significant overlapping functions but that Lrp6 plays a more crucial role, at
least during embryogenesis. This is consistent with observations that
overexpression of Lrp6 exhibits significantly stronger activity than Lrp5 in
Xenopus (Tamai et al.,
2000
) and mammalian cells
(Holmen et al., 2002
). It is
possible that Lrp6 has a higher affinity for Wnts or a stronger signaling
efficacy than Lrp5, or both.
Arrow/Lrp5/Lrp6: members of the LDLR family
Arrow/Lrp5/Lrp6 is a subfamily of the LDL receptor (LDLR) family
(Fig. 2), which has diverse
roles in metabolism and development (Herz
and Bock, 2002). Human LRP5 was isolated through its homology to
LDLR (Dong et al., 1998
;
Hey et al., 1998
;
Kim et al., 1998
). Human LRP6
was identified by its homology to LRP5
(Brown et al., 1998
).
Arrow/LRP5/LRP6 are type I single-span transmembrane proteins with 1678, 1615
and 1613 amino acid residues, respectively. LRP5 and LRP6 share 73% and 64%
identity in extracellular and intracellular domains, respectively, whereas
Arrow is equally related (40% identical) to LRP5 and LRP6
(Box 2,
Box 3, Fig. S1 at
http://dev.biologists.org/supplemental/).
Indeed, LRP6 substitutes for Arrow during Wg signaling in cultured
Drosophila cells (Schweizer and
Varmus, 2003
), and constitutively activated Arrow (discussed
later) activates Wnt/ß-catenin signaling in mammalian cells and Xenopus
embryos (Tamai et al.,
2004
).
|
Arrow/Lrp5/Lrp6: Wnt co-receptors?
The simplest model to account for the role of Arrow/Lrp5/Lrp6 in Wnt
signaling is that Arrow/Lrp5/Lrp6 are Wnt coreceptors. Nevertheless, a few
issues concerning this view remain:
|
|
|
Box 3. Features of Arrow/Lrp5/Lrp6 intracellular domains
Arrow/Lrp5/Lrp6 intracellular domains (see Fig. S1 at
http://dev.biologists.org/supplemental/)
have 209, 207 and 218 amino acid residues, respectively, and are rich in
prolines and serines (15-20% each). They lack any recognizable catalytic
motifs and share no sequence similarity with other LDLR proteins. Identifiable
are scattered conserved regions, including five reiterated PPP(S/T)P motifs
(see Fig. S1), which are the Axin-binding sites and are essential for Lrp6
signaling function (Tamai et al.,
2004
Noticeably absent in the intracellular domains of Arrow/Lrp5/Lrp6 is the
NPxY motif (x, any amino acid), which is present in all other LDLR proteins
(Fig. 2) and mediates
interactions with the endocytic apparatus (for receptor internalization)
and/or cytoplasmic signaling/scaffolding proteins in signal transduction
(Herz and Bock, 2002
|
Further experiments will be needed to substantiate any Fz-Lrp5/Lrp6 association. First, can Wnt-Lrp5/Lrp6 affinities be measured? Second, can an in vitro Wnt-Fz-Lrp5/Lrp6 (extracellular domains) complex be observed for Wnt and Fz proteins other than just Wnt1 and Fz8? Third, can a complex between wild-type Fz and Lrp5/Lrp6 be detected at the plasma membrane? Finally, although the simplest version of the coreceptor model is that Lrp5/Lrp6 and Fz bind independently to Wnt, it remains to be examined whether Fz binding to Wnt enhances the Wnt-Lrp5/Lrp6 interaction, and/or vice versa. Answering these questions may not be simple, however, because Wnt-Fz specificity itself is not well understood, and, with the possible exception of a few Wnts, it is not clear which of the 19 Wnts and 10 Fzs engage Lrp5/Lrp6-dependent Wnt/ß-catenin signaling.
Mechanisms of Arrow/Lrp5/Lrp6 signaling
Arrow/Lrp5/Lrp6 have a key signaling role
Given that Wnt/ß-catenin signaling requires both Fz and
Arrow/Lrp5/Lrp6, an obvious question to ask is what role do these two distinct
receptors play in Wnt signal transduction? A mutant Lrp6 protein lacking the
intracellular domain is completely inactive, and in fact blocks Wnt and Fz
signaling in a dominant-negative fashion
(Tamai et al., 2000)
(Fig. 3). Conversely, mutant
Arrow/Lrp5/Lrp6 proteins that lack the extracellular domain (but are anchored
on the membrane), referred to here as Arrow/Lrp5/Lrp6
N
(Fig. 3), activate
ß-catenin signaling constitutively in mammalian cells
(Mao et al., 2001a
;
Mao et al., 2001b
;
Liu et al., 2003
) and in
Xenopus embryos (Tamai et al.,
2004
), suggesting that Arrow/Lrp5/Lrp6 have a signaling capacity
that is normally suppressed by the extracellular domain. The Lrp5
intracellular domain anchored to the plasma membrane via myristylation (a
covalent lipid modification that targets proteins to the plasma membrane) is
also constitutively active (Fig.
3) (Mao et al.,
2001b
). Thus, Arrow/Lrp5/Lrp6 is a key signaling receptor for the
Wnt/ß-catenin pathway.
Arrow/Lrp5/Lrp6 bind Axin
An important insight into the function of Lrp5 came from the finding that
the Lrp5 intracellular domain binds Axin in both yeast two-hybrid and co-IP
assays (Mao et al., 2001b), an
observation that has been extended to Arrow
(Tolwinski et al., 2003
) and
Lrp6 (Liu et al., 2003
;
Tamai et al., 2004
). As
mentioned previously, Axin is a scaffolding protein that contains binding
sites for Apc, ß-catenin, Gsk3, Ck1 and possibly other proteins
(Polakis, 2002
;
Kikuchi, 1999
)
(Fig. 4). Axin nucleates this
Axin complex, resulting in ß-catenin phosphorylation and degradation
(Fig. 1). Thus, the binding
between the Arrow/Lrp5/Lrp6 intracellular domain and Axin permits the Wnt
co-receptors to directly control ß-catenin phosphorylation and
degradation. The domain of Axin involved in binding Arrow/Lrp5 has only been
mapped via the yeast two-hybrid assay and remains poorly defined
(Fig. 4). The DIX domain of
Axin is necessary, but not sufficient, for the Axin-Arrow/Lrp5 interaction,
whereas the RGS domain may be inhibitory to it
(Mao et al., 2001b
;
Tolwinski et al., 2003
).
|
A recent study discovered that a PPP(S/T)P motif, which is reiterated five
times in Arrow/Lrp5/Lrp6 intracellular domains
(Box 3, see Fig. S1), is the
minimal module that is necessary and sufficient for Lrp6 signaling function in
mammalian cells and Xenopus embryos
(Tamai et al., 2004). When a
single PPPSP motif is transferred artificially to a truncated LDLR protein
(which has no role in Wnt/ß-catenin signaling) it becomes phosphorylated
and can fully activate Wnt/ß-catenin signaling
(Tamai et al., 2004
)
(Fig. 3). Importantly, Axin
preferentially binds to the phosphorylated PPPSP motif, whose phosphorylation
in Lrp6 is rapidly induced by Wnt (Tamai
et al., 2004
). It was thus proposed that Wnt activates Lrp6
signaling by inducing Lrp6 phosphorylation at the PPP(S/T)P motifs, which
serve as inducible docking sites for Axin, thereby recruiting Axin to the
plasma membrane (Fig. 5). This
model is also likely to apply to Arrow and Lrp5, which share conserved
PPP(S/T)P motifs (see Fig. S1 at
http://dev.biologists.org/supplemental/).
The phosphorylation-dependent activation of Lrp6 and its inducible recruitment
of Axin is reminiscent of other types of transmembrane signaling, such as that
by tyrosine kinase receptors and cytokine receptors
(Pawson and Scott, 1997
).
|
Gsk3 and Lrp5/Lrp6-Axin binding
In mammalian cells, Gsk3 overexpression enhances the Lrp5/Lrp6-Axin
interaction in co-IP experiments (Mao et
al., 2001b; Liu et al.,
2003
). This finding is puzzling as Gsk3 antagonizes
Wnt/ß-catenin signaling. In Drosophila mutant embryos lacking
Gsk3/zw3, Wg recruitment of Axin to the plasma membrane and Wg
signaling to Axin can occur (Cliffe et
al., 2003
; Tolwinski et al.,
2003
), implying that the Arrow-Axin association is not defective
in the absence of Gsk3. Then why does Gsk3 overexpression enhance
Lrp5/Lrp6-Axin interaction in mammalian cells? One possibility may be that
Lrp5/Lrp6 signaling destabilizes Axin (see below), whereas Gsk3 can
phosphorylate and stabilize Axin (Willert
et al., 1999
; Yamamoto et
al., 1999
). An increase in the level of Axin following Gsk3
overexpression may explain `enhanced' Lrp5/Lrp6-Axin binding. Another
possibility is that Lrp5/Lrp6 preferentially interact with Axin phosphorylated
by or complexed with Gsk3 (Mao et al.,
2001b
). Additional explanations may include that Gsk3
overexpression mimicks the action of the PPPSP kinase.
Consequences of Arrow/Lrp5/Lrp6-Axin binding
How does Arrow/Lrp5/Lrp6 activation and binding to Axin initiate
ß-catenin signaling? One possibility is that Arrow/Lrp5/Lrp6 binding to
Axin promotes Axin degradation. Indeed, Wnt stimulation
(Willert et al., 1999;
Yamamoto et al., 1999
), Wg
overexpression (Tolwinski et al.,
2003
), and Lrp5
N overexpression
(Mao et al., 2001b
), all
induce Axin degradation in mammalian and Drosophila cells (see also
Cliffe et al., 2003
). These
studies demonstrated reductions in Axin protein level after 2-4 hours of Wnt
stimulation (or longer when transfection or transgenic experiments are
involved). However, ß-catenin stabilization is detectable within 30
minutes of Wnt stimulation and can thus occur before an obvious reduction in
levels of Axin (Willert et al.,
1999
). One explanation, according to a recent theoretical and
experimental analysis (Lee et al.,
2003
), is that a slight decrease in Axin protein level may have a
significant effect on ß-catenin phosphorylation and degradation.
It is also possible that Arrow/Lrp5/Lrp6 binding inhibits the activity of
the Axin complex, by altering its component composition. This may be important
in the early phase of Wnt signaling when Axin degradation is insignificant. In
any event, one should keep in mind that, in mammalian cells,
Wnt/ß-catenin signaling induces the expression of an Axin homolog, Axin2
(also known as Axil/Conductin) (Behrens et
al., 1998; Yamamoto et al.,
1998
; Yan et al.,
2001
; Jho et al.,
2002
; Leung et al.,
2002
; Lustig et al.,
2002
). Thus, a reduction in the Axin protein level is likely to be
accompanied by an increase in the level of Axin2 during Wnt signaling, thereby
complicating the Axin degradation scenario. How Arrow/Lrp5/Lrp6 promotes Axin
degradation or inhibits Axin function is unknown, but their recruitment of
Axin to near the plasma membrane appears essential. Indeed, the Lrp5
intracellular domain, although capable of binding Axin, is incapable of
signaling in mammalian cells unless it is anchored to the plasma membrane
(Mao et al., 2001b
).
Fz and Dishevelled: an unresolved mystery
Overexpression of Arrow/Lrp5/Lrp6N, or even a single PPPSP motif
(tethered to the LDLR; Fig. 3),
constitutively activates the ß-catenin pathway
(Mao et al., 2001a
;
Mao et al., 2001b
;
Liu et al., 2003
;
Tamai et al., 2004
), probably
in a Wnt- and Fz-independent manner. However, this is difficult to verify as a
cell completely lacking Fz proteins may not exist. Wnt and Fz might normally
function to activate the signaling activity of Arrow/Lrp5/Lrp6. Given the
activated nature of the truncated Arrow/Lrp5/Lrp6
N, a scenario in which
Wnt/Fz induces post-translational cleavage of Arrow/Lrp5/Lrp6 is attractive
yet lacks experimental evidence. If Arrow/Lrp5/Lrp6 were to be activated in
this way, the cleavage would have to occur extracellularly because an
Lrp5/Lrp6 intracellular domain that is not anchored to the membrane is
inactive (Mao et al.,
2001b
).
Fz function remains a mystery. Fz is thought to have a signaling role
because its intracellular regions are required for Wnt/ß-catenin
signaling (Umbhauer et al.,
2000). Fz proteins also play a key part in
ß-catenin-independent signaling, such as in the PCP pathway, in
Ca2+/PKC (protein kinase C) signaling, and perhaps in other
pathways (Adler, 2002
;
Strutt, 2003
;
Veeman et al., 2003
).
Although some Fz functions, such as PKC activation, can be blocked by
pharmacological inhibitors of the trimeric G proteins, whether Fz function
during Wnt/ß-catenin signaling relies on G proteins remains debatable
(reviewed by Malbon et al.,
2001
). A protein that is required for most, if not all, Fz
functions is Dishevelled (Dsh in Drosophila and Xenopus, and
Dvl1-3 in mammals), another mysterious protein that is genetically defined
downstream of Fz in both Wnt/ß-catenin and PCP pathways
(Boutros and Mlodzik, 1999
),
and that may also be required for Fz activation of PKC
(Sheldahl et al., 2003
).
Dsh/Dvl is a modular scaffolding protein that contains a DIX domain (which
also exists in Axin), a PDZ domain (a domain discovered in PSD,
Discs-large, and ZO1 proteins) and a DEP domain (a
domain discovered in Dishevelled, Egl-10, and
Pleckstrin proteins) (Boutros
and Mlodzik, 1999
; Wharton,
2003
). Dsh/Dvl is recruited to the plasma membrane upon
overexpression of a number of different Fz proteins
(Axelrod et al., 1998
;
Boutros et al., 2000
;
Rothbacher et al., 2000
;
Umbhauer et al., 2000
), and
may bind directly to the Fz carboxyl-terminal region via the PDZ domain
(Chen et al., 2003
;
Wong et al., 2003
). However,
Fz recruitment of Dsh/Dvl to the plasma membrane does not correlate fully with
the activation of Wnt/ß-catenin signaling
(Axelrod et al., 1998
;
Rothbacher et al., 2000
;
Umbhauer et al., 2000
), and
Dsh is not localized near the plasma membrane in Wg-responsive cells in fly
embryos (Axelrod et al., 1998
;
Axelrod, 2001
;
Cliffe et al., 2003
).
Thus, it remains unclear how Fz and Dsh/Dvl fit into the scenario in which
Arrow/Lrp5/Lrp6 binding to Axin initiates ß-catenin signaling. Three
models can be proposed. Dsh appears to be epistatic to, or downstream of,
Arrow, because Dsh overexpression activates ß-catenin signaling in
arrow mutants (Wehrli et al.,
2000) and the constitutively active Dfz2-Arrow fusion protein is
inactive in dsh mutants
(Tolwinski et al., 2003
). In
addition, Dsh/Dvl can associate with and inhibit Axin
(Fagotto et al., 1999
;
Kishida et al., 1999b
;
Li et al., 1999
;
Smalley et al., 1999
;
Salic et al., 2000
).
Therefore, one scenario, referred to here as the `co-recruitment' model, is
that Fz and Arrow/Lrp5/Lrp6 recruit Dsh/Dvl and Axin into the coreceptor
complex, respectively, thereby bringing Dsh/Dvl and Axin into proximity for
effective Axin inhibition or degradation
(Fig. 5A). However, a lack of
correlation between Dsh/Dvl plasma membrane localization and Wg/Wnt signaling
poses difficulties for this model, although it is possible that a small
fraction of Dvl/Dsh recruited to the membrane, albeit undetectable, is
sufficient for signaling. This model implies that Dvl/Dsh has a key role in
Axin inhibition or degradation, and could account for the finding that
overexpression of Dsh activates ß-catenin signaling in arrow
mutant flies (i.e. via inhibiting or degrading Axin in the cytoplasm).
The second scenario, referred to here as the `vesicle-transport' model
(Cliffe et al., 2003)
(Fig. 5B), is based on the
observations that Dsh/Dvl and Axin, upon overexpression, are co-localized in
intracellular `dots' that may represent `vesicles'
(Axelrod et al., 1998
;
Fagotto et al., 1999
; Kishida
et al., 1999; Smalley et al.,
1999
; Axelrod,
2001
; Cliffe et al.,
2003
), and that Axin recruitment to the plasma membrane requires
Dsh (Cliffe et al., 2003
).
This model proposes that Dsh/Dvl, through association with vesicles and Axin,
shuttles Axin to the plasma membrane, where it becomes associated with
Arrow/Lrp5/Lrp6. This view is consistent with the observation that the Dvl DIX
domain, which is essential for Dsh/Dvl function in ß-catenin signaling,
harbors phospholipid-binding activity and mediates vesicle association
(Capelluto et al., 2002
), but
it does not easily explain how, in Drosophila, Dsh overexpression
activates ß-catenin signaling in arrow mutants.
The third scenario, which perhaps can be referred to as a `parallel
signaling' model (Fig. 5C),
implies that Fz-Dsh/Dvl-Axin and Arrow/Lrp5/Lrp6-Axin represent two parallel
branches: overactivation of either branch is sufficient to activate
ß-catenin signaling, whereas simultaneous activation of both is required
under physiological conditions. This model, which can explain why Dsh
overexpression bypasses Arrow function, is based on observations that
Lrp5/Lrp6N signaling does not seem to be affected by depletion of
Dvl/Dsh proteins in mammalian and Drosophila cells [from short
interfering RNA (siRNA) or RNA interference (RNAi) experiments]
(Li et al., 2002
;
Schweizer and Varmus, 2003
).
These observations apparently contradict the finding in Drosophila
embryos that signaling by the constitutively active Dfz2-Arrow fusion protein
(Fig. 3) requires Dsh
(Tolwinski et al., 2003
),
although it is possible that the mechanisms by which Lrp5/Lrp6
N and the
Dfz2-Arrow fusion protein become constitutively active may be different.
However, depletion of the three Dvl proteins or Dsh through siRNA/RNAi is
unlikely to be complete, rendering the interpretation less straightforward.
Whether Arrow
N, which is constitutively active in mammalian cells and
Xenopus embryos (Tamai et al.,
2004
), can activate ß-catenin signaling in dsh
mutant flies will be a key test. Given the possible Fz-Lrp5/Lrp6 and
Dsh/Dvl-Axin interactions, these two parallel branches, if they exist, may
nonetheless operate in physical proximity.
Finally, because Wnt activates Lrp6 signaling by inducing Lrp6
phosphorylation at the PPP(S/T)P motif
(Tamai et al., 2004), it
could be possible that Fz/Dsh signaling acts by activating or recruiting the
PPPSP kinase to phosphorylate Lrp6. We consider this scenario to be less
likely as Dsh overexpression can activate ß-catenin signaling in the
absence of Arrow function (Wehrli et al.,
2000
).
Wnt/ß-catenin signaling in worms
Wnt signaling is essential for many aspects of nematode development.
However, some Wnt pathways in worms are organized differently to those in
Drosophila and vertebrates
(Korswagen, 2002).
Nonetheless, a canonical Wnt/ß-catenin signaling pathway controlling
neuronal migration was discovered in worms that involves Wnt, Fz, Dsh, Axin,
Apc, Gsk3 and ß-catenin in a similar way as in flies and vertebrates
(Korswagen et al., 2002
).
Perplexingly however, no Arrow/Lrp5/Lrp6 homologs have been identified in the
worm genome, although other Lrp genes (such as Lrp1) exist. Thus either a
functional homolog of Arrow/Lrp5/Lrp6 has yet to be discovered, or nematodes
use other means for Wnt/Fz/Dsh to activate ß-catenin signaling in the
absence of Arrow/Lrp5/Lrp6-Axin interaction. This latter possibility shares
some resemblance to the `parallel signaling' model discussed above.
Regulation of Arrow/Lrp5/Lrp6
Dickkopf and Wise
Lrp5/Lrp6 are subjected to modulation by secreted antagonistic/modulatory
ligands in vertebrates and by other types of regulations
(Box 4). Two families of such
ligands have been identified: the Dickkopf (Dkk) family and the Wise family,
which antagonize Wnt/ß-catenin signaling through interactions with
Lrp5/Lrp6. Dkk and Wise homologs have not been found in invertebrate
genomes.
The Dkk family
Wnt signaling is required for posterior patterning in vertebrates; thus,
inhibition of Wnt signaling permits anterior development
(Niehrs, 1999).
Xenopus Dkk1 was isolated as a head-inducing molecule and behaves as
an antagonist for Wnt signaling (Glinka et
al., 1998
). Genetic analysis of
Dkk1/ mice, which lack head formation, is
consistent with this view (Mukhopadhyay et
al., 2001
). Distinct from several families of secreted Wnt
antagonists that bind Wnts, including the sFRP (secreted Frizzled-related
protein) family, Wif1 (Wnt inhibitory factor 1) and Xenopus Cerberus
(Semenov and He, 2003
), Dkk1
does not bind Wnt but is a high affinity ligand for Lrp6
(Kd=0.3-0.5 nM) and Lrp5 (Bafico
et al., 2001
; Mao et al.,
2001a
; Semenov et al.,
2001
). Dkk1 disrupts the Fz-Lrp5/Lrp6 complex formation induced by
Wnt1 in vitro (Semenov et al.,
2001
), suggesting that Dkk1 inhibits Wnt signaling by preventing
Fz-Lrp5/Lrp6 complex formation. The Dkk1-Lrp5/Lrp6 antagonistic relationship
is supported by mouse genetic studies
(MacDonald et al., 2004
).
Thus, reducing the dosage of Lrp5 or Lrp6 can significantly rescue phenotypes
associated with a loss of Dkk1 function, and vise versa. For example, while
Dkk1/ mutant mice lack head formation and
die during embryogenesis, Dkk1/;
Lrp6+/ mice have extensive head development and can
survive to postnatal stages (MacDonald et
al., 2004
).
By inhibiting Lrp5/Lrp6, Dkk1 appears to be a specific antagonist for
Wnt/ß-catenin signaling (Semenov et
al., 2001), and is thus distinct from sFRPs, Wif1 and Cerberus,
which may antagonize multiple Wnt pathways
(Semenov and He, 2003
). Of
the two conserved cysteine-rich domains of Dkk1 (see Fig. S2 at
http://dev.biologists.org/supplemental/),
the carboxyl one is essential for its binding to Lrp6 and its antagonization
of Wnt signaling, whereas the amino terminal one may exert some undefined
regulatory roles (Brott and Sokol,
2002
; Li et al.,
2002
; Mao and Niehrs,
2003
). Dkk1 may interact with a region encompassing the third and
fourth YWTD ß-propeller-EGF-like domains of Lrp6, which is distinct from
the Wnt-binding region (Mao et al.,
2001a
; Itasaki et al.,
2003
) (Fig. 6).
In Xenopus and mammals, the Dkk family includes Dkk1, Dkk2, Dkk3
and Dkk4 (see Fig. S2 at
http://dev.biologists.org/supplemental/),
which exhibit distinct and dynamic expression patterns
(Glinka et al., 1998;
Monaghan et al., 1999
) and may
have distinct properties. Dkk1 and Dkk4 are antagonists for Wnt signaling
(Krupnik et al., 1999
;
Brott and Sokol, 2002
;
Mao and Niehrs, 2003
), whereas
Dkk2 can, paradoxically, inhibit or activate (albeit weakly) ß-catenin
signaling, depending on the experimental assays employed
(Wu et al., 2000
;
Brott and Sokol, 2002
;
Li et al., 2002
). Whether Dkk2
can function as a Wnt agonist in vivo remains to be seen. Dkk3 neither binds
Lrp5 or Lrp6, nor affects Wnt signaling
(Krupnik et al., 1999
;
Mao and Niehrs, 2003
).
Kremen: a Dkk1 co-receptor?
Dkk1 also binds to vertebrate Kremen (Krm) 1 and Krm2, two related
single-pass transmembrane proteins (Mao et
al., 2002) (see Fig. S2 at
http://dev.biologists.org/supplemental/).
In mammalian cells, either Krm1 or Krm2 can cooperate with Dkk1 in the
inhibition of Wnt-Fz-Lrp6 function (Mao et
al., 2002
). Drosophila has no Dkk or Krm homologs,
although ectopic expression of vertebrate Dkk1 and Krm2
together, but not either of these genes alone, results in inhibition of Wg
signaling (Mao et al., 2002
).
In addition, antisense knockdown of both Krm1 and Krm2 (but not either
individually) in Xenopus results in deficient head development,
similar to phenotypes of embryos with no or reduced Dkk1 function
(Davidson et al., 2002
). Thus,
Krm1 and Krm2 appear to have redundant roles in Dkk1 function. Because Dkk1
can stimulate Lrp6 internalization upon Krm2 overexpression
(Mao et al., 2002
), it was
proposed that Dkk1, by binding both Lrp6 and Krm, induces Lrp6 internalization
from the cell surface, thereby attenuating Wnt signaling. Perplexingly
however, the Krm intracellular domain is neither conserved nor required for
any of these functions (Mao et al.,
2002
), which raises the question of how can Krm have a key
function in Lrp6 internalization? This internalization model is different,
although not mutually exclusive, from a model in which Dkk1 functions by
preventing Fz-Lrp6 complex formation
(Semenov et al., 2001
).
The Wise family
Wise was identified in Xenopus embryo assays as a secreted
molecule with dual properties somewhat similar to Dkk2. Wise is an antagonist
for Xwnt8/ß-catenin signaling, but on its own can weakly activate
ß-catenin signaling (Itasaki et al.,
2003). Wise binds to the Lrp6 extracellular domain in co-IP
experiments, in particular to the first two YWTD ß-propeller-EGF-like
domains (Fig. 6), the same
region that Wnt appears to bind, and can compete with Xwnt8 for Lrp6 binding
(Itasaki et al., 2003
). Wise
belongs to a large family of secreted `cysteine-knot' domain-containing
proteins (see Fig. S2 at
http://dev.biologists.org/supplemental/),
which include members that bind and antagonize BMPs (bone morphogenetic
proteins) (Hsu et al., 1998
;
Pearce et al., 1999
;
Piccolo et al., 1999
). Indeed,
Wise was also isolated as a BMP inhibitor
(Laurikkala et al., 2003
).
Thus, Wise appears to be a multifunctional inhibitor for both
Wnt/ß-catenin and BMP signaling. This property is somewhat similar to the
Xenopus protein Cerberus, which antagonizes signaling by Wnt, BMP and
Nodal (Piccolo et al.,
1999
).
LRP5 in human diseases
Bone density disorders
WNT signaling is not only essential for embryogenesis, but also for
postnatal development and tissue homeostasis. This is illustrated by LRP5
mutations that underlie familial osteoporosis, high bone density syndromes and
ocular disorders (Gong et al.,
2001; Boyden et al.,
2002
; Little et al.,
2002
). Children with autosomal-recessive osteoporosis-pseudoglioma
syndrome (OPPG) have low bone mass and are prone to bone fractures
(Gong et al., 2001
). Most of
these children suffer a loss of LRP5 function due to nonsense or frame-shift
mutations in the LRP5 extracellular domain
(Gong et al., 2001
)
(Fig. 6). Remarkably, several
groups of autosomal-dominant bone disorders, characterized by high bone
density traits, are also associated with LRP5 mutations, which are missense in
nature and clustered in the first ß-propeller region of LRP5
(Boyden et al., 2002
;
Little et al., 2002
;
Van Wesenbeeck et al., 2003
)
(Fig. 6). This is reflected in
mice. Lrp5/ mice exhibit low bone density
and frequent bone fractures reminiscent of OPPG patients
(Kato et al., 2002
), and
transgenic mice expressing LRP5 (G171V), a mutation from high bone density
patients, had increased bone mass (Babij et
al., 2003
). Thus, loss-of-function mutations of LRP5 lead to low
bone densities whereas `gain-of-function' mutations cause high bone mass.
These studies identify LRP5 as a central player and an ideal therapeutic
target in bone mass regulation and in associated diseases such as osteoporosis
(Patel and Karsenty, 2002
).
Nevertheless, several key issues remain unresolved:
Box 4. Regulation of Arrow/Lrp5/Lrp6 expression and membrane
trafficking
In Drosophila, arrow expression, like that of Dfz1 and
Dfz2 (Bhanot et al.,
1996
In order for Arrow/Lrp5/Lrp6 maturation and trafficking to the plasma
membrane where they function, the Drosophila Boca protein and its
mouse homolog Mesd, which are chaperones residing in the endoplasmic reticulum
(ER), are required (Culi and Mann,
2003
|
It is of interest to discuss here sclerosteosis, another rare
autosomal-recessive bone disorder characterized by skeletal overgrowth and
high bone density. Sclerosterosis is a progressive bone dysplasia associated
with loss-of-function mutations of a secreted protein referred to as SOST
(Balemans et al., 2001;
Brunkow et al., 2001
).
Intriguingly, SOST is most related to WISE (38% identical; see Fig. S2 at
http://dev.biologists.org/supplemental/),
which antagonizes LRP6 via binding to the region containing the first and
second YWTD ß-propeller domains (Fig.
6) (Itasaki et al.,
2003
). Although SOST, like Wise, can also bind and antagonize BMP
(Laurikkala et al., 2003
;
Kusu et al., 2003
), it is
tempting to speculate that SOST binds and antagonizes LRP5 in bone growth
regulation, and that LRP5 mutations associated with high bone densities
prevent/reduce SOST-LRP5 interaction.
Ocular disorders
OPPG patients also suffer, in addition to low bone mass, from severe
disruption of ocular structures due to a failure of regression of the primary
vitreal vasculature (the temporary capillary networks that normally regress
during development) (Gong et al.,
2001). This phenotype is recapitulated in
Lrp5/ mice, possibly because of a lack of
capillary endothelial apoptosis in the eye
(Kato et al., 2002
). This may
be due to defects in ocular macrophages, which express Lrp5 and are required
for the induction of capillary cell death
(Kato et al., 2002
).
Another hereditary ocular disorder, the autosomal-dominant form of familial
exudative vitreoretinopathy (FEVR), which is characterized by the premature
arrest of retinal angiogenesis/vasculogenesis, is also associated with
LRP5 mutations (Toomes et al.,
2004). Loss-of-function mutations in one LRP5 chromosomal
copy are associated with this disease (Fig.
6), presumably due to haploinsufficiency
(Toomes et al., 2004
). These
patients, like the obligate OPPG carriers (parents of OPPG patients)
(Gong et al., 2001
), also
exhibit low bone mass (Toomes et al.,
2004
). Interestingly, some autosomal-dominant FEVR families harbor
loss-of-function mutations in the frizzled 4 (FZD4) gene
(Robitaille et al., 2002
),
which, like LRP5, is located in the chromosomal 11q13 region.
Therefore, FEVR is associated with a deficiency in either LRP5 or FZD4
function, providing genetic evidence outside Drosophila that LRP5 and
FZD cooperate in the same signaling pathway. In summary, LRP5 (and FZD)
function is important for multiple stages of retinal angiogenesis and
associated diseases, presumably because it mediates signaling by a WNT (or
non-WNT) ligand.
Cholesterol and glucose metabolism
LRP5 is also involved in lipid metabolism. LRP5 binds apolipoprotein E
(APOE), and LRP5 expression is upregulated in the liver of
Ldlr/ mice
(Kim et al., 1998). Indeed,
Lrp5/ mice fed on a high-fat diet exhibit
increased plasma cholesterol levels relative to normal mice
(Fujino et al., 2003
), and
mutants for both ApoE and Lrp5
(ApoE/;
Lrp5/) show hypercholesterolemia, impaired fat
tolerance and advanced atherosclerosis
(Magoori et al., 2003
). The
role of LRP5 in cholesterol metabolism appears analogous to the classical LDLR
function and is probably WNT independent
(Magoori et al., 2003
).
Concluding remarks
Although the role of Arrow/Lrp5/Lrp6 in Wnt/ß-catenin signaling during development is established, the underlying molecular mechanism remains relatively poorly defined. Unresolved questions concern how Wnt molecules interact, functionally and/or physically, with Fz and Arrow/Lrp5/Lrp6, how Wnt activates Arrow/Lrp5/Lrp6 by phopshorylation to regulate the Axin complex, and how Fz receptors and Dsh/Dvl proteins operate. Such studies are likely to identify additional signaling components at the plasma membrane or intracellularly that will bridge major gaps in our understanding of transmembrane signaling by Wnt proteins.
Although Dkk and Wise families of Lrp5/Lrp6 ligands/antagonists are only
found in vertebrates, the high similarity between the entire Arrow and
Lrp5/Lrp6 extracellular domains implies that other evolutionarily conserved
ligands may yet be discovered. An area that requires more investigation is the
cell biological aspect of Wnt signaling, including the biogenesis,
trafficking, localization and endocytosis of Fz and Arrow/Lrp5/Lrp6 during Wnt
signaling. On the translational side, the specific function of LRP5 in bone
mass regulation provides an ideal therapeutic target for treatment of bone
disorders, but this will critically rely on our understanding of LRP5, its
putative ligand (a WNT or non-WNT), and antagonists such as DKK, WISE or
others during bone accrual. In addition, the roles of LRP5 in lipid metabolism
and other physiological regulations, such as in enhancing insulin secretion
(Fujino et al., 2003), may
have significant medical implications. In this regard, molecules that can
stimulate LRP5 expression or activity may be used as therapeutics for
osteoporosis, high cholesterol associated diseases and diabetes.
ACKNOWLEDGMENTS
We thank R. Korswagen, N. Tolwinski and S. Blacklow for discussion, B. Skarnes for communication before publication, and referees for constructive suggestions.
Footnotes
Supplemental data available online
It was recently reported that Axin has a nuclear-cytoplasmic shuttling role
in the regulation of ß-catenin subcellular localization
(Cong and Varmus, 2004).
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