Cloning and characterization of low-density lipoprotein receptor (LDLR)-related protein (LRP) family genes

A novel member of the low density lipoprotein receptor (LDLR) gene family has been identified and characterized. This gene, termed LDL receptor-related protein 6 (LRP6), encodes a transmembrane protein that has 71% identity and is structurally similar to the protein encoded by LRP5, a proposed candidate gene for type 1 diabetes located on human chromosome 11q13. LRP6 maps to human chromosome 12p11-p13. Mouse Lrp6 encodes a protein that has 98% identity to human LRP6 and maps to chromosome 6. Unlike other members of the LDLR family, LRP6 and LRP5 display a unique pattern of four epidermal growth factor (EGF) and three LDLR repeats in the extracellular domain. The cytoplasmic domain of LRP6 is not similar to other members of the LDLR family, while comparison with LRP5 reveals proline-rich motifs that may mediate protein-protein interactions. Thus, it is likely that LRP6 and LRP5 comprise a new class of the LDLR family (Brown, 1998).

A gene encoding a novel transmembrane protein has been identified by DNA sequence analysis within the insulin-dependent diabetes mellitus (IDDM) locus IDDM4 on chromosome 11q13. Based on its chromosomal position, this gene is a candidate for conferring susceptibility to diabetes. The gene, termed low-density lipoprotein receptor related protein 5 (LRP5), encodes a protein of 1615 amino acids that contains conserved modules which are characteristic of the low-density lipoprotein (LDL) receptor family. These modules include a putative signal peptide for protein export, four epidermal growth factor (EGF) repeats with associated spacer domains, three LDL-receptor (LDLR) repeats, a single transmembrane spanning domain, and a cytoplasmic domain. The encoded protein has a unique organization of EGF and LDLR repeats; therefore, LRP5 likely represents a new category of the LDLR family. Both human and mouse LRP5 cDNAs have been isolated and the encoded mature proteins are 95% identical, indicating a high degree of evolutionary conservation (Hey, 1998).

The isolation and characterization of rabbit and human cDNAs reveals a new low density lipoprotein receptor (LDLR)-related protein (LRP) designated as LRP5. Human LRP5 cDNA encodes a 1,616-amino acid type I membrane-like protein with three ligand binding repeats in its extracellular region. LDLR-deficient cells transduced by recombinant adenovirus containing human LRP5 exhibit increased binding of apolipoprotein E (apoE)-enriched beta-migrating very low density lipoprotein. Northern blotting and in situ hybridization have revealed a high level of LRP5 expression in hepatocytes and the adrenal gland cortex. In LDLR-deficient Watanabe heritable hyperlipidemic rabbits, LRP5 mRNA is increased in the liver and accumulates in cholesterol-laden foam cells of atherosclerotic lesions (Kim, 1998).

Molecular cloning and initial functional characterization of a novel member of the low density lipoprotein receptor (LDLR) gene family is reported. The cDNA was isolated from a human osteoblast cDNA library and encodes a 1,615 amino acids protein designated as LR3. It has, in the extracellular region, a cluster of three LDLR ligand binding repeats at a juxtamembrane position and four EGF precursor homology domains separated by YWTD spacer repeats. The entire ectodomain shares the same modular organization with the middle portion of the extracellular regions of two LDLR family members, LDLR-related protein (LRP), and gp330/megalin. LR3 mRNA is expressed in most of the adult and fetal tissues examined. The highest expression level is seen in aorta. In human osteosarcoma cells examined, LR3 mRNA is highly enriched in TE85 cells, moderately expressed in MG63 cells and primary human osteoblasts, and undetectable in SaOS-2 cells. NIH 3T3 cells transfected with either full length LR3 or its ectodomain show significantly increased proliferation, whereas transfection of intracellular domain has no proliferative effect. It is predicted that LR3 is a multi-functional protein with potential mitogenic activity (Dong, 1998).

A context-dependent combination of Wnt receptors controls axis elongation and leg development in a short germ insect

Short germ embryos elongate their primary body axis by consecutively adding segments from a posteriorly located growth zone. Wnt signalling is required for axis elongation in short germ arthropods, including Tribolium castaneum, but the precise functions of the different Wnt receptors involved in this process are unclear. This study analysed the individual and combinatorial functions of the three Wnt receptors, Frizzled-1 (Tc-Fz1), Frizzled-2 (Tc-Fz2) and Frizzled-4 (Tc-Fz4), and their co-receptor Arrow (Tc-Arr) in the beetle Tribolium. Knockdown of gene function and expression analyses revealed that Frizzled-dependent Wnt signalling occurs anteriorly in the growth zone in the presegmental region (PSR). Simultaneous functional knockdown of the Wnt receptors Tc-fz1 and Tc-fz2 via RNAi resulted in collapse of the growth zone and impairment of embryonic axis elongation. Although posterior cells of the growth zone were not completely abolished, Wnt signalling within the PSR controls axial elongation at the level of pair-rule patterning, Wnt5 signalling and FGF signalling. These results identify the PSR in Tribolium as an integral tissue required for the axial elongation process, reminiscent of the presomitic mesoderm in vertebrates. Knockdown of Tc-fz1 alone interfered with the formation of the proximo-distal and the dorso-ventral axes during leg development, whereas no effect was observed with single Tc-fz2 or Tc-fz4 RNAi knockdowns. Tc-Arr was identied as an obligatory Wnt co-receptor for axis elongation, leg distalisation and segmentation (Beermann, 2012).

The Tc-fz2 expression pattern and the Tc-fz1/2 double RNAi phenotype identify the region between the posterior growth zone and the new segments as crucial for axial elongation (AE). This tissue develops in a similar position to the presomitic mesoderm (PSM) in vertebrates and is called the presegmental region (PSR) in Tribolium. In both Drosophila and Tribolium, Fz1 and Fz2 function redundantly: in Drosophila during segmentation and in Tribolium within the PSR, the axis elongation phenotype was only seen when Tc-Fz1 and Tc-Fz2 were depleted simultaneously. Since Wnt signalling drives segmentation from within the PSR, reception of the Wnt signal in this tissue by the Tc-Fz1 and Tc-Fz2 receptors is necessary for this process (Beermann, 2012).

Marker gene analysis showed that the posterior growth zone is not completely abolished in Tc-fz1/2RNAi embryos, as was previously shown for Tc-arrowRNAi embryos. The markers Eve, odd and wnt8 are still expressed in posterior cells of the growth zone. The persistent expression of the marker gene caudal proves the identity of these cells as posterior cells. Because the complete wild-type expression pattern of caudal is not retained in Tc-fz1/2RNAi embryos, the terminal-most tissues could still contribute to axis elongation. The absence of the hindgut marker brachyenteron in strongly affected Tc-fz1/2RNAi embryos is in accordance with the loss of the hindgut in fz1/2RNAi larval cuticles. In less strongly affected Tc-fz1/2RNAi embryos, byn expression is still detectable. The results show that the transition of the pair-rule genes from the primary to the segmental phase depends on Wnt signalling. As a consequence of this loss of Wnt signalling, AE does not occur. In contrast to Tc-wnt8, Tc-wnt5 expression is completely abolished in strong Tc-fz1/2RNAi embryos. This finding could be explained by an autoregulatory loop in the PSR involving Frizzled and Wnt5 (Beermann, 2012).

In vertebrates, coordinated cell movements in the PSM under the control of FGF signalling are responsible for AE. Because signalling pathways are connected to other signalling pathways, it is hypothesised that FGF signalling might be involved in AE in Tribolium as well. Indeed, the prominent wild-type FGF expression domain is missing in Tc-fz1/2RNAi embryos, indicating that FGF signalling depends on Wnt signalling in Tribolium and might itself be crucially required for AE. It is striking that in both vertebrates and insects, a tissue immediately posterior to the last somites or segment formed - the PSM and the PSR, respectively - fulfils analogous functions in the axial elongation process (Beermann, 2012).

In Drosophila and Tribolium, wingless is required for segmentation as well as for distalisation and dorso-ventral patterning of the appendages. For Tribolium, it was argued that the leg patterning function of wingless is only required during later embryonic stages (Beermann, 2012).

This study shows that Tc-fz1 function is required for both proximo-distal (PD) and dorso-ventral (DV) axis formation in the leg. Because both the PD and the DV leg phenotypes are the result of parental RNAi, a distinct time-dependency for Tc-fz1 function could not be verified. Rather, the data support concentration-dependent regulation of the PD and DV axes during leg development. The Tc-fz1RNAi leg phenotype strongly resembles the winglessRNAi phenotypes described for Tribolium and Drosophila. Based on bristle mapping, it is proposed that the best candidate ligand for Tc-Fz1 in appendage formation is Tc-Wnt1 (Beermann, 2012).

Remarkably, Tc-fz1/2RNAi larvae lacking most of the abdominal segments develop normal legs. Here, the reduced Tc-fz1 function seems to be partially compensated. This result is in contrast to Tc-arrowRNAi, in which leg formation is severely affected in larvae with a mild AE defect. Why does the knockdown of Tc-fz1 in the Tc-fz1/2 double RNAi experiment interfere only mildly with leg formation? AE is apparently very sensitive to depletion of Tc-fz1/2 function. The dose of dsRNA required for an AE phenotype hardly affects Tc-Fz1-dependent leg patterning at all. Presumably, in the double knockdown embryos, the concentration of Tc-fz1 transcripts does not sink below a critical threshold. In contrast, AE and leg patterning require similar amounts of the co-receptor Arrow. These results support a dosage dependency of Wnt receptors and co-receptors during embryogenesis (Beermann, 2012).

In larvae with weak Tc-fz1 RNAi phenotypes, lesions occur in the proximal leg, affecting the joints of the trochanter - sites of Tc-fz4 expression - and leading to a fusion of coxa and trochanter. Tc-Fz4, therefore, supports Tc-Fz1 in transducing the Wnt signal in the proximal leg. This assumption is corroborated by the finding that Tc-fz1/4 double RNAi enhances the frequency and the phenotype of this leg phenotype. However, Tc-Fz4 on its own is dispensable in the leg; Tc-Fz1 appears to be the crucial partner. At present, the precise function of Tc-Fz1/Fz4 in the dorso-proximal leg is speculative. The hypothesised leg differentiation center at the coxa/trochanter boundary, postulated for Tenebrio, is an intriguing possibility. Larvae with the strongest Tc-fz1RNAi phenotype display loss of distal structures, including the pretarsal claw. Because Tc-fz4 is not expressed distal to the tibiotarsal region, Tc-Fz1 seems to be the single primary Wnt transducer in the distal appendage. The ligand for this signal is probably distal Wnt1, based on the leg phenotype of wgRNAi larvae. Depending on their conformation, Frizzled receptor proteins have been proposed to signal via different downstream signalling pathways and could thus serve as multifunctional signal transducers depending on, e.g. ligand availability. In this way, a variety of different biological outcomes can be controlled by a small number of Wnt receptors (Beermann, 2012).

Ligands of low-density lipoprotein receptor-related proteins

A novel secreted molecule, Wise, has been isolated by a functional screen for activities that alter the anteroposterior character of neuralized Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of ß-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonizes the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor, lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling (Itasaki, 2003).

The predicted Wise protein consists of 206 amino acids and contains a cysteine knot-like domain found in a number of growth factors, as well as in Slit, mucin and CCN (Cef10/Cyr61, CTGF and Nov) family members. Among these, the C-terminal domain of the CCN family members shows the highest homology to Wise, but other motifs conserved within the CCN family are absent in Wise. Hence, Wise is related to but not a member of the CCN family. A homology search revealed that Wise shows the highest amino acid identity (38%) to Sclerostin (SOST), identified by positional cloning of the gene mutated in sclerosteosis. There are a number of EST sequences homologous to Wise in zebrafish, mouse and human databases, but none was found in the Drosophila or C. elegans genomes (Itasaki, 2003).

Role of low-density lipoprotein receptor (LDLR)-related protein (LRP) family genes in Wnt signaling

The Frizzled (Fz) family of serpentine receptors function as Wnt receptors, but how Fz proteins transduce signaling is not understood. Drosophila arrow encodes a transmembrane protein that is homologous to two members of the mammalian low-density lipoprotein receptor (LDLR)-related protein (LRP) family, LRP5 and LRP6. LRP6 functions as a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6 activates Wnt-Fz signaling, and induces Wnt responsive genes, dorsal axis duplication and neural crest formation. An LRP6 mutant lacking the carboxyl intracellular domain blocks signaling by Wnt or Wnt-Fz, but not by Dishevelled or beta-catenin, and inhibits neural crest development. The extracellular domain of LRP6 binds Wnt-1 and associates with Fz in a Wnt-dependent manner. These results indicate that LRP6 may be a component of the Wnt receptor complex (Tamai, 2000).

Human LRP5 and LRP6 share 71% amino-acid identity, and together with Arrow, form a distinct subgroup of the LRP family. Arrow, LRP5 and LRP6 each contain an extracellular domain with EGF (epidermal growth factor) repeats and LDLR repeats, followed by a transmembrane region and a cytoplasmic domain lacking recognizable catalytic motifs. An lrp6 mutation in mice results in pleiotropic defects recapitulating some, but not all, of the wnt mutant phenotype (Pinson, 2000). To study LRP5/LRP6 involvement in Wnt signaling, their function was examined in Wnt-induced axis and neural crest formation in Xenopus embryos (Tamai, 2000).

Wnt/beta-catenin signaling induces dorsal axis formation through activation of responsive genes, including nodal-related 3 (Xnr3) and siamois (sia). Ventral injection of LRP6 RNA into four-cell stage embryos results in dorsal axis duplication in a dose-dependent manner. In animal pole explants, LRP6 induces Xnr3/sia, but not brachyury (Xbra) expression, which is activated by mesoderm inducers like activin or basic fibroblast growth factor (bFGF). These results indicate that overexpression of LRP6 may specifically activate Wnt signaling. To examine whether LRP6 mediates Wnt effect, RNAs for LRP6 and Wnt-5a were co-injected. Neither Wnt-5a nor a low dose of LRP6 alone exhibits any effect, but Wnt-5a plus LRP6 synergistically induce axis duplication and ectopic Xnr3 expression in the embryo, and activate Xnr3/sia in explants. Synergy is also observed between Wnt-5a and hFz5, and between LRP6 and hFz5. Although LRP5 alone does not induce axes, co-injecting LRP5 and Wnt-5a does. LDLR alone or in combination with Wnt-5a does not induce axes or Xnr3/sia. Although Wnt-5a-hFz5 can induce complete axes including head and the notochord, Wnt-5a-LRP6 or LRP6 alone (higher doses) induce trunk axis with muscle and neural tissues but lacking head and the notochord. This may be explained by quantitative or qualitative differences between Wnt-5a/LRP6 and Wnt-5a/hFz5 co-injections (Tamai, 2000).

Because ectopic Wnt expression enhances, whereas lack of Wnt signaling inhibits, neural crest formation, the effect of LRP6 on neural crest development was analyzed. LDLR injection has no effect, but LRP6 expression significantly expands neural crest progenitors in the injected half of the embryo, as determined by the expression of a crest-specific marker, slug. Thus, overexpression of LRP6 also mimics Wnt signaling during neural crest formation (Tamai, 2000).

To distinguish whether LRP6 functions in Wnt-responding or Wnt-producing cells, Wnt-5a and LRP6 were injected separately into neighboring blastomeres at the four-cell stage. Induction of secondary axes in embryos and of Xnr3/sia in explants occurs even when Wnt-5a and LRP6 are expressed in different cells. Therefore, LRP6 is probably involved in responding to, rather than in enhancing, the production or secretion of the Wnt ligand (Tamai, 2000).

LRP6deltaC, which has most of its cytoplasmic domain deleted, was generated to inhibit the function of endogenous LRP6, which is expressed maternally and throughout embryogenesis. LRP6deltaC does not, either alone or in combination with Wnt-5a, induce axes or activate Xnr3/sia, but inhibits axis duplication and Xnr3/sia induction by the wild-type LRP6. This inhibition is counteracted by an increasing amount of co-injected LRP6. These data suggest that LRP6deltaC is a dominant interfering mutant for LRP6 or related molecules, and that LRP6 cytoplasmic domain is required for Wnt signaling. LRP6deltaC inhibits Xnr3/sia induction by several Wnt molecules, including Wnt-1, Wnt-2, Wnt-3a and Wnt-8. LRP6deltaC also inhibits Wnt-5a signaling through hFz5, showing that hFz5, and probably other endogenous Fz molecules mediating Wnt-1 or Wnt-8 signaling, depend on LRP6 or related proteins. LRP6deltaC does not affect Xbra induction by activin or bFGF, and thus interferes specifically with Wnt signaling (Tamai, 2000).

LRP6deltaC injected dorsally at the four-cell stage does not perturb endogenous axis formation. This may mean that the dorsal beta-catenin pathway is activated by mechanisms other than Wnt stimulation; alternatively, the dorsal Wnt-Fz signaling may occur early before LRP6deltaC can interfere. However, LRP6deltaC inhibits neural crest development as examined by slug expression, and suppresses ectopic crest formation induced by Wnt-3a DNA. Furthermore, co-injection of LRP6 rescues LRP6deltaC inhibition of crest formation. Thus, LRP6 or a related molecule is required for Wnt-dependent neural crest formation in vivo (Tamai, 2000).

In the current model of Wnt/beta-catenin signaling, Wnt stimulation of a Fz receptor activates the intracellular protein Dishevelled (Dsh or Dvl), thereby antagonizing the inhibitory action of the Axin/GSK-3 complex and stabilizing beta-catenin, which together with the transcription factor TCF/LEF activates responsive genes. To position LRP6 in this cascade, relationships between LRP6 and other Wnt signaling components were tested. LRP6deltaC inhibits Xnr3/sia induction by Wnts, but not by Dsh or beta-catenin, suggesting that LRP6deltaC interfers with Wnt signaling upstream of Dsh function. Supporting this epistasis, LRP6 induction of Xnr3/sia is antagonized by Axin, by a dominant-negative TCF, deltaNTCF, and by a dominant-negative Dsh, mDvl2-DIX. LRP6 activity is also inhibited by frizzled-related protein (FRP), a secreted Wnt antagonist, implying that LRP6 activation of Wnt signaling relies on endogenous Wnt molecules. Alternatively, FRP may directly inhibit LRP6. Thus, LRP6 acts between the extracellular Wnt, FRP and intracellular Dsh, possibly as a co-receptor for Wnt molecules (Tamai, 2000).

To function as a Wnt co-receptor, LRP6 should bind Wnt or Fz or both. Used to examine the issue were a secreted form of mFz8, mFz8CRD-IgG, comprising the cysteine-rich domain (CRD) of mFz8 N-terminal extracellular region fused with the immunoglobulin-gamma (IgG) Fc epitope, and a secreted LRP6N-Myc, consisting of the LRP6 extracellular domain tagged by the Myc epitope. mFz8CRD-IgG and LRP6N-Myc proteins were incubated with or without Wnt-1. mFz8CRD-IgG co-precipitates LRP6N-Myc only in the presence of Wnt-1; the secreted IgG fusion partner fails to do so regardless of Wnt-1. A reciprocal precipitation was also performed using secreted LRP6N-IgG and mFz8CRD-Myc, which were generated by swapping the two epitopes. LRP6N-IgG co-precipitates mFz8CRD-Myc, again only in the presence of Wnt-1, whereas the control IgG does not. LRP6N-IgG also co-precipitates Wnt-1-Myc, a tagged Wnt-1 protein. These results suggest that the extracellular domain of LRP6 can bind Wnt-1 and form a complex with Fz in a Wnt-dependent fashion (Tamai, 2000).

Thus, in two developmental processes dependent on the Wnt pathway in Xenopus -- secondary axis and neural crest formation -- LRP6 activates Wnt signaling, but a dominant-negative LRP6 inhibits Wnt signaling, providing compelling evidence that LRP6 is critical in Wnt signal transduction. LRP6 functions upstream of Dsh in Wnt-responding cells, synergizes with either Wnt or Fz, and importantly, is able to bind Wnt-1 and to associate with Fz in a Wnt-dependent manner. The simplest interpretation of these findings is that LRP6 is a component of the Wnt-Fz receptor complex. Genetic studies of arrow in Drosophila and lrp6 in mice (Pinson, 2000) strongly support this hypothesis. The binding data also raise the possibility that Wnt-induced formation of the Fz-LRP6 complex assembles LRP6, Fz and their associated proteins, thereby initiating cytoplasmic signaling. Consistent with this notion, Wnt signal transduction requires intracellular regions of both Fz and LRP6, which harbours candidate protein-protein interaction motifs. Notably, arrow does not exhibit fz planar polarity phenotype, implying that Arrow-LRP6 may specify Wnt-Fz signaling towards the beta-catenin pathway. How Fz, LRP6 and proteoglycan molecules such as Dally interact to mediate Wnt recognition/specificity and signal transduction remains to be studied. In addition, whether other LRPs and LRP-binding proteins participate in or modulate different Wnt-Fz signaling pathways needs evaluation (Tamai, 2000).

To understand how the Wnt coreceptor LRP-5 is involved in transducing the canonical Wnt signals, Axin was identified as a protein that interacts with the intracellular domain of LRP-5. LRP-5, when expressed in fibroblast cells, shows no effect on the canonical Wnt signaling pathway by itself, but instead acts synergistically with Wnt. In contrast, LRP-5 mutants that lack the extracellular domain function as constitutively active forms that bind Axin and that induce LEF-1 activation by destabilizing Axin and stabilizing beta-catenin. Addition of Wnt causes the translocation of Axin to the membrane and enhances the interaction between Axin and LRP-5. In addition, the LRP-5 sequences involved in interactions with Axin are required for LEF-1 activation. Thus, it is concluded that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway (J. Mao, 2001).

Thus, expression of the cytoplasmic domain of LRP-5, with or without the transmembrane domain, acts as a constitutive activator of the Wnt pathway, activating LEF-1 transcription and stabilizing ß-catenin. This result suggests that the extracellular domain inhibits the function of the intracellular domain. Binding of a Wnt ligand presumably overcomes this inhibition, perhaps by inducing a conformational change in LRP-5 or through interactions with the Frizzled receptor. Intriguingly, a construct containing the intracellular domain and the transmembrane region (LRPC2) was significantly more active than one containing just the intracellular domain (LRPC3), indicating that recruitment of Axin to the membrane is important for the activation of the Wnt pathway by LRP-5. The finding that a LRPC3 variant containing a myristylation signal shows an activity similar to LRPC2 lends further supports to this idea. One of the roles that the translocation plays is Axin destabilization, which has previously been shown to be induced by Wnt, because expression of LRPC2, but not LRPC3, caused Axin degradation. Together with the results showing that Wnt stimulation causes Axin to be recruited to the membrane via LRP-5, it is proposed that Wnt induces Axin destabilization at least in part by stimulating the interaction of LRP-5 with Axin. Although the precise mechanism by which activated LRP-5 causes the destabilization of Axin is not clear, it is reasonable to conclude that Axin destabilization contributes to the stabilization of ß-catenin (J. Mao, 2001).

The degradation of Axin is not the only means of inhibiting its function. This is consistent with the results that a construct that does not degrade Axin (LRPC3) can still stimulate LEF-1 transcription. Thus, part of the mechanism by which the intracellular domain of LRP-5 inhibits the function of Axin may simply involve the binding of this region to Axin, thus preventing it from participating in the degradation of ß-catenin. The region necessary for binding Axin and for stimulating LEF-1 transcription was narrowed down to 40 C-terminal amino acids in LRP-5. This region contains three copies of a motif PPT/SP, which is conserved in human and mouse LRP-5 and LRP-6 and Drosophila Arrow. Although removal of the very C-terminal motif shows little effect, elimination of two of these motifs reduces both Axin binding and LEF-1 stimulation but still retains some activity. Truncation of an additional 10 amino acids, including the third motif, abolishes Axin binding and transcriptional activation. This repeated motif could function as a phosphorylation site for a serine/threonine kinase. In this light, it is interesting that the wild-type GSK3 strongly stimulates the binding of Axin to LRP-5, whereas a kinase-dead GSK3 does not. While the intracellular domain of LRP-5 cannot be phosphorylated by GSK3 when immunoprecipitated LRPC2 and GSK were tested in an in vitro kinase assay, GSK3 may stimulate an additional kinase. Alternatively, the phosphorylation of Axin by GSK3 might cause it to bind LRP-5 more effectively. For instance, the phosphorylation of Axin by GSK3 enhances Axin's ability to bind ß-catenin and to bind GSK3 itself. Given that GSK3 promotes ß-catenin degradation, while the LRP/Axin interaction has the opposite result, the stimulatory effect of GSK3 on the interaction between Axin and LRP-5 is somewhat surprising. However, such an effect may allow LRP-5 to interact only with the Axin molecules that are associated with GSK3. In this way, LRP-5 could specifically direct the degradation of only those (GSK3-associated) Axin molecules that would otherwise participate in the degradation of ß-catenin (J. Mao, 2001).

Wnt-3a-induced binding of Axin to LRP-5 occurs within 4 min of the addition of ligand. This ligand-induced process occurs much sooner than the other processes that have thus far been reported. Wnt proteins induce the dephosphorylation of Axin and Dvl within 30 min, and the degradation of Axin starting at approximately 2 hr. Thus, the rapid stimulation of the interaction of LRP-5 and Axin by Wnt-3a suggests that the interaction between LRP-5 and Axin might be one of the first events in the Wnt canonical pathway (J. Mao, 2001).

In summary, a model is proposed describing the involvement of LRP proteins in the canonical Wnt signaling pathway. In this model, it is suggested that LRP, when activated by Wnt proteins, recruits Axin to the membranes. The translocation of Axin to the membrane prevents it from participating in the degradation of ß-catenin and plays an important role in the destabilization of Axin. LRP-6, a close homolog of LRP-5, may act in the same way as LRP-5, because a LRP-6 mutant that is equivalent to LRPC2 can also bind Axin and constitutively activates LEF-1. Thus, binding to Axin and activation of the canonical Wnt signaling pathway may occur with other LRP-5 homologs, including Arrow (J. Mao, 2001).

Although many components of the Wnt signaling pathway have been identified, little is known about how Wnts and their cognate Frizzled receptors signal to downstream effector molecules. Evidence is presented that a new member of the low-density lipoprotein (LDL)-receptor-related protein family, LRP6, is critical for Wnt signaling in mice. Embryos homozygous for an insertion mutation in the LRP6 gene exhibit developmental defects that are a striking composite of those caused by mutations in individual Wnt genes. Furthermore, a genetic enhancement of a Wnt mutant phenotype has been shown in mice lacking one functional copy of LRP6. These results support a broad role for LRP6 in the transduction of several Wnt signals in mammals (Pinson, 2000).

In a screen for recessive lethal phenotypes in mice caused by gene trap insertions in cell-surface proteins, an insertion mutation was recovered that joined the first 321 amino acids of the LRP6 protein in-frame with the betageo reporter gene. Embryos homozygous for the insertion in LRP6 die at birth, and exhibit a variety of severe developmental abnormalities including a truncation of the axial skeleton, limb defects, microophthalmia and malformation of the urogenital system. Northern blot analysis shows a complete absence of LRP6 transcripts in LRP6-/- embryos and embryonic fibroblasts. Southern blot analysis indicates a simple insertion event in which LRP6 exons downstream of the vector are retained. On the basis of betageo reporter activity, which accurately reports endogenous gene expression, the LRP6 gene appears to be expressed in all cells of the developing embryo (Pinson, 2000).

The specific developmental defects that are observed in homozygous embryos are remarkably similar to mice carrying mutations in Wnt genes, specifically Wnt-3a, Wnt-1 and Wnt-7a. Both the targeted null mutation and a classical hypomorphic allele of Wnt-3a, vestigial tail (vt), cause caudal truncations of the body axis. A similar axial truncation was observed in LRP6-/- neonates in which vertebrae caudal to the lumbar regions are absent. This was first evident as a reduction in the size of the tailbud at 8.5 days of development, with the loss of paraxial mesoderm and caudal somites at later stages. Sections through the tailbud of LRP6-/- embryos revealed an excess of neural tissue and a corresponding loss of paraxial mesoderm, similar to what has been observed in the targeted Wnt-3a mutant. As in vt mutants, Wnt-3a transcripts are reduced in the tailbud. By 10.5 days of development, Wnt-3a transcripts are completely absent from the tail, probably reflecting the depletion of mesodermal precursors at this stage. The extent of axial truncations in vt and Wnt-3a null mutants is sensitive to the dose of Wnt-3a. On the basis of the level at which the axial skeleton is truncated, it is concluded that LRP6-/- embryos display a severely hypomorphic Wnt-3a phenotype (Pinson, 2000).

To determine whether the vestigial tail phenotype is modified by the dose of LRP6, vt;LRP6 double heterozygous animals were crossed to vt/vt animals and embryos were collected at 11.5 and 13.5 days of development. The expected ratio of phenotypic classes was observed: half showed a normal tail length (corresponding to the vt/+;+/+ and vt/+;LRP6-/+ genotypes) and half exhibited a truncation of the tail (representing the vt/ vt;+/+ and vt/vt;LRP6-/+ genotypes). The tail defects in vt/vt embryos were clearly more pronounced among all eight embryos that inherited the LRP6 mutant allele. The enhancement of the vestigial tail phenotype upon elimination of one functional copy of the LRP6 gene provides genetic evidence that LRP6 and Wnt-3a function in the same pathway (Pinson, 2000).

Mutant embryos were examined for mid/hindbrain defects, a hallmark of Wnt-1-deficient mice. The phenotypes of a classical Wnt-1 allele, swaying, and a targeted mutation of Wnt-1 have variable expressivity, exhibiting a range of malformations of the midbrain and cerebellum. About one-half of the LRP6 mutants exhibit neural tube closure defects (exencephaly and/or spina bifida), and among those embryos that were not exencephalic morphological changes at the mid/hindbrain junction were observed. At 10.5 days, the mid/hindbrain boundary is less distinct in LRP6 mutant embryos, and by 14.5 days, a deletion of the caudal midbrain is evident. At birth, the inferior colliculus is absent and the cerebellum is disorganized. Furthermore, an elevation and expansion of Wnt-1 transcripts observed in swaying embryos is also seen in LRP6-/- embryos. Thus, LRP6-deficient mice phenocopy the less severe class of mid/hindbrain defects observed in Wnt-1 mutant mice (Pinson, 2000).

LRP6 mutant embryos display both anteroposterior and dorsoventral patterning defects in the limbs. These defects are reminiscent of those observed in Wnt-7a mutant mice that fail to maintain Shh expression in the zone of polarizing activity (ZPA), leading to the variable loss of posterior digits. Wnt-7a is also required to establish dorsal cell fates in the limb: in its absence, a biventral limb is formed. Because hindlimb defects in the LRP6 mutants may be exacerbated by a general disruption of caudal development, an analysis was focused on forelimb development. The forelimbs of LRP6 -/- embryos show a loss of one or more posterior digits that is presaged at earlier stages by a gradual loss of Shh expression. Moreover, some of the remaining digits contained ectopic tendons diagnostic of a ventralized limb. A distinguishing feature of the LRP6 limb phenotype not observed in Wnt-7a mutants was a failure to maintain the apical ectodermal ridge (AER), as judged by discontinuous expression of Fgf8. Discontinuity of the AER may explain the variable loss of anterior digits observed in LRP6 mutant embryos. The added complexity may indicate that, in addition to Wnt-7a, LRP6 mediates signaling by other Wnts in the limb (Pinson, 2000).

Clearly, LRP6 mutant mice do not exhibit all of the Wnt phenotypes reported to date (for example, the early embryonic lethality associated with Wnt-3 mutants and the limb outgrowth defects in Wnt-5a mutants; therefore, LRP6 may be involved in signaling by only a subset of vertebrate Wnts. Furthermore, the defects in LRP6 mutant embryos are generally less severe than those exhibited by individual Wnt mutants. Notably, a second highly related family member, LRP5, is widely expressed in embryos and may limit the range and severity of Wnt phenotypes observed in LRP6-/- mice (Pinson, 2000).

Members of the LDL receptor family are classically known for their involvement in the endocytic pathway, mediating the uptake through clathrin-coated pits of macromolecules such as apolipoproteins, lipases and proteases. Mice carrying mutations in two members of the LDL receptor family (the VLDL and ApoER2 receptors) have been shown to phenocopy mouse mutations in reelin or disabled , providing the first indication that LDL receptors can participate in specific signaling pathways. Given the central importance of the LDL receptor family in the transport of lipoproteins and other macromolecules into the cell, the relationship between nutrient uptake and the response of cells to developmental signals such as Wnts warrants further investigation (Pinson, 2000).

Wnt glycoproteins have been implicated in diverse processes during embryonic patterning in metazoa. They signal through frizzled-type seven-transmembrane-domain receptors to stabilize beta-catenin. Wnt signalling is antagonized by the extracellular Wnt inhibitor dickkopf1 (dkk1), which is a member of a multigene family. dkk1 was initially identified as a head inducer in Xenopus embryos but the mechanism by which it blocks Wnt signalling is unknown. LDL-receptor-related protein 6 (LRP6) is required during Wnt/beta-catenin signalling in Drosophila, Xenopus and mouse, possibly acting as a co-receptor for Wnt. LRP6 is a specific, high-affinity receptor for Dkk1 and Dkk2. Dkk1 blocks LRP6-mediated Wnt/beta-catenin signalling by interacting with domains that are distinct from those required for Wnt/Frizzled interaction. dkk1 and LRP6 interact antagonistically during embryonic head induction in Xenopus where LRP6 promotes the posteriorizing role of Wnt/beta-catenin signalling. Thus, DKKs inhibit Wnt co-receptor function, exemplifying the modulation of LRP signalling by antagonists (B. Mao, 2001).

The differentiation of epithelial cells and fiber cells from the anterior and posterior compartments of the lens vesicle, respectively, give the mammalian lens its distinctive polarity. While much progress has been made in understanding the molecular basis of fiber differentiation, little is known about factors that govern the differentiation of the epithelium. Members of the Wnt growth factor family appear to be key regulators of epithelial differentiation in various organ systems. Wnts are ligands for Frizzled receptors and can activate several signaling pathways, of which the best understood is the Wnt/ß-catenin pathway. The presence of LDL-related protein coreceptors (LRPs) 5 or 6 has been shown to be a requirement for Wnt signaling through the ß-catenin pathway. To access the role of this signaling pathway in the lens, mice were analyzed with a null mutation of lrp6. These mice have small eyes and aberrant lenses, characterized by an incompletely formed anterior epithelium resulting in extrusion of the lens fibers into the overlying corneal stroma. Multiple Wnts, including 5a, 5b, 7a, 7b, 8a, 8b, and Frizzled receptors 1, 2, 3, 4, and 6, are detected in the lens. Expression of these molecules is generally present throughout the lens epithelium and extended into the transitional zone, where early fiber elongation occurs. In addition to both LRP5 and LRP6, the expression was shown of other molecules involved in Wnt signaling and its regulation, including Dishevelleds, Dickkopfs, and secreted Frizzled-related proteins. Taken together, these results indicate a role for Wnt signaling in regulating the differentiation and behavior of lens cells (Stump, 2003).

Current models of canonical Wnt signaling assume that a pathway is active if β-catenin becomes nuclearly localized and Wnt target genes are transcribed. In Xenopus, maternal LRP6 is essential in such a pathway, playing a pivotal role in causing expression of the organizer genes siamois and Xnr3, and in establishing the dorsal axis. Evidence iis provided that LRP6 acts by degrading axin protein during the early cleavage stage of development. In the full-grown oocyte, before maturation, axin levels are also regulated by Wnt11 and LRP6. In the oocyte, Wnt11 and/or LRP6 regulates axin to maintain β-catenin at a low level, while in the embryo, asymmetrical Wnt11/LRP6 signaling stabilizes β-catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality (Kofron, 2007)

Wnt and Dickkopf (Dkk) regulate the stabilization of beta-catenin antagonistically in the Wnt signaling pathway; however, the molecular mechanism is not clear. In this study, Wnt3a was foud to act in parallel to induce the caveolin-dependent internalization of low-density-lipoprotein receptor-related protein 6 (LRP6), as well as the phosphorylation of LRP6 and the recruitment of Axin to LRP6 on the cell surface membrane. The phosphorylation and internalization of LRP6 occurred independently of one another, and both were necessary for the accumulation of beta-catenin. In contrast, Dkk1, which inhibits Wnt3a-dependent stabilization of beta-catenin, induced the internalization of LRP6 with clathrin. Knockdown of clathrin suppressed the Dkk1-dependent inhibition of the Wnt3a response. Furthermore, Dkk1 reduced the distribution of LRP6 in the lipid raft fraction where caveolin is associated. These results indicate that Wnt3a and Dkk1 shunt LRP6 to distinct internalization pathways in order to activate and inhibit the beta-catenin signaling, respectively (Yamamoto, 2008).

R-spondin interacts with LRP6 to promote Wnt signaling

R-spondin proteins are newly identified secreted molecules that activate beta-catenin signaling. However, the mechanism of R-spondin action and its relationship with Wnt signaling remain unclear. This stud shows that human R-spondin1 (hRspo1) is a high affinity ligand for the Wnt co-receptor LRP6 (K(d) = 1.2 nm). hRspo1 induces glycogen synthase kinase 3-dependent phosphorylation and activation of LRP6. DKK1, an LRP6 antagonist, inhibits hRspo1-induced LRP6 phosphorylation. hRspo1 synergizes with Frizzled5 in Xenopus axis induction assays and induces the phosphorylation of Dishevelled, a cytoplasmic component downstream of Frizzled function. This study reveals interesting similarity and distinction between Wnt and R-spondin signaling (Wei, 2007).

The vertebrate embryonic vasculature develops from angioblasts, which are specified from mesodermal precursors and develop in close association with blood cells. The signals that regulate embryonic vasculogenesis and angiogenesis are incompletely understood. This study shows that R-spondin 3 (Rspo3), a member of a novel family of secreted proteins in vertebrates that interact with LRP6 and activate Wnt/β-catenin signaling, plays a key role in these processes. In Xenopus embryos, morpholino antisense knockdown of Rspo3 induces vascular defects because Rspo3 is essential for regulating the balance between angioblast and blood cell specification. In mice, targeted disruption of Rspo3 leads to embryonic lethality caused by vascular defects. Specifically in the placenta, remodeling of the vascular plexus is impaired. In human endothelial cells, R-spondin signaling promotes proliferation and sprouting angiogenesis in vitro, indicating that Rspo3 can regulate endothelial cells directly. This study shows that vascular endothelial growth factor is an immediate early response gene and a mediator of R-spondin signaling. The results identify Rspo3 as a novel, evolutionarily conserved angiogenic factor in embryogenesis (Kazanskaya, 2009).

Surrogate Wnt agonists that phenocopy canonical Wnt and beta-catenin signalling

Wnt proteins (see Drosophila Wingless) modulate cell proliferation and differentiation and the self-renewal of stem cells by inducing β-catenin-dependent signalling through the Wnt receptor frizzled (FZD; see Drosophila Frizzled) and the co-receptors LRP5 and LRP6 (see Drosophila Arrow) to regulate cell fate decisions and the growth and repair of several tissues. As a result of their acylation, Wnt proteins are very hydrophobic and require detergents for purification, which presents major obstacles to the preparation and application of recombinant Wnt proteins. This hydrophobicity has hindered the determination of the molecular mechanisms of Wnt signalling activation. This study develop surrogate Wnt agonists, water-soluble FZD-LRP5/LRP6 heterodimerizers, with FZD5/FZD8-specific and broadly FZD-reactive binding domains. These Wnt agonists elicit a characteristic β-catenin signalling response in a FZD-selective fashion and enhance the osteogenic lineage commitment of primary mouse and human mesenchymal stem cells. These surrogates demonstrate that canonical Wnt signalling can be activated by bi-specific ligands that induce receptor heterodimerization. Furthermore, these easily produced, non-lipidated Wnt surrogate agonists facilitate functional studies of Wnt signalling and the exploration of Wnt agonists for translational applications in regenerative medicine (Janda, 2017).

Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation

Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor-related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, this study shows that Wnt treatment quickly induces plasma membrane-associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. It is proposed that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and beta-catenin stabilization (Bilic, 2007).

The scaffold protein Dvl was previously thought to act downstream of LRP6 because dsh overexpression activates ß-catenin signaling in Drosophila LRP6 (arrow) mutants and because the constitutively active Dfz2-Arrow fusion protein is inactive in dsh mutants. The explanation for this discrepancy may be that overexpressing Dsh/Dvl leads to artificial sequestration of Axin or that the protein has multiple functions in the Wnt pathway (Bilic, 2007).

Taken together, the results suggest that Dvl-mediated co-aggregation triggers LRP6 phosphorylation by CK1γ. In this model, upon Wnt signaling Dvl aggregates form at the plasma membrane, where they co-cluster LRP6 with other pathway components including Fz, Axin, and GSK3ß, in a 'LRP6-signalosome.' The role of Wnt would be to bridge LRP6 and Fz, which copolymerize on a Dvl platform. Clustering of LRP6 then provides a high local receptor concentration that triggers phosphorylation by CK1γ and Axin recruitment (Bilic, 2007).

Predictions of this model are as follows: (1) artificial oligomerization of LRP6 should activate the receptor and (2) oligomerized LRP6 should signal independent of Dvl. Indeed, forced oligomerization of LRP6 using a synthetic multimerizer is sufficient to induce Wnt signaling, and this oligomerization bypasses the need for Dvl. (3) Constitutively active LRP6 should signal independently of Dvl because its self-aggregation should bypass the need for Dvl polymers. This is also the case as shown in reporter assays with Dvl siRNA knockdown, which supports previous findings. (4) If LRP6 aggregation is a prerequisite for phosphorylation by CK1γ rather than its consequence, LRP6 aggregates should form even when the kinase is blocked. This is the case: Nonphosphorylated LRP6 aggregates were observed in response to Wnt treatment in cells transfected with dominant-negative CK1γ. The model of LRP6-signalosomes not only provides a mechanism for Wnt signal transduction but may also be relevant for the understanding of intracellular transport of maternal Wnt determinants in the fertilized Xenopus egg (Bilic, 2007).

Lrps and gastrulation

Recent work has identified LDL receptor-related family members, Lrp5 and Lrp6, as co-receptors for the transduction of Wnt signals. Analysis of mice carrying mutations in both Lrp5 and Lrp6 demonstrates that the functions of these genes are redundant and are essential for gastrulation. Lrp5;Lrp6 double homozygous mutants fail to establish a primitive streak, although the anterior visceral endoderm and anterior epiblast fates are specified. Thus, Lrp5 and Lrp6 are required for posterior patterning of the epiblast, consistent with a role in transducing Wnt signals in the early embryo. Interestingly, Lrp5+/-;Lrp6-/- embryos die shortly after gastrulation and exhibit an accumulation of cells at the primitive streak and a selective loss of paraxial mesoderm. A similar phenotype is observed in Fgf8 and Fgfr1 mutant embryos and provides genetic evidence in support of a molecular link between the Fgf and Wnt signaling pathways in patterning nascent mesoderm. Lrp5+/-;Lrp6-/- embryos also display an expansion of anterior primitive streak derivatives and anterior neurectoderm that correlates with increased Nodal expression in these embryos. The effect of reducing, but not eliminating, Wnt signaling in Lrp5+/-;Lrp6-/- mutant embryos provides important insight into the interplay between Wnt, Fgf and Nodal signals in patterning the early mouse embryo (Kelly, 2004).

Wnt signaling regulates β-catenin-mediated gene transcription and planar cell polarity (PCP). The Wnt co-receptor, Lrp6, is required for signaling along the β-catenin arm. Lrp6 downregulation (by morpholino injection) or overexpression in Xenopus embryos disrupts convergent extension, a hallmark feature of Wnt/PCP components. In embryos with decreased Lrp6 levels, cells of the dorsal marginal zone (DMZ), which undergoes extensive cellular rearrangements during gastrulation, exhibit decreased length:width ratios, decreased migration, and increased numbers of transient cytoplasmic protrusions. Lrp6 opposes Wnt11 activity and localizes to the posterior edge of migrating DMZ cells; Lrp6 downregulation enhances cortical and nuclear localization of Dsh and phospho-JNK, respectively. Taken together, these data suggest that Lrp6 inhibits Wnt/PCP signaling. Finally, the region of the Lrp6 protein with Wnt/PCP activity was localized to a stretch of 36 amino acids, distinct from regions required for Wnt/β-catenin signaling. A model is proposed in which Lrp6 plays a critical role in the switch from Wnt/PCP to Wnt/β-catenin signaling (Tahinci, 2007)

Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/βcatenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a 'head inducer' during gastrulation. Lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/βcatenin pathway is overactivated, and dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a βcatenin independent fashion when modulating gastrulation movements. The glypican 4/6 homolog Knypek (Kny) binds to Dkk1, and they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. Indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the βcatenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. These data therefore indicate that Dkk1 regulates gastrulation movement through interaction the Frizzled coreceptor transmembrane protein LRP5/6 and Kny and coordinated modulations of Wnt/βcatenin and Wnt/PCP pathways (Caneparo, 2007).

Intracellular transport of LDL receptor family members

Specification of embryonic polarity and pattern formation in multicellular organisms requires inductive signals from neighboring cells. One approach toward understanding these interactions is to study mutations that disrupt development. mesd, a gene identified in the mesoderm development (mesd) deletion interval on mouse chromosome 7, is essential for specification of embryonic polarity and mesoderm induction. MESD functions in the endoplasmic reticulum as a specific chaperone for LRP5 and LRP6, which in conjunction with Frizzled, are coreceptors for canonical WNT signal transduction. Disruption of embryonic polarity and mesoderm differentiation in mesd-deficient embryos likely results from a primary defect in WNT signaling. However, phenotypic differences between mesd-deficient and wnt3 minus embryos suggest that MESD may function on related members of the low-density lipoprotein receptor (LDLR) family, whose members mediate diverse cellular processes ranging from cargo transport to signaling (Hsieh, 2003).

Lrp6-mediated canonical Wnt signaling is required for lip formation and fusion

Neither the mechanisms that govern lip morphogenesis nor the cause of cleft lip are well understood. Four paired prominences give rise to the vertebrate face: the medial nasal (mnp), lateral nasal (lnp), maxillary (maxp) and mandibular (manp) prominences, which are derived from the frontonasal prominence and the first pharyngeal (or branchial) arch. Mutations of the Tgfbeta/Bmp and Fgf pathways can result in cleft palate by diminishing proliferation or increasing apoptosis in orofacial primordia. This study reports that genetic inactivation of Lrp6, a co-receptor of the Wnt/beta-catenin signaling pathway, leads to cleft lip with cleft palate. The activity of a Wnt signaling reporter is blocked in the orofacial primordia by Lrp6 deletion in mice. The morphological dynamic that is required for normal lip formation and fusion is disrupted in these mutants. The expression of the homeobox genes Msx1 and Msx2 is dramatically reduced in the mutants, which prevents the outgrowth of orofacial primordia, especially in the fusion site. that Msx1 and Msx2 (but not their potential regulator Bmp4) are the downstream targets of the Wnt/beta-catenin signaling pathway during lip formation and fusion. By contrast, a 'fusion-resistant' gene, Raldh3 (also known as Aldh1a3), that encodes a retinoic acid-synthesizing enzyme is ectopically expressed in the upper lip primordia of Lrp6-deficient embryos, indicating a region-specific role of the Wnt/beta-catenin signaling pathway in repressing retinoic acid signaling. Thus, the Lrp6-mediated Wnt signaling pathway is required for lip development by orchestrating two distinctively different morphogenetic movements (Song, 2009).

Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors

Wnt ligands signal through β-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through β-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. This study demonstrates that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). Ror2 Ser 864 was identified as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, evidence is provided that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways (Grumolato, 2010).

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