LDL-receptor-related protein 6 is a receptor for Dickkopf proteins

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. Here we show that LRP6 (ref. 7) 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.     

LDL-receptor-related proteins in Wnt signal transduction

The Wnt family of secreted signalling molecules are essential in embryo development and tumour formation. The Frizzled (Fz) family of serpentine receptors function as Wnt receptors, but how Fz proteins transduce signalling is not understood. In Drosophila, arrow phenocopies the wingless (DWnt-1) phenotype, and 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 (refs 12-15). Here we report that LRP6 functions as a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6 activated Wnt-Fz signalling, and induced Wnt responsive genes, dorsal axis duplication and neural crest formation. An LRP6 mutant lacking the carboxyl intracellular domain blocked signalling by Wnt or Wnt-Fz, but not by Dishevelled or beta-catenin, and inhibited neural crest development. The extracellular domain of LRP6 bound Wnt-1 and associated with Fz in a Wnt-dependent manner. Our results indicate that LRP6 may be a component of the Wnt receptor complex.     

A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation

Signalling by the Wnt family of secreted lipoproteins has essential functions in development and disease. The canonical Wnt/beta-catenin pathway requires a single-span transmembrane receptor, low-density lipoprotein (LDL)-receptor-related protein 6 (LRP6), whose phosphorylation at multiple PPPSP motifs is induced upon stimulation by Wnt and is critical for signal transduction. The kinase responsible for LRP6 phosphorylation has not been identified. Here we provide biochemical and genetic evidence for a 'dual-kinase' mechanism for LRP6 phosphorylation and activation. Glycogen synthase kinase 3 (GSK3), which is known for its inhibitory role in Wnt signalling through the promotion of beta-catenin phosphorylation and degradation, mediates the phosphorylation and activation of LRP6. We show that Wnt induces sequential phosphorylation of LRP6 by GSK3 and casein kinase 1, and this dual phosphorylation promotes the engagement of LRP6 with the scaffolding protein Axin. We show further that a membrane-associated form of GSK3, in contrast with cytosolic GSK3, stimulates Wnt signalling and Xenopus axis duplication. Our results identify two key kinases mediating Wnt co-receptor activation, reveal an unexpected and intricate logic of Wnt/beta-catenin signalling, and illustrate GSK3 as a genuine switch that dictates both on and off states of a pivotal regulatory pathway.     

Kremen proteins are Dickkopf receptors that regulate Wnt/beta-catenin signalling

The Wnt family of secreted glycoproteins mediate cell cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the beta-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular beta-catenin. Wnt/beta-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. Here we show that the transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/beta-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates.     

The DIX domain targets dishevelled to actin stress fibres and vesicular membranes

Colorectal cancer results from mutations in components of the Wnt pathway that regulate beta-catenin levels. Dishevelled (Dvl or Dsh) signals downstream of Wnt receptors and stabilizes beta-catenin during cell proliferation and embryonic axis formation. Moreover, Dvl contributes to cytoskeletal reorganization during gastrulation and mitotic spindle orientation during asymmetric cell division. Dvl belongs to a family of eukaryotic signalling proteins that contain a conserved 85-residue module of unknown structure and biological function called the DIX domain. Here we show that the DIX domain mediates targeting to actin stress fibres and cytoplasmic vesicles in vivo. Neighbouring interaction sites for actin and phospholipid are identified between two helices by nuclear magnetic resonance spectroscopy (NMR). Mutation of the actin-binding motif abolishes the cytoskeletal localization of Dvl, but enhances Wnt/beta-catenin signalling and axis induction in Xenopus. By contrast, mutation of the phospholipid interaction site disrupts vesicular association of Dvl, Dvl phosphorylation, and Wnt/beta-catenin pathway activation. We propose that partitioning of Dvl into cytoskeletal and vesicular pools by the DIX domain represents a point of divergence in Wnt signalling.     

arrow encodes an LDL-receptor-related protein essential for Wingless signalling

The Wnt family of secreted molecules functions in cell-fate determination and morphogenesis during development in both vertebrates and invertebrates (reviewed in ref. 1). Drosophila Wingless is a founding member of this family, and many components of its signal transduction cascade have been identified, including the Frizzled class of receptor. But the mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. Here we describe a gene that is necessary for all Wingless signalling events in Drosophila. We show that arrow gene function is essential in cells receiving Wingless input and that it acts upstream of Dishevelled. arrow encodes a single-pass transmembrane protein, indicating that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, our data suggests a new and conserved function for this LRP subfamily in Wingless/Wnt signal reception.     

A new secreted protein that binds to Wnt proteins and inhibits their activities

The Wnt proteins constitute a large family of extracellular signalling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. Here we describe Wnt-inhibitory factor-1 (WIF-1), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1 is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm, notochord, branchial arches and neural crest derivatives. We use Xenopus embryos to show that WIF-1 overexpression affects somitogenesis (the generation of trunk mesoderm segments), in agreement with its normal expression in paraxial mesoderm. In vitro, WIF-1 binds to Drosophila Wingless and Xenopus Wnt8 produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled-related proteins, our results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity.     

IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis

Insulin-like growth-factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs). Although some of the actions of IGFBPs have been reported to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown. Here we report a previously unknown function for IGFBP-4 as a cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation in vitro, and knockdown of Igfbp4 attenuated cardiomyogenesis both in vitro and in vivo. The cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signalling. IGFBP-4 physically interacted with a Wnt receptor, Frizzled 8 (Frz8), and a Wnt co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Although IGF-independent, the cardiogenic effect of IGFBP-4 was attenuated by IGFs through IGFBP-4 sequestration. IGFBP-4 is therefore an inhibitor of the canonical Wnt signalling required for cardiogenesis and provides a molecular link between IGF signalling and Wnt signalling.     

Casein kinase I transduces Wnt signals.

The Wnt signalling cascade is essential for the development of both invertebrates and vertebrates, and is altered during tumorigenesis. Although a general framework for Wnt signalling has been elucidated, not all of the components have been identified. Here we describe a serine kinase, casein kinase I (CKI), which was isolated by expression cloning in Xenopus embryos. CKI reproduces several properties of Wnt signals, including generation of complete dorsal axes, stabilization of beta-catenin and induction of genes that are direct targets of Wnt signals. Dominant-negative forms of CKI and a pharmacological blocker of CKI inhibited Wnt signals in Xenopus. Inhibiting CKI in Caenorhabditis elegans generated worms with a mom phenotype, indicative of a loss of Wnt signals. In addition, CKI bound to and increased the phosphorylation of dishevelled, a known component of the Wnt pathway. These data indicate that CKI may be a conserved component of the Wnt pathway.     

An LDL-receptor-related protein mediates Wnt signalling in mice

Wnt genes comprise a large family of secreted polypeptides that are expressed in spatially and tissue-restricted patterns during vertebrate embryonic development. Mutational analysis in mice has shown the importance of Wnts in controlling diverse developmental processes such as patterning of the body axis, central nervous system and limbs, and the regulation of inductive events during organogenesis. Although many components of the Wnt signalling pathway have been identified, little is known about how Wnts and their cognate Frizzled receptors signal to downstream effector molecules. Here we present evidence that a new member of the low-density lipoprotein (LDL)-receptor-related protein family, LRP6 (ref. 3), is critical for Wnt signalling 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, we show a genetic enhancement of a Wnt mutant phenotype in mice lacking one functional copy of LRP6. Together, our results support a broad role for LRP6 in the transduction of several Wnt signals in mammals.     

Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis

Formation of the vertebrate heart requires a complex interplay of several temporally regulated signalling cascades. In Xenopus laevis, cardiac specification occurs during gastrulation and requires signals from the dorsal lip and underlying endoderm. Among known Xenopus Wnt genes, only Wnt-11 shows a spatiotemporal pattern of expression that correlates with cardiac specification, which indicates that Wnt-11 may be involved in heart development. Here we show, through loss- and gain-of-function experiments, that XWnt-11 is required for heart formation in Xenopus embryos and is sufficient to induce a contractile phenotype in embryonic explants. Treating the mouse embryonic carcinoma stem cell line P19 with murine Wnt-11 conditioned medium triggers cardiogenesis, which indicates that the function of Wnt-11 in heart development has been conserved in higher vertebrates. XWnt-11 mediates this effect by non-canonical Wnt signalling, which is independent of beta-catenin and involves protein kinase C and Jun amino-terminal kinase. Our results indicate that the cardiac developmental program requires non-canonical Wnt signal transduction.     

Frizzled-7 signalling controls tissue separation during Xenopus gastrulation.

Cell signalling through Frizzled receptors has evolved to considerable complexity within the metazoans. The Frizzled-dependent signalling cascade comprises several branches, whose differential activation depends on specific Wnt ligands, Frizzled receptor isoforms and the cellular context. In Xenopus laevis embryos, the canonical beta-catenin pathway contributes to the establishment of the dorsal-ventral axis. A different branch, referred to as the planar cell polarity pathway, is essential for cell polarization during elongation of the axial mesoderm by convergent extension. Here we demonstrate that a third branch of the cascade is independent of Dishevelled function and involves signalling through trimeric G proteins and protein kinase C (PKC). During gastrulation, Frizzled-7 (Fz7)-dependent PKC signalling controls cell-sorting behaviour in the mesoderm. Loss of zygotic Fz7 function results in the inability of involuted anterior mesoderm to separate from the ectoderm, which leads to severe gastrulation defects. This result provides a developmentally relevant in vivo function for the Fz/PKC pathway in vertebrates.     

Regulation of Lethal giant larvae by Dishevelled

The establishment of polarity in many cell types depends on Lgl, the tumour suppressor product of lethal giant larvae, which is involved in basolateral protein targeting. The conserved complex of Par3, Par6 and atypical protein kinase C phosphorylates and inactivates Lgl at the apical surface; however, the signalling mechanisms that coordinate cell polarization in development are not well defined. Here we show that a vertebrate homologue of Lgl associates with Dishevelled, an essential mediator of Wnt signalling, and that Dishevelled regulates the localization of Lgl in Xenopus ectoderm and Drosophila follicular epithelium. We show that both Lgl and Dsh are required for normal apical-basal polarity of Xenopus ectodermal cells. In addition, we show that the Wnt receptor Frizzled 8, but not Frizzled 7, causes Lgl to dissociate from the cortex with the concomitant loss of its activity in vivo. These findings suggest a molecular basis for the regulation of cell polarity by Frizzled and Dishevelled.     

ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner

R-spondin proteins strongly potentiate Wnt signalling and function as stem-cell growth factors. Despite the biological and therapeutic significance, the molecular mechanism of R-spondin action remains unclear. Here we show that the cell-surface transmembrane E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) and its homologue ring finger 43 (RNF43) are negative feedback regulators of Wnt signalling. ZNRF3 is associated with the Wnt receptor complex, and inhibits Wnt signalling by promoting the turnover of frizzled and LRP6. Inhibition of ZNRF3 enhances Wnt/β-catenin signalling and disrupts Wnt/planar cell polarity signalling in vivo. Notably, R-spondin mimics ZNRF3 inhibition by increasing the membrane level of Wnt receptors. Mechanistically, R-spondin interacts with the extracellular domain of ZNRF3 and induces the association between ZNRF3 and LGR4, which results in membrane clearance of ZNRF3. These data suggest that R-spondin enhances Wnt signalling by inhibiting ZNRF3. Our study provides new mechanistic insights into the regulation of Wnt receptor turnover, and reveals ZNRF3 as a tractable target for therapeutic exploration.     

氨甲环酸与Wnt信号通路中含kringle结构域蛋白的结合对新生小鼠骨密度的影响

最近研究发现Wnt信号通路在骨形成过程中发挥重要作用Wnt受体如脂蛋白相关蛋白5（lrp5）和孤独受体（Ror2）的缺失或突变导致骨的不正常发育．Dkk是一个分泌型规范Wnt信号系统的抑制剂，通过与脂蛋白相关蛋白5和最近新发现的一种含kringle结构域的蛋白kremen形成三聚体复合物．这种复合物随即被细胞内吞，从而导致细胞表面Wnt受体脂蛋白相关蛋白5水平迅速下降，从而达到抑制Wnt信号通路的日地．通过对kremen和Ror2蛋白序列分析发现kremen和Ror2的胞外部分均含有一个结构上能与赖氨酸结合的kringle结构域．通过给怀孕母鼠注射一种赖氨酸类似物——氨甲环酸来研究kremen和Ror2的kringle结构域上的赖氨酸结合位点被占据对小鼠骨发育的作用．但是，研究结果表明AMCA组和对照组之间的骨密度并没有显著差异，揭示赖氨酸结合位点不参与骨的发育调控．     

Wnt signalling regulates adult hippocampal neurogenesis

The generation of new neurons from neural stem cells is restricted to two regions of the adult mammalian central nervous system: the subventricular zone of the lateral ventricle, and the subgranular zone of the hippocampal dentate gyrus. In both regions, signals provided by the microenvironment regulate the maintenance, proliferation and neuronal fate commitment of the local stem cell population. The identity of these signals is largely unknown. Here we show that adult hippocampal stem/progenitor cells (AHPs) express receptors and signalling components for Wnt proteins, which are key regulators of neural stem cell behaviour in embryonic development. We also show that the Wnt/beta-catenin pathway is active and that Wnt3 is expressed in the hippocampal neurogenic niche. Overexpression of Wnt3 is sufficient to increase neurogenesis from AHPs in vitro and in vivo. By contrast, blockade of Wnt signalling reduces neurogenesis from AHPs in vitro and abolishes neurogenesis almost completely in vivo. Our data show that Wnt signalling is a principal regulator of adult hippocampal neurogenesis and provide evidence that Wnt proteins have a role in adult hippocampal function.