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.     

The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors

Mammalian Toll-like receptors (TLRs) function as sensors of infection and induce the activation of innate and adaptive immune responses. Upon recognizing conserved pathogen-associated molecular products, TLRs activate host defence responses through their intracellular signalling domain, the Toll/interleukin-1 receptor (TIR) domain, and the downstream adaptor protein MyD88 (refs 1-3). Although members of the TLR and the interleukin-1 (IL-1) receptor families all signal through MyD88, the signalling pathways induced by individual receptors differ. TIRAP, an adaptor protein in the TLR signalling pathway, has been identified and shown to function downstream of TLR4 (refs 4, 5). Here we report the generation of mice deficient in the Tirap gene. TIRAP-deficient mice respond normally to the TLR5, TLR7 and TLR9 ligands, as well as to IL-1 and IL-18, but have defects in cytokine production and in activation of the nuclear factor NF-kappaB and mitogen-activated protein kinases in response to lipopolysaccharide, a ligand for TLR4. In addition, TIRAP-deficient mice are also impaired in their responses to ligands for TLR2, TLR1 and TLR6. Thus, TIRAP is differentially involved in signalling by members of the TLR family and may account for specificity in the downstream signalling of individual TLRs.     

Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction

Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus.     

Chondroitin proteoglycans are involved in cell division of Caenorhabditis elegans

Glycosaminoglycans such as heparan sulphate and chondroitin sulphate are extracellular sugar chains involved in intercellular signalling. Disruptions of genes encoding enzymes that mediate glycosaminoglycan biosynthesis have severe consequences in Drosophila and mice. Mutations in the Drosophila gene sugarless, which encodes a UDP-glucose dehydrogenase, impairs developmental signalling through the Wnt family member Wingless, and signalling by the fibroblast growth factor and Hedgehog pathways. Heparan sulphate is involved in these pathways, but little is known about the involvement of chondroitin. Undersulphated and oversulphated chondroitin sulphate chains have been implicated in other biological processes, however, including adhesion of erythrocytes infected with malaria parasite to human placenta and regulation of neural development. To investigate chondroitin functions, we cloned a chondroitin synthase homologue of Caenorhabditis elegans and depleted expression of its product by RNA-mediated interference and deletion mutagenesis. Here we report that blocking chondroitin synthesis results in cytokinesis defects in early embryogenesis. Reversion of cytokinesis is often observed in chondroitin-depleted embryos, and cell division eventually stops, resulting in early embryonic death. Our findings show that chondroitin is required for embryonic cytokinesis and cell division.     

TLR signalling augments macrophage bactericidal activity through mitochondrial ROS

Reactive oxygen species (ROS) are essential components of the innate immune response against intracellular bacteria and it is thought that professional phagocytes generate ROS primarily via the phagosomal NADPH oxidase machinery. However, recent studies have suggested that mitochondrial ROS (mROS) also contribute to mouse macrophage bactericidal activity, although the mechanisms linking innate immune signalling to mitochondria for mROS generation remain unclear. Here we demonstrate that engagement of a subset of Toll-like receptors (TLR1, TLR2 and TLR4) results in the recruitment of mitochondria to macrophage phagosomes and augments mROS production. This response involves translocation of a TLR signalling adaptor, tumour necrosis factor receptor-associated factor 6 (TRAF6), to mitochondria, where it engages the protein ECSIT (evolutionarily conserved signalling intermediate in Toll pathways), which is implicated in mitochondrial respiratory chain assembly. Interaction with TRAF6 leads to ECSIT ubiquitination and enrichment at the mitochondrial periphery, resulting in increased mitochondrial and cellular ROS generation. ECSIT- and TRAF6-depleted macrophages have decreased levels of TLR-induced ROS and are significantly impaired in their ability to kill intracellular bacteria. Additionally, reducing macrophage mROS levels by expressing catalase in mitochondria results in defective bacterial killing, confirming the role of mROS in bactericidal activity. These results reveal a novel pathway linking innate immune signalling to mitochondria, implicate mROS as an important component of antibacterial responses and further establish mitochondria as hubs for innate immune signalling.     

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.     

Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity

Fungal infections are increasing worldwide due to the marked rise in immunodeficiencies including AIDS; however, immune responses to fungi are poorly understood. Dectin-1 is the major mammalian pattern recognition receptor for the fungal component zymosan. Dectin-1 represents the prototype of innate non-Toll-like receptors (TLRs) containing immunoreceptor tyrosine-based activation motifs (ITAMs) related to those of adaptive antigen receptors. Here we identify Card9 as a key transducer of Dectin-1 signalling. Although being dispensable for TLR/MyD88-induced responses, Card9 controls Dectin-1-mediated myeloid cell activation, cytokine production and innate anti-fungal immunity. Card9 couples to Bcl10 and regulates Bcl10-Malt1-mediated NF-kappaB activation induced by zymosan. Yet, Card9 is dispensable for antigen receptor signalling that uses Carma1 as a link to Bcl10-Malt1. Thus, our results define a novel innate immune pathway and indicate that evolutionarily distinct ITAM receptors in innate and adaptive immune cells use diverse adaptor proteins to engage selectively the conserved Bcl10-Malt1 module.     

The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens.

Macrophages orchestrate innate immunity by phagocytosing pathogens and coordinating inflammatory responses. Effective defence requires the host to discriminate between different pathogens. The specificity of innate immune recognition in Drosophila is mediated by the Toll family of receptors; Toll mediates anti-fungal responses, whereas 18-wheeler mediates anti-bacterial defence. A large number of Toll homologues have been identified in mammals, and Toll-like receptor 4 is critical in responses to Gram-negative bacteria. Here we show that Toll-like receptor 2 is recruited specifically to macrophage phagosomes containing yeast, and that a point mutation in the receptor abrogates inflammatory responses to yeast and Gram-positive bacteria, but not to Gram-negative bacteria. Thus, during the phagocytosis of pathogens, two classes of innate immune receptors cooperate to mediate host defence: phagocytic receptors, such as the mannose receptor, signal particle internalization, and the Toll-like receptors sample the contents of the vacuole and trigger an inflammatory response appropriate to defence against the specific organism.     

Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli

The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. We devised a double-stranded RNA interference-based screen in macrophage-like Drosophila S2 cells, and have defined 34 gene products involved in phagocytosis. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. Here we show that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. Our results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis.     

Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains.

Members of the Frizzled family of seven-pass transmembrane proteins serve as receptors for Wnt signalling proteins. Wnt proteins have important roles in the differentiation and patterning of diverse tissues during animal development, and inappropriate activation of Wnt signalling pathways is a key feature of many cancers. An extracellular cysteine-rich domain (CRD) at the amino terminus of Frizzled proteins binds Wnt proteins, as do homologous domains in soluble proteins-termed secreted Frizzled-related proteins-that function as antagonists of Wnt signalling. Recently, an LDL-receptor-related protein has been shown to function as a co-receptor for Wnt proteins and to bind to a Frizzled CRD in a Wnt-dependent manner. To investigate the molecular nature of the Wnt signalling complex, we determined the crystal structures of the CRDs from mouse Frizzled 8 and secreted Frizzled-related protein 3. Here we show a previously unknown protein fold, and the design and interpretation of CRD mutations that identify a Wnt-binding site. CRDs exhibit a conserved dimer interface that may be a feature of Wnt signalling. This work provides a framework for studies of homologous CRDs in proteins including muscle-specific kinase and Smoothened, a component of the Hedgehog signalling pathway.     

Dally cooperates with Drosophila Frizzled 2 to transduce Wingless signalling.

The Drosophila wingless gene (wg) encodes a protein of the Wnt family and is a critical regulator in many developmental processes. Biochemical studies have indicated that heparan sulphate proteoglycans, consisting of a protein core to which heparan sulphate glycosaminoglycans are attached, are important for Wg function. Here we show that, consistent with these findings, the Drosophila gene sulfateless (sfl), which encodes a homologue of vertebrate heparan sulphate N-deacetylase/N-sulphotransferase (an enzyme needed for the modification of heparan sulphate) is essential for Wg signalling. We have identified the product of division abnormally delayed (dally), a glycosyl-phosphatidyl inositol (GPI)-linked glypican, as a heparan sulphate proteoglycan molecule involved in Wg signalling. Our results indicate that Dally may act as a co-receptor for Wg, and that Dally, together with Drosophila Frizzled 2, modulates both short- and long-range activities of Wg.     

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction.

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.     

Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6

Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.     

Distortion of proximodistal information causes JNK-dependent apoptosis in Drosophila wing.

Distinct and evolutionarily conserved signal-transduction cascades mediate the survival or death of cells during development. The c-Jun amino-terminal kinases (JNKs) of the mitogen-activated protein kinase superfamily are involved in apoptotic signalling in various cultured cells. However, the role of the JNK pathway in development is less well understood. In Drosophila, Decapentaplegic (Dpp; a homologue of transforming growth factor-beta) and Wingless (Wg; a Wnt homologue) proteins are secretory morphogens that act cooperatively to induce formation of the proximodistal axis of appendages. Here we show that either decreased Dpp signalling in the distal wing cells or increased Dpp signalling in the proximal wing cells causes apoptosis. Inappropriate levels of Dpp signalling lead to aberrant morphogenesis in the respective wing zones, and these apoptotic zones are also determined by the strength of the Wg signal. Our results indicate that distortion of the positional information determined by Dpp and Wg signalling gradients leads to activation of the JNK apoptotic pathway, and the consequent induction of cell death thereby maintains normal morphogenesis.     

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-mediated axon guidance via the Drosophila Derailed receptor

In nervous systems with bilateral symmetry, many neurons project axons across the midline to the opposite side. In each segment of the Drosophila embryonic nervous system, axons that display this projection pattern choose one of two distinct tracts: the anterior or posterior commissure. Commissure choice is controlled by Derailed, an atypical receptor tyrosine kinase expressed on axons projecting in the anterior commissure. Here we show that Derailed keeps these axons out of the posterior commissure by acting as a receptor for Wnt5, a member of the Wnt family of secreted signalling molecules. Our results reveal an unexpected role in axon guidance for a Wnt family member, and show that the Derailed receptor is an essential component of Wnt signalling in these guidance events.     

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.     

Mesodermal Wnt2b signalling positively regulates liver specification

Endodermal organs such as the lung, liver and pancreas emerge at precise locations along the primitive gut tube. Although several signalling pathways have been implicated in liver formation, so far no single gene has been identified that exclusively regulates liver specification. In zebrafish, the onset of liver specification is marked by the localized endodermal expression of hhex and prox1 at 22 hours post fertilization. Here we used a screen for mutations affecting endodermal organ morphogenesis to identify a unique phenotype: prometheus (prt) mutants exhibit profound, though transient, defects in liver specification. Positional cloning reveals that prt encodes a previously unidentified Wnt2b homologue. prt/wnt2bb is expressed in restricted bilateral domains in the lateral plate mesoderm directly adjacent to the liver-forming endoderm. Mosaic analyses show the requirement for Prt/Wnt2bb in the lateral plate mesoderm, in agreement with the inductive properties of Wnt signalling. Taken together, these data reveal an unexpected positive role for Wnt signalling in liver specification, and indicate a possible common theme for the localized formation of endodermal organs along the gut tube.