ABSTRACT
R-spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R-spondins bind to the orphan G-protein-coupled receptors LGR4 and LGR5 by their Furin domains. Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R-spondin-mediated Wnt/ß-catenin and Wnt/PCP signalling. R-spondin-triggered ß-catenin signalling requires Clathrin, while Wnt3a-mediated ß-catenin signalling requires Caveolin-mediated endocytosis, suggesting that internalization has a mechanistic role in R-spondin signalling.
Subject(s)
Receptors, G-Protein-Coupled/metabolism , Thrombospondins/metabolism , Wnt Signaling Pathway , Xenopus Proteins/metabolism , Animals , Cell Line , Clathrin/metabolism , Endocytosis , Gene Expression Regulation , HEK293 Cells , Hep G2 Cells , Humans , Mice , Protein Binding , Receptors, G-Protein-Coupled/genetics , Xenopus/genetics , Xenopus/metabolism , Xenopus Proteins/geneticsABSTRACT
The R-Spondin (Rspo) family of secreted Wnt modulators is involved in development and disease and holds therapeutic promise as stem cell growth factors. Despite growing biological importance, their mechanism of action is poorly understood. Here, we show that Rspo3 binds syndecan 4 (Sdc4) and that together they activate Wnt/PCP signaling. In Xenopus embryos, Sdc4 and Rspo3 are essential for two Wnt/PCP-driven processes-gastrulation movements and head cartilage morphogenesis. Rspo3/PCP signaling during gastrulation requires Wnt5a and is transduced via Fz7, Dvl, and JNK. Rspo3 functions by inducing Sdc4-dependent, clathrin-mediated endocytosis. We show that this internalization is essential for PCP signal transduction, suggesting that endocytosis of Wnt-receptor complexes is a key mechanism by which R-spondins promote Wnt signaling.
Subject(s)
Clathrin/metabolism , Endocytosis/physiology , Morphogenesis/physiology , Signal Transduction/physiology , Syndecan-4/metabolism , Thrombospondins/metabolism , Wnt Proteins/metabolism , Xenopus Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cartilage/physiology , Dishevelled Proteins , Gastrulation , HEK293 Cells , Humans , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Syndecan-4/genetics , Thrombospondins/genetics , Wnt Proteins/genetics , Wnt-5a Protein , Xenopus Proteins/genetics , Xenopus laevis/anatomy & histology , Xenopus laevis/embryologyABSTRACT
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.
Subject(s)
Casein Kinase I/metabolism , Cytoplasm/metabolism , Signal Transduction , Wnt Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Axin Protein , Body Patterning , Casein Kinase I/genetics , Cell Line , Cell Membrane/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Humans , Low Density Lipoprotein Receptor-Related Protein-6 , Molecular Sequence Data , Phosphorylation , Phosphothreonine/metabolism , Protein Binding , Receptors, Cell Surface/metabolism , Receptors, LDL/chemistry , Receptors, LDL/genetics , Receptors, LDL/metabolism , Repressor Proteins/metabolism , Substrate Specificity , Xenopus/embryology , Xenopus/genetics , Xenopus Proteins , beta Catenin/metabolismABSTRACT
We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/beta-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-beta pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/beta-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/beta-catenin cascade.
Subject(s)
Muscle Development , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Signal Transduction , Xenopus Proteins/metabolism , Xenopus/embryology , Animals , Blotting, Western , Cell Line , Culture Techniques/methods , Cytoskeletal Proteins , Extracellular Matrix Proteins , Genes, Reporter , HeLa Cells , Humans , Immunohistochemistry , In Situ Hybridization , Intercellular Signaling Peptides and Proteins , Luciferases/metabolism , Microinjections , Molecular Sequence Data , Neoplasm Proteins/chemistry , RNA/biosynthesis , RNA, Small Interfering/metabolism , Recombinant Proteins/metabolism , Trans-Activators , Wnt Proteins , Xenopus/genetics , Xenopus Proteins/chemistry , beta CateninABSTRACT
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.
