RESUMO
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.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismo , Via de Sinalização Wnt , Proteínas de Xenopus/metabolismo , Animais , Linhagem Celular , Clatrina/metabolismo , Endocitose , Regulação da Expressão Gênica , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMO
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.
Assuntos
Clatrina/metabolismo , Endocitose/fisiologia , Morfogênese/fisiologia , Transdução de Sinais/fisiologia , Sindecana-4/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cartilagem/fisiologia , Proteínas Desgrenhadas , Gastrulação , Células HEK293 , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sindecana-4/genética , Trombospondinas/genética , Proteínas Wnt/genética , Proteína Wnt-5a , Proteínas de Xenopus/genética , Xenopus laevis/anatomia & histologia , Xenopus laevis/embriologiaRESUMO
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.
Assuntos
Caseína Quinase I/metabolismo , Citoplasma/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteína Axina , Padronização Corporal , Caseína Quinase I/genética , Linhagem Celular , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Dados de Sequência Molecular , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Receptores de LDL/química , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Repressoras/metabolismo , Especificidade por Substrato , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus , beta Catenina/metabolismoRESUMO
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.
Assuntos
Desenvolvimento Muscular , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Animais , Western Blotting , Linhagem Celular , Técnicas de Cultura/métodos , Proteínas do Citoesqueleto , Proteínas da Matriz Extracelular , Genes Reporter , Células HeLa , Humanos , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular , Luciferases/metabolismo , Microinjeções , Dados de Sequência Molecular , Proteínas de Neoplasias/química , RNA/biossíntese , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/metabolismo , Transativadores , Proteínas Wnt , Xenopus/genética , Proteínas de Xenopus/química , beta CateninaRESUMO
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.
