Wnt/?-catenin signaling plays an important role in embryonic development and adult tissue homeostasis. When Wnt ligands bind to the receptor complex, LRP5/6 coreceptors are activated by phosphorylation and concomitantly endocytosed. In vertebrates, Wnt ligands induce caveolin-dependent endocytosis of LRP6 to relay signal downstream, whereas antagonists such as Dickkopf promote clathrin-dependent endocytosis, leading to inhibition. However, little is known about how LRP6 is directed to different internalization mechanisms, and how caveolin-dependent endocytosis is mediated. In an RNAi screen, we identified the Rab GTPase RAB8B as being required for Wnt/?-catenin signaling. RAB8B depletion reduces LRP6 activity, ?-catenin accumulation, and induction of Wnt target genes, whereas RAB8B overexpression promotes LRP6 activity and internalization and rescues inhibition of caveolar endocytosis. In Xenopus laevis and Danio rerio, RAB8B morphants show lower Wnt activity during embryonic development. Our results implicate RAB8B as an essential evolutionary conserved component of Wnt/?-catenin signaling through regulation of LRP6 activity and endocytosis.
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/?-catenin and Wnt/PCP signalling. R-spondin-triggered ?-catenin signalling requires Clathrin, while Wnt3a-mediated ?-catenin signalling requires Caveolin-mediated endocytosis, suggesting that internalization has a mechanistic role in R-spondin signalling.
Wnt/?-catenin signaling is fundamental in embryogenesis and tissue homeostasis in metazoans. Upon Wnt stimulation, cognate coreceptors LRP5 and LRP6 ([LRP5/6] low-density lipoprotein receptor-related proteins 5 and 6) are activated via phosphorylation at key residues. Although several kinases have been implicated, the LRP5/6 activation mechanism remains unclear. Here, we report that transmembrane protein 198 (TMEM198), a previously uncharacterized seven-transmembrane protein, is able to specifically activate LRP6 in transducing Wnt signaling. TMEM198 associates with LRP6 and recruits casein kinase family proteins, via the cytoplasmic domain, to phosphorylate key residues important for LRP6 activation. In mammalian cells, TMEM198 is required for Wnt signaling and casein kinase 1-induced LRP6 phosphorylation. During Xenopus embryogenesis, maternal and zygotic tmem198 mRNAs are widely distributed in the ectoderm and mesoderm. TMEM198 is required for Wnt-mediated neural crest formation, antero-posterior patterning, and particularly engrailed-2 expression in Xenopus embryos. Thus, our results identified TMEM198 as a membrane scaffold protein that promotes LRP6 phosphorylation and Wnt signaling activation.
Colorectal cancer (CRC) is a complex disease related to environmental and genetic risk factors. Several studies have shown that susceptibility to complex diseases can be mediated by ancestral alleles. Using RNAi screening, CTNNBL1 was identified as a putative regulator of the Wnt signaling pathway, which plays a key role in colorectal carcinogenesis. Recently, single nucleotide polymorphisms (SNPs) in CTNNBL1 have been associated with obesity, a known risk factor for CRC. We investigated whether genetic variation in CTNNBL1 affects susceptibility to CRC and tested for signals of recent selection. We applied a tagging SNP approach that cover all known common variation in CTNNBL1 (allele frequency >5%; r(2)>0.8). A case-control study was carried out using two well-characterized study populations: a hospital-based Czech population composed of 751 sporadic cases and 755 controls and a family/early onset-based German population (697 cases and 644 controls). Genotyping was performed using allele specific PCR based TaqMan® assays (Applied Biosystems, Weiterstadt, Germany). In the Czech cohort, containing sporadic cases, the ancestral alleles of three SNPs showed evidence of association with CRC: rs2344481 (OR 1.44, 95%CI 1.06-1.95, dominant model), rs2281148 (OR 0.59, 95%CI 0.36-0.96, dominant model) and rs2235460 (OR 1.38, 95%CI 1.01-1.89, AA vs. GG). The associations were less prominent in the family/early onset-based German cohort. Data derived from several databases and statistical tests consistently pointed to a likely shaping of CTNNBL1 by positive selection. Further studies are needed to identify the actual function of CTNNBL1 and to validate the association results in other populations.
Wnt/beta-catenin signaling is important in stem cell biology, embryonic development, and disease, including cancer. However, the mechanism of Wnt signal transmission, notably how the receptors are activated, remains incompletely understood. We found that the prorenin receptor (PRR) is a component of the Wnt receptor complex. PRR functions in a renin-independent manner as an adaptor between Wnt receptors and the vacuolar H+-adenosine triphosphatase (V-ATPase) complex. Moreover, PRR and V-ATPase were required to mediate Wnt signaling during antero-posterior patterning of Xenopus early central nervous system development. The results reveal an unsuspected role for the prorenin receptor, V-ATPase activity, and acidification during Wnt/beta-catenin signaling.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.