The Wnt/β-catenin signaling system plays essential roles in embryonic development and in the self-renewal and maintenance of adult stem cells. R-spondins (RSPOs) are a group of secreted proteins that enhance Wnt/β-catenin signaling and have pleiotropic functions in development and stem cell growth.LGR5, an orphan receptor of the G protein-coupled receptor (GPCR) superfamily, is specifically expressed in stem cells of the intestinal crypt and hair follicle. Knockout of LGR5 in the mouse results in neonatal lethality.LGR4, a receptor closely related to LGR5, also has essential roles in development, as its knockout leads to reduced viability and retarded growth. Overexpression of both receptors has been reported in several types of cancer. Here we demonstrate that LGR4 and LGR5 bind the R-spondins with high affinity and mediate the potentiation of Wnt/β-catenin signaling by enhancing Wntinduced LRP6 phosphorylation. Interestingly, neither receptor is coupled to heterotrimeric G proteins or to β-arrestin when stimulated by the R-spondins, indicating a unique mechanism of action. The findings provide a basis for stem cell-specific effects of Wnt/ β-catenin signaling and for the broad range of functions LGR4, LGR5, and the R-spondins have in normal and malignant growth.cell signaling | stem cell control | gastrointestinal growth | colon cancer A dult stem cells are specialized, undifferentiated cells that are capable of self-renewal and of generating all cell types of the tissue in which they reside. They are generally identified and traced by one marker, or a set of markers, that is specifically expressed in these cells. These markers are likely to constitute some of the important processes that ultimately define stem cells, and thus hold key information to the understanding of stem cell biology. Nevertheless, for the majority of these markers, the functions remain unknown. LGR5 (leucine-rich repeat containing G protein-coupled receptor 5) has been identified and validated as a marker of the crypt basal columnar stem cells along the gastrointestinal tract and of the bulge stem cells in the hair follicle (1-2). This receptor, also known as HG38, GPR49, and FEX, was first reported by us as an orphan receptor (HG38) with homology to the glycoprotein hormone receptor subfamily of the class A rhodopsin-like seven transmembrane (7-TM) domain, G proteincoupled receptors (GPCRs) (3-5).LGR5 is closely related to two other receptors, LGR4 and LGR6 (∼50% identity between each other), and together the trio (LGR4-6) forms a structurally distinct group of 7-TM receptors that have a substantially large N-terminal extracellular domain (ECD) composed of 17 leucinerich repeats (6). Knockout of LGR5 in the mouse leads to total neonatal lethality accompanied by ankyloglossia and gastrointestinal distension (7). Loss of LGR4 results in reduced viability with developmental defects in many organs, including the kidney (8, 9), testis (10, 11), eye (12), bone (13), skin (14), and gall bladder (15). LGR4 and LGR5 are also overexpressed in sev...

Cachexia, characterized by muscle wasting, is a major contributor to cancer-related mortality. However, the key cachexins that mediate cancer-induced muscle wasting remain elusive. Here, we show that tumor-released extracellular Hsp70 and Hsp90 are responsible for tumor’s capacity to induce muscle wasting. We detected high-level constitutive release of Hsp70 and Hsp90 associated with extracellular vesicles (EVs) from diverse cachexia-inducing tumor cells, resulting in elevated serum levels in mice. Neutralizing extracellular Hsp70/90 or silencing Hsp70/90 expression in tumor cells abrogates tumor-induced muscle catabolism and wasting in cultured myotubes and in mice. Conversely, administration of recombinant Hsp70 and Hsp90 recapitulates the catabolic effects of tumor. In addition, tumor-released Hsp70/90-expressing EVs are necessary and sufficient for tumor-induced muscle wasting. Further, Hsp70 and Hsp90 induce muscle catabolism by activating TLR4, and are responsible for elevation of circulating cytokines. These findings identify tumor-released circulating Hsp70 and Hsp90 as key cachexins causing muscle wasting in mice.

LGR5, a seven-transmembrane domain receptor of the rhodopsin family, is a Wnt target gene and a bona fide marker of adult stem cells in the gastrointestinal tract and hair follicle bulge. Recently, we and others demonstrated that LGR5 and its homologues function as receptors of the R-spondin family of stem cell factors to potentiate Wnt/␤-catenin signaling. However, the mechanism of how LGR5 enhances the signaling output remains unclear. Here we report that following costimulation with the ligands R-spondin1 and Wnt3a, LGR5 interacts and forms a supercomplex with the Wnt coreceptors LRP6 and Fzd5 which is rapidly internalized and then degraded. Internalization of LGR5 is mediated through a dynamin-and clathrin-dependent pathway. Inhibition of this endocytic process has no effect on LGR5 signaling. Deletion of the C-terminal tail of LGR5 maintains its ability to interact with LRP6, yet this LGR5 mutant exhibits increased signaling activity and a decreased rate of endocytosis in response to R-spondin1 compared to the wild-type receptor. This study provides direct evidence that LGR5 becomes part of the Wnt signaling complex at the membrane level to enhance Wnt/␤-catenin signaling. However, internalization of LGR5 does not appear to be essential for potentiating the canonical Wnt signaling pathway.