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Unlike LGR4, LGR5 potentiates Wnt-β-catenin signaling without sequestering E3 ligases.

ABSTRACT: LGR4 and LGR5 encode two homologous receptors with critical, yet distinct, roles in organ development and adult stem cell survival. Both receptors are coexpressed in intestinal crypt stem cells, bind to R-spondins (RSPOs) with high affinity, and potentiate Wnt-β-catenin signaling, presumably by the same mechanism: forming RSPO-bridged complexes with the E3 ligases RNF43 and ZNRF3 to inhibit ubiquitylation of Wnt receptors. However, direct evidence for RSPO-bound, full-length LGR5 interacting with these E3 ligases in whole cells has not been reported, and only LGR4 is essential for the self-renewal of intestinal stem cells. Here, we examined the mechanisms of action of LGR4 and LGR5 in parallel using coimmunoprecipitation, proximity ligation, competition binding, and time-resolved FRET assays in whole cells. Full-length LGR4 formed a tight complex with ZNRF3 and RNF43 even without RSPO, whereas LGR5 did not interact with either E3 ligase with or without RSPO. Domain-swapping experiments with LGR4 and LGR5 revealed that the seven-transmembrane domain of LGR4 conferred interaction with the E3 ligases. Native LGR4 and LGR5 existed as dimers on the cell surface, and LGR5 interacted with both FZD and LRP6 of the Wnt signalosome to enhance LRP6 phosphorylation and potentiate Wnt-β-catenin signaling. These findings provide a molecular basis for the weaker activity of LGR5 in the potentiation of Wnt signaling that may underlie the distinct roles of LGR4 and LGR5 in organ development, as well as the self-renewal and fitness of adult stem cells.

SUBMITTER: Park S

PROVIDER: S-EPMC7905944 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of putative stem cell origin that predominantly occurs in children and young adults. Although most patients with localized ES can be cured with intensive therapy, the clinical course is variable and up to one third of patients relapse following initial remission. Unfortunately, little is yet known about the biologic features that distinguish low-risk from high-risk disease or the mechanisms of ES disease progression. Recent reports have suggested that putative cancer stem cells exist in ES and may contribute to an aggressive phenotype. The cell surface receptor leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a somatic stem cell marker that functions as an oncogene in several human cancers, most notably colorectal carcinoma. LGR5 is a receptor for the R-spondin (RSPO) family of ligands and RSPO-mediated activation of LGR5 potentiates Wnt/β-catenin signaling, contributing to stem cell proliferation and self-renewal. Given its presumed stem cell origin, we investigated whether LGR5 contributes to ES pathogenesis. We found that LGR5 is expressed by ES and that its expression is relatively increased in cells and tumors that display a more aggressive phenotype. In particular, LGR5 expression was increased in putative cancer stem cells. We also found that neural crest-derived stem cells express LGR5, raising the possibility that expression of LGR5 may be a feature of ES cells of origin. LGR5-high ES cells showed nuclear localization of β-catenin and robust activation of TCF reporter activity when exposed to Wnt ligand and this was potentiated by RSPO. However, modulation of LGR5 or exposure to RSPO had no impact on proliferation confirming that Wnt/β-catenin signaling in ES cells does not recapitulate signaling in epithelial cells. Together these studies show that the RSPO-LGR5-Wnt-β-catenin axis is present and active in ES and may contribute to tumor pathogenesis.

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Unlike LGR4, LGR5 potentiates Wnt-β-catenin signaling without sequestering E3 ligases.

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