RSPO–LGR4 functions via IQGAP1 to potentiate Wnt signaling

Abstract

R-spondins (RSPOs) and their receptor leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) play pleiotropic roles in normal and cancer development as well as the survival of adult stem cells through potentiation of Wnt signaling. Current evidence indicates that RSPO-LGR4 functions to elevate levels of Wnt receptors through direct inhibition of two membrane-bound E3 ligases (RNF43 and ZNRF3), which otherwise ubiquitinate Wnt receptors for degradation. Whether RSPO-LGR4 is coupled to intracellular signaling proteins to regulate Wnt pathways remains unknown. We identified the intracellular scaffold protein IQ motif containing GTPase-activating protein 1 (IQGAP1) as an LGR4-interacting protein that mediates RSPO-LGR4's interaction with the Wnt signalosome. IQGAP1 binds to and modulates the activities of a plethora of signaling molecules, including MAP kinases, Rho GTPases, and components of the Wnt signaling pathways. Interaction of LGR4 with IQGAP1 brings RSPO-LGR4 to the Wnt signaling complex through enhanced IQGAP1-DVL interaction following RSPO stimulation. In this configuration, RSPO-LGR4-IQGAP1 potentiates β-catenin-dependent signaling by promoting MEK1/2-medidated phosphorylation of LRP5/6 as well as β-catenin-independent signaling through regulation of actin dynamics. Overall, these findings reveal that RSPO-LGR4 not only induces the clearance of RNF43/ZNRF3 to increase Wnt receptor levels but also recruits IQGAP1 into the Wnt signaling complex, leading to potent and robust potentiation of both the canonical and noncanonical pathways of Wnt signaling.cell signaling | receptor activation | adhesion | migration T he four R-spondins (RSPO1-4) and three related leucinerich repeat-containing G-protein coupled receptors, LGR4, LGR5, and LGR6 (LGR4-6), constitute a ligand-receptor system that plays critical roles in development, stem cell survival, and oncogenesis (1-6). Mutations of RSPO1 and RSPO4 affect sex development and nail formation (7-9), respectively, and haplotype insufficiency of LGR4 in humans is associated with several diseases and other traits (10). In the mouse, knockouts of LGR4 or RSPOs are presented with severe developmental abnormalities, including neonatal/embryonic lethality accompanied by hypoplasia and defects in tubule elongation and branching in the kidney, lung, mammary gland, and testis (11-16).LGR5 and LGR6 are markers of adult stem cells in the intestine and other select solid tissues (2).LGR4 is frequently coexpressed with LGR5 or LGR6 and is required for the survival of crypt stem cells in the gut (4, 17). Recurrent, gain-of-expression gene fusions of RSPO2 (to EIF3E) and RSPO3 (to PTPRK) occur in a subset of colorectal cancers (18). In mouse mammary tumor virus-induced mouse models of breast and colon cancer, RSPO2 and RSPO3 were identified as two of the most commonly activated alleles (19-21). Ectopic expression of RSPO2/3 in mouse mammary epithelial cells led to an increase in invasiveness in vitro and in tumor formation and metastasis in vivo (20,22). LGR4 is up-reg...

Targeting Wnts at the Source—New Mechanisms, New Biomarkers, New Drugs

“…This complex results in the inhibition and membrane clearance of these E3 ubiquitin ligases, leading to increased Frizzled and LRP5/6 proteins on the cell surface (43,54). The LGR RSPO complex may also enhances b-catenin-dependent signaling by promoting MEK1/2-mediated phosphorylation of LRP5/6 and b-catenin-independent signaling through regulation of actin dynamics (55).…”

R-spondin 2 promotes proliferation and migration via the Wnt/β-catenin pathway in human hepatocellular carcinoma

“…A number of previous studies have indicated that RSPOs may potentiate Wnt signaling via stimulation of the leucine-rich repeat-containing G-protein coupled receptors (LGR) 4, LGR5 and LGR6 (16)(17)(18). Wnt proteins, comprising a large family of extracellular, lipid-modified glycoproteins, are crucial for embryonic development and cell proliferation, regulation, differentiation, survival and tissue homeostasis in adults (19,20).…”

“…It has been revealed that canonical Wnt/β-catenin signaling serves an important role in numerous cancer types, including lung, breast, brain, colorectal and liver tumors (21)(22)(23). Interaction between RSPOs and LGR4, as well as the intracellular signaling proteins, promotes phosphorylation of LRP5/6, stabilizes β-catenin expression and promotes its transcriptional activity (18). Activation of Wnt/β-catenin results in the increased expression level of its target genes, including cyclin D1 and c-Myc, which are important for driving tumorigenesis in numerous types of cancer (24).…”

CAF-like state in primary skin fibroblasts with constitutional BRCA1 epimutation sheds new light on tumor suppressor deficiency-related changes in healthy tissue

“…In contrast, a reduction of RSPO3 expression, which is the consequence of the mosaic RSPO3 deletion in our proband's cells, 9 was already linked to the opposite of our findings, namely a reduction in cell proliferation and migration, in RSPO3 knockdown cancer cells. 47,48 The expression of NREP in BRCA1 mosMe was unaffected by the deletion 9 and no connection of this gene to cancerogenesis is known to date. It is beyond the scope of our present study, but remains to be determined, whether the deletions of RSPO3 and NREP co-exist with the BRCA1 epimutation in the same cells of the BRCA1 mosMe fibroblasts.…”