{"gene":"RSPO2","run_date":"2026-04-28T20:42:06","timeline":{"discoveries":[{"year":2014,"finding":"RSPO2 interacts with LGR5 to stabilize membrane-associated ZNRF3, thereby inhibiting Wnt/β-catenin signaling in a LGR5-dependent manner in colorectal cancer cells","method":"Co-immunoprecipitation, overexpression/knockdown with Wnt reporter assays, epistasis with LGR5 depletion","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP and epistasis in multiple CRC cell lines, replicated functional phenotype","pmids":["24476626"],"is_preprint":false},{"year":2017,"finding":"RSPO2 physically interacts with Fzd7 and promotes ZNRF3-mediated ubiquitination and degradation of cell-surface Fzd7, thereby suppressing PKC/ERK noncanonical Wnt signaling and antagonizing Wnt5a-driven cell migration","method":"Co-immunoprecipitation, ubiquitination assay, cell migration/invasion assays, epistasis with ZNRF3 and Fzd7","journal":"Cancer letters","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, ubiquitination assay, and functional epistasis with defined phenotypic readout","pmids":["28600110"],"is_preprint":false},{"year":2022,"finding":"RSPO2 and RANKL signal through LGR4 to promote osteoclastic premetastatic niche formation; RSPO2/RANKL-LGR4 modulates DKK1 via Gαq and β-catenin signaling, and DKK1 facilitates osteoclast precursor recruitment by suppressing LRP5 and upregulating Rnasek","method":"GPCR ligand/agonist screening, Co-IP, knockdown/overexpression, decoy receptor (soluble LGR4 ECD) in mouse bone metastasis model","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — unbiased screening plus Co-IP, in vivo rescue with decoy receptor, multiple orthogonal methods","pmids":["34847079"],"is_preprint":false},{"year":2022,"finding":"RSPO2 maintains AML self-renewal by inhibiting BMP receptor signaling in a WNT-independent manner; autocrine RSPO2 prevents differentiation of normal hematopoietic stem cells and primary AML cells","method":"RSPO2 knockdown/overexpression, BMP signaling assays, AML xenograft mouse models with survival readout","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function with defined cellular phenotype, in vivo xenograft validation, multiple orthogonal assays","pmids":["34407399"],"is_preprint":false},{"year":2022,"finding":"RSPO2 is secreted by a subset of CD142+ adipogenic progenitor cells and inhibits maturation of early adipogenic progenitors through the receptor LGR4, leading to adipose tissue hypertrophy and insulin resistance","method":"Single-cell RNA-sequencing, loss-of-function mouse models, receptor identification (Lgr4), metabolic phenotyping","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 2 — scRNA-seq combined with in vivo mouse models and receptor epistasis, multiple orthogonal methods","pmids":["35027768"],"is_preprint":false},{"year":2020,"finding":"Rspo2 inhibits FGF signaling upstream of Mek1 via its thrombospondin type 1 (TSP1) domain, downregulating FGF target genes tbxt/brachyury and cdx4 during Xenopus mesoderm formation; this is distinct from its Furin-like domain-mediated Wnt activity","method":"Xenopus ectoderm explant elongation assay, ERK1 phosphorylation assay, domain deletion/mutagenesis, morpholino depletion","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1 — domain mutagenesis with in vitro biochemical assay and in vivo phenotypic rescue, multiple orthogonal methods","pmids":["32366679"],"is_preprint":false},{"year":2021,"finding":"Rspo2 inhibits TCF3/TCF7L1 phosphorylation to antagonize Wnt signaling during Xenopus anteroposterior axis specification, independently of binding to RNF43/ZNRF3 and LGR4/5","method":"Xenopus gain/loss-of-function, domain binding assays (RNF43/ZNRF3 and LGR4/5 binding-deficient mutants), TCF3 phosphorylation assays, epistasis with TCF3 depletion","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 — domain mutagenesis with phosphorylation biochemistry and epistasis rescue, multiple orthogonal approaches","pmids":["34183732"],"is_preprint":false},{"year":2018,"finding":"Rspo2 promotes acetylcholine receptor (AChR) clustering and neuromuscular junction (NMJ) formation via its receptor Lgr5; motor neuron-derived Rspo2 plays the major role in AChR clustering while muscle-derived Rspo2 normalizes ultrastructural NMJ features","method":"Tissue-specific rescue of Rspo2-/- mice (SMN-specific and muscle-specific expression), immunofluorescence, electron microscopy, gene expression analysis","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 — genetic rescue with tissue-specific expression in knockout mice, ultrastructural and functional readouts","pmids":["30206360"],"is_preprint":false},{"year":2022,"finding":"RSPO2 promotes ovarian cancer growth and metastasis through dual receptor signaling: (1) RSPO2-LGR4 interaction prevents endocytic degradation of LGR4 and promotes LGR4-mediated translocation of Src to the plasma membrane; (2) RSPO2 directly binds integrin β3 as a ligand, enhancing integrin stability; both mechanisms potentiate FAK/Src autophosphorylation","method":"Co-immunoprecipitation, proximity ligation assay, receptor pulldown, phosphorylation assays, knockdown/overexpression","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and functional phosphorylation assays in single study with multiple orthogonal methods","pmids":["36217544"],"is_preprint":false},{"year":2020,"finding":"A HH-Foxf1-Rspo2 signaling axis governs tracheal cartilage development: HH/Gli signaling maintains Foxf1 expression in splanchnic mesoderm, which in turn supports Rspo2 expression, and Rspo2 potentiates Wnt signaling required for Sox9+ chondrocyte progenitor specification","method":"Multiple HH/Gli mouse mutants, lineage tracing, Foxf1 conditional knockout, Rspo2 expression analysis","journal":"Disease models & mechanisms","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with multiple mouse mutant lines and lineage tracing, independently validated pathway placement","pmids":["33328171"],"is_preprint":false},{"year":2020,"finding":"Wnt9b and RSPO2 cooperate to activate canonical Wnt/β-catenin signaling during mouse facial morphogenesis; compound Wnt9b;Rspo2 double knockout mice display more severe facial defects than either single mutant","method":"Compound mouse knockout, ex vivo facial explants, gene expression analysis","journal":"Frontiers in cell and developmental biology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with compound knockout and rescue experiments in vivo","pmids":["32457899"],"is_preprint":false},{"year":2024,"finding":"KAT6A regulates Rspo2 transcription in hippocampal CA3 neurons via histone acetylation; KAT6A deficiency reduces RSPO2-Wnt signaling leading to impaired synaptic plasticity and memory deficits; restoring RSPO2 in CA3 rescues Wnt signaling and cognitive deficits in Kat6a mutant mice","method":"KAT6A conditional knockout, RSPO2 conditional knockout in excitatory neurons, AAV-mediated RSPO2 re-expression in CA3, Wnt signaling assays, behavioral tests","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 — genetic loss-of-function plus rescue with defined molecular (Wnt signaling) and behavioral phenotypes","pmids":["38758792"],"is_preprint":false},{"year":2022,"finding":"RSPO2 overexpression suppresses ectopic ossification in a mouse Achilles tendon puncture model by suppressing chondrogenic differentiation; RSPO2 is induced by NF-κB signaling in response to inflammatory stimulation and mechanical loading","method":"RSPO2 overexpression in mouse tendon puncture model, chondrogenic differentiation assays in human ligament cells, NF-κB inhibitor experiments","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo overexpression with defined phenotypic readout and mechanistic follow-up, single study","pmids":["35984875"],"is_preprint":false},{"year":2021,"finding":"miR-181a targets RSPO2 mRNA in mesenchymal stromal cells during chondrogenesis, leading to reduced WNT signaling and increased BMP signaling (phospho-SMAD1/5/9 and SOX9 accumulation), establishing RSPO2 as a coordinator of WNT-BMP signaling crosstalk","method":"miR reporter assay (luciferase), miR-181a mimic/inhibitor transfection, Western blot for SMAD1/5/9 phosphorylation and SOX9","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — luciferase reporter plus downstream signaling validation with multiple readouts in single study","pmids":["34778258"],"is_preprint":false},{"year":2016,"finding":"RSPO2 inhibits PPAR-γ nuclear translocation and reduces PPAR-γ binding to the CD36 promoter (assessed by ChIP), thereby suppressing CD36 expression, reducing oxLDL uptake, and inhibiting macrophage apoptosis","method":"Flow cytometry, Western blot, chromatin immunoprecipitation (ChIP), Rspo2 knockdown/overexpression in macrophages","journal":"Molecular medicine reports","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus functional assays, single study with multiple orthogonal methods","pmids":["27571704"],"is_preprint":false},{"year":2024,"finding":"Calcitriol induces Rspo2 expression via vitamin D receptor (VDR) binding to a region ~15 kb upstream of Rspo2; deletion of this VDR-binding site by CRISPR/Cas9 abolishes calcitriol-mediated Rspo2 induction and MuSK phosphorylation at the neuromuscular junction","method":"ChIP-seq of VDR, CRISPR/Cas9 deletion of VDR-binding site, RNA-seq, MuSK phosphorylation assay, Chrne KO mouse model","journal":"Neurotherapeutics","confidence":"High","confidence_rationale":"Tier 1 — ChIP-seq plus CRISPR functional deletion with biochemical (MuSK phosphorylation) and in vivo phenotypic readouts","pmids":["38233267"],"is_preprint":false},{"year":2021,"finding":"RSPO2 knockdown in oocytes disrupts granulosa cell development and oocyte competence; RSPO2 (expressed in oocytes) physically interacts with GDF9:BMP15 heterodimers and its effects involve crosstalk between CTNNB1- and SMAD2-dependent pathways in granulosa cells","method":"Conditional oocyte-specific Rspo2 knockout, Co-immunoprecipitation (RSPO2 with GDF9:BMP15), transcriptomic analysis, pathway inhibitor experiments","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 — conditional knockout plus Co-IP with defined downstream pathway analysis, single study","pmids":["40492505"],"is_preprint":false},{"year":2021,"finding":"RSPO2 silence in nasopharyngeal carcinoma increases ZNRF3 expression and reduces Hedgehog/Gli1 signaling; ZNRF3 knockdown or Gli1 overexpression reverses the anti-tumor effects of RSPO2 depletion, placing RSPO2 upstream of ZNRF3/Hedgehog-Gli1","method":"siRNA knockdown, epistasis rescue by ZNRF3 knockdown and Gli1 overexpression, in vivo xenograft","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis rescue experiments with defined pathway placement, single study","pmids":["34273374"],"is_preprint":false},{"year":2025,"finding":"PALMD interacts with RSPO2 and facilitates its secretion, enabling paracrine activation of Wnt/β-catenin signaling to promote colorectal cancer stemness and tumor growth; pharmacologic or antibody blockade of RSPO2 attenuates PALMD-mediated Wnt activation","method":"Co-immunoprecipitation (PALMD-RSPO2), gene knockdown/overexpression, Wnt reporter assay, organoid models, in vivo xenograft","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional epistasis with rescue, multiple orthogonal systems; preprint only","pmids":[],"is_preprint":true},{"year":2025,"finding":"A mutant RSPO2 furin domain retains high-affinity binding to LGR4/5/6 but lacks Wnt/β-catenin signaling activity; this property was used to create drug-conjugated peptibodies that deliver cytotoxins specifically to LGR4/5/6-positive cancer cells","method":"Binding affinity assays, TOPFLASH Wnt reporter assay, mutagenesis of furin domain, in vitro cytotoxicity, in vivo xenograft","journal":"Journal of medicinal chemistry","confidence":"High","confidence_rationale":"Tier 1 — furin domain mutagenesis with binding and signaling assays, validated in vitro and in vivo","pmids":["41954225"],"is_preprint":false},{"year":2017,"finding":"RSPO2 activates the β-catenin pathway through GPR48/LGR4, inducing phosphorylation of ERK, LRP6, and GSK3β (serine 9); downregulation of GPR48/LGR4 decreases β-catenin pathway activity in thyroid cancer cells","method":"Exogenous RSPO2 treatment, LGR4 knockdown, Western blot for phospho-ERK/LRP6/GSK3β and β-catenin","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — receptor-ligand gain/loss-of-function with downstream phosphorylation readouts, single study","pmids":["29383135"],"is_preprint":false},{"year":2021,"finding":"DNA methylation of RSPO2 promoter at -758/-749 and -563/-553 CpG regions controls RSPO2 transcription by regulating occupancy of transcription factor E2F1; hypomethylation facilitates E2F1 binding and increases RSPO2 expression in granulosa cells","method":"Bisulfite sequencing, DNMT1 knockdown, DNMT inhibitor treatment, ChIP for E2F1 at RSPO2 promoter, luciferase reporter assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus luciferase reporter with methylation manipulation, single study","pmids":["34175894"],"is_preprint":false}],"current_model":"RSPO2 is a secreted protein that potentiates Wnt/β-catenin signaling primarily by binding LGR4/5/6 receptors and ZNRF3/RNF43 E3 ubiquitin ligases (via its furin-like domains) to prevent Frizzled degradation, but also acts context-dependently as a Wnt antagonist (by inhibiting TCF3 phosphorylation), an FGF antagonist (via its TSP1 domain), and a BMP antagonist to support stem cell self-renewal, while additionally engaging integrin β3 and LGR4 to activate FAK/Src signaling, and its transcription is regulated by VDR, C/EBPβ, E2F1, and promoter methylation."},"narrative":{"teleology":[{"year":2014,"claim":"Resolving whether RSPO2 is solely a Wnt agonist, this study revealed that RSPO2 can stabilize ZNRF3 at the membrane via LGR5 to inhibit rather than activate Wnt/β-catenin signaling in colorectal cancer cells, establishing context-dependent duality.","evidence":"Reciprocal Co-IP and Wnt reporter epistasis with LGR5 knockdown in multiple CRC cell lines","pmids":["24476626"],"confidence":"High","gaps":["Whether this inhibitory mode operates in normal colonic epithelium in vivo","Structural basis for how LGR5 engagement switches RSPO2 from agonist to antagonist"]},{"year":2016,"claim":"Extended RSPO2's functional reach beyond canonical Wnt by showing it inhibits PPARγ nuclear translocation and PPARγ-dependent CD36 transcription, reducing oxidized LDL uptake in macrophages.","evidence":"ChIP for PPARγ at CD36 promoter plus knockdown/overexpression in macrophages","pmids":["27571704"],"confidence":"Medium","gaps":["Which RSPO2 receptor mediates PPARγ inhibition","Whether this is Wnt/β-catenin-dependent or represents a distinct signaling branch"]},{"year":2017,"claim":"Demonstrated that RSPO2 engages noncanonical Wnt signaling by directly binding Fzd7 and promoting its ZNRF3-mediated ubiquitination and degradation, thereby suppressing PKC/ERK-dependent cell migration.","evidence":"Reciprocal Co-IP, ubiquitination assays, and epistasis with ZNRF3/Fzd7 in cancer cell migration assays","pmids":["28600110"],"confidence":"High","gaps":["Whether other Frizzled family members are similarly targeted","In vivo relevance of RSPO2–Fzd7 antagonism in tumor metastasis"]},{"year":2017,"claim":"Established the RSPO2–LGR4 axis in canonical Wnt activation by showing RSPO2 signals through GPR48/LGR4 to phosphorylate ERK, LRP6, and GSK3β(Ser9) in thyroid cancer cells.","evidence":"Exogenous RSPO2 treatment with LGR4 knockdown and phospho-Western blots","pmids":["29383135"],"confidence":"Medium","gaps":["Whether LGR4 and LGR5 mediate opposing RSPO2 outputs within the same cell","Contribution of ZNRF3/RNF43 in this thyroid cancer context"]},{"year":2018,"claim":"Identified a developmental role for RSPO2 at the neuromuscular junction, showing motor neuron–derived Rspo2 signals via Lgr5 to promote AChR clustering and proper NMJ ultrastructure.","evidence":"Tissue-specific genetic rescue of Rspo2−/− mice with immunofluorescence and electron microscopy","pmids":["30206360"],"confidence":"High","gaps":["Whether Rspo2-Lgr5 at the NMJ acts through canonical Wnt or a distinct pathway","Role of muscle-derived Rspo2 beyond ultrastructural normalization"]},{"year":2020,"claim":"Revealed a Wnt-independent function: the TSP1 domain of Rspo2 inhibits FGF/Mek1/ERK signaling to regulate mesoderm patterning, mechanistically separating this from the furin domain–mediated Wnt activity.","evidence":"Domain deletion/mutagenesis in Xenopus explants with ERK phosphorylation assays","pmids":["32366679"],"confidence":"High","gaps":["Identity of the receptor for the TSP1 domain","Whether TSP1-mediated FGF antagonism operates in mammalian systems"]},{"year":2020,"claim":"Placed Rspo2 in a HH–Foxf1 transcriptional hierarchy governing tracheal cartilage, and showed genetic cooperation between Rspo2 and Wnt9b for facial morphogenesis via compound knockout synergy.","evidence":"Multiple HH/Gli and Foxf1 conditional mouse mutants with lineage tracing; Wnt9b;Rspo2 compound knockout mice","pmids":["33328171","32457899"],"confidence":"High","gaps":["Whether RSPO2 acts directly on Sox9+ chondrocyte progenitors or via an intermediate cell type","Relative contributions of RSPO2 versus other R-spondins in craniofacial development"]},{"year":2021,"claim":"Uncovered a second Wnt-antagonizing mechanism: Rspo2 inhibits TCF3 phosphorylation independently of RNF43/ZNRF3 and LGR4/5 binding, establishing a distinct mode of Wnt antagonism during anteroposterior axis specification.","evidence":"Binding-deficient RSPO2 mutants with TCF3 phosphorylation assays and epistasis in Xenopus","pmids":["34183732"],"confidence":"High","gaps":["The kinase whose activity on TCF3 is blocked by RSPO2","Whether this TCF3-dependent mechanism operates in mammals"]},{"year":2021,"claim":"Defined transcriptional regulation of RSPO2: E2F1 occupancy at the RSPO2 promoter is controlled by DNA methylation at specific CpG sites, providing an epigenetic on/off switch for RSPO2 expression in granulosa cells.","evidence":"Bisulfite sequencing, DNMT1 knockdown, ChIP for E2F1, luciferase reporter","pmids":["34175894"],"confidence":"Medium","gaps":["Whether this methylation-dependent regulation applies outside the ovarian context","Upstream signals that trigger RSPO2 promoter demethylation"]},{"year":2021,"claim":"Established a Wnt-independent role for RSPO2 in stem cell biology by showing that autocrine RSPO2 inhibits BMP receptor signaling to maintain AML self-renewal, and separately showed RSPO2 in oocytes interacts with GDF9:BMP15 heterodimers to coordinate Wnt and SMAD2 signaling in granulosa cells.","evidence":"RSPO2 knockdown/overexpression with BMP signaling assays and AML xenograft survival; oocyte-specific conditional knockout with Co-IP of RSPO2–GDF9:BMP15","pmids":["34407399","40492505"],"confidence":"High","gaps":["Molecular mechanism by which RSPO2 inhibits BMP receptors","Whether BMP antagonism requires LGR receptors or is receptor-independent"]},{"year":2022,"claim":"Identified RSPO2 as a paracrine inhibitor of adipogenic progenitor maturation via LGR4, linking it to adipose tissue remodeling and systemic insulin resistance.","evidence":"scRNA-seq of adipose tissue, Rspo2 loss-of-function mouse models with metabolic phenotyping and Lgr4 epistasis","pmids":["35027768"],"confidence":"High","gaps":["Whether RSPO2-LGR4 acts through Wnt, BMP, or another downstream pathway in adipocyte progenitors","Therapeutic potential of blocking RSPO2 for metabolic disease"]},{"year":2022,"claim":"Expanded RSPO2's receptor repertoire beyond LGR/ZNRF3 by showing it directly binds integrin β3 and stabilizes it, while simultaneously preventing LGR4 endocytic degradation to activate FAK/Src signaling in ovarian cancer.","evidence":"Co-IP, proximity ligation assay, receptor pulldown, and phosphorylation assays in ovarian cancer cells","pmids":["36217544"],"confidence":"Medium","gaps":["Structural basis for RSPO2–integrin β3 binding","Whether integrin β3 engagement is a general feature of RSPO2 or specific to ovarian cancer"]},{"year":2022,"claim":"Demonstrated that RSPO2/RANKL co-signal through LGR4 via Gαq and β-catenin to induce DKK1 and promote osteoclastic premetastatic niche formation, revealing a cooperative ligand-receptor mechanism.","evidence":"GPCR agonist screening, Co-IP, soluble LGR4 decoy receptor in mouse bone metastasis model","pmids":["34847079"],"confidence":"High","gaps":["Whether RSPO2 and RANKL form a ternary complex on LGR4 or signal sequentially","Relevance to human bone metastasis"]},{"year":2024,"claim":"Identified VDR as a direct transcriptional regulator of Rspo2 via a distal enhancer ~15 kb upstream; deletion of this site abolished calcitriol-mediated Rspo2 induction and downstream MuSK phosphorylation at the NMJ.","evidence":"ChIP-seq of VDR, CRISPR/Cas9 enhancer deletion, MuSK phosphorylation assay","pmids":["38233267"],"confidence":"High","gaps":["Whether VDR-dependent RSPO2 regulation operates in tissues beyond skeletal muscle","Therapeutic implications for NMJ diseases"]},{"year":2024,"claim":"Placed RSPO2 downstream of the chromatin modifier KAT6A in hippocampal neurons, showing that KAT6A-dependent histone acetylation drives Rspo2 transcription to sustain Wnt signaling required for synaptic plasticity and memory.","evidence":"KAT6A and RSPO2 conditional knockouts in excitatory neurons, AAV-mediated RSPO2 rescue in CA3, behavioral tests","pmids":["38758792"],"confidence":"High","gaps":["Whether RSPO2 acts through LGR4 or LGR5 in hippocampal neurons","Whether other R-spondins compensate when RSPO2 is lost in the brain"]},{"year":2025,"claim":"Structure-function dissection showed that a mutant furin domain retains LGR4/5/6 binding but loses Wnt-activating capacity, enabling design of signaling-dead RSPO2 peptibodies for targeted drug delivery to LGR-positive cancers.","evidence":"Furin domain mutagenesis with binding affinity and TOPFLASH assays, in vitro cytotoxicity, xenograft efficacy","pmids":["41954225"],"confidence":"High","gaps":["Crystal structure of the signaling-dead mutant bound to LGR","Long-term in vivo safety and pharmacokinetics"]},{"year":null,"claim":"Key unresolved questions include the structural basis for how RSPO2 switches between Wnt agonist and antagonist outputs depending on LGR receptor identity, the receptor mediating TSP1-domain FGF antagonism, and the direct mechanism of BMP receptor inhibition.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal structure of RSPO2 in complex with LGR5 versus LGR4 to explain opposing signaling outcomes","Identity of the TSP1 domain receptor remains unknown","Mechanism by which RSPO2 inhibits BMP receptor signaling is undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,2,4,5,7,8,19]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,3,5,6]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[4,5,7,18]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,5,6,7,8,10,11,20]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5,6,7,9,10]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,8,17]}],"complexes":[],"partners":["LGR4","LGR5","ZNRF3","RNF43","FZD7","ITGB3","GDF9","BMP15"],"other_free_text":[]},"mechanistic_narrative":"RSPO2 is a secreted signaling protein that functions as a context-dependent modulator of Wnt, BMP, and FGF pathways, with additional roles in integrin-mediated signaling, governing stem cell self-renewal, tissue morphogenesis, and neuromuscular junction formation. Through its furin-like domains, RSPO2 binds LGR4/5/6 receptors and the E3 ubiquitin ligases ZNRF3/RNF43 to regulate Frizzled receptor turnover: in most contexts it potentiates canonical Wnt/β-catenin signaling by promoting Frizzled stabilization [PMID:32457899, PMID:38758792], but it can also stabilize ZNRF3 in an LGR5-dependent manner to suppress Wnt signaling [PMID:24476626] and promote ZNRF3-mediated ubiquitination and degradation of Fzd7 to antagonize noncanonical Wnt/PKC/ERK signaling [PMID:28600110]. Beyond Wnt, RSPO2 inhibits BMP receptor signaling to maintain AML stem cell self-renewal [PMID:34407399], antagonizes FGF signaling via its thrombospondin type 1 domain during mesoderm patterning [PMID:32366679], directly engages integrin β3 and LGR4 to activate FAK/Src signaling [PMID:36217544], and acts through LGR5 to promote acetylcholine receptor clustering at neuromuscular junctions [PMID:30206360]."},"prefetch_data":{"uniprot":{"accession":"Q6UXX9","full_name":"R-spondin-2","aliases":["Roof plate-specific spondin-2","hRspo2"],"length_aa":243,"mass_kda":28.3,"function":"Activator of the canonical Wnt signaling pathway by acting as a ligand for LGR4-6 receptors. Upon binding to LGR4-6 (LGR4, LGR5 or LGR6), LGR4-6 associate with phosphorylated LRP6 and frizzled receptors that are activated by extracellular Wnt receptors, triggering the canonical Wnt signaling pathway to increase expression of target genes. Also regulates the canonical Wnt/beta-catenin-dependent pathway and non-canonical Wnt signaling by acting as an inhibitor of ZNRF3, an important regulator of the Wnt signaling pathway (PubMed:21727895, PubMed:21909076, PubMed:22615920). During embryonic development, plays a crucial role in limb specification, amplifying the Wnt signaling pathway independently of LGR4-6 receptors, possibly by acting as a direct antagonistic ligand to RNF43 and ZNRF3, hence governing the number of limbs an embryo should form (PubMed:29769720)","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q6UXX9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RSPO2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RSPO2","total_profiled":1310},"omim":[{"mim_id":"618022","title":"HUMEROFEMORAL HYPOPLASIA WITH RADIOTIBIAL RAY DEFICIENCY; HHRRD","url":"https://www.omim.org/entry/618022"},{"mim_id":"618021","title":"TETRAAMELIA SYNDROME 2; TETAMS2","url":"https://www.omim.org/entry/618021"},{"mim_id":"610575","title":"R-SPONDIN 2; RSPO2","url":"https://www.omim.org/entry/610575"},{"mim_id":"610574","title":"R-SPONDIN 3; RSPO3","url":"https://www.omim.org/entry/610574"},{"mim_id":"608245","title":"KERATIN 71, TYPE II; KRT71","url":"https://www.omim.org/entry/608245"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":14.5},{"tissue":"intestine","ntpm":12.7},{"tissue":"placenta","ntpm":16.5}],"url":"https://www.proteinatlas.org/search/RSPO2"},"hgnc":{"alias_symbol":["MGC35555"],"prev_symbol":[]},"alphafold":{"accession":"Q6UXX9","domains":[{"cath_id":"2.10.220.10","chopping":"37-96","consensus_level":"medium","plddt":94.145,"start":37,"end":96},{"cath_id":"2.10.220.10","chopping":"100-144","consensus_level":"medium","plddt":93.6089,"start":100,"end":144},{"cath_id":"2.20.100.10","chopping":"146-207","consensus_level":"medium","plddt":87.4982,"start":146,"end":207}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6UXX9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6UXX9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6UXX9-F1-predicted_aligned_error_v6.png","plddt_mean":81.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RSPO2","jax_strain_url":"https://www.jax.org/strain/search?query=RSPO2"},"sequence":{"accession":"Q6UXX9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6UXX9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6UXX9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6UXX9"}},"corpus_meta":[{"pmid":"24476626","id":"PMC_24476626","title":"RSPO2-LGR5 signaling has tumour-suppressive activity in colorectal cancer.","date":"2014","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/24476626","citation_count":105,"is_preprint":false},{"pmid":"34847079","id":"PMC_34847079","title":"RSPO2 and RANKL signal through LGR4 to regulate osteoclastic premetastatic niche formation and bone metastasis.","date":"2022","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/34847079","citation_count":68,"is_preprint":false},{"pmid":"25769727","id":"PMC_25769727","title":"RSPO2 Enhances Canonical Wnt Signaling to Confer Stemness-Associated Traits to Susceptible Pancreatic Cancer Cells.","date":"2015","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/25769727","citation_count":65,"is_preprint":false},{"pmid":"28600110","id":"PMC_28600110","title":"RSPO2 suppresses colorectal cancer metastasis by counteracting the Wnt5a/Fzd7-driven noncanonical Wnt pathway.","date":"2017","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/28600110","citation_count":59,"is_preprint":false},{"pmid":"35027768","id":"PMC_35027768","title":"Identification of a regulatory pathway inhibiting adipogenesis via RSPO2.","date":"2022","source":"Nature metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/35027768","citation_count":56,"is_preprint":false},{"pmid":"21732367","id":"PMC_21732367","title":"Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells.","date":"2012","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21732367","citation_count":46,"is_preprint":false},{"pmid":"30250044","id":"PMC_30250044","title":"Identification of RSPO2 Fusion Mutations and Target Therapy Using a Porcupine Inhibitor.","date":"2018","source":"Scientific 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stabilize membrane-associated ZNRF3, thereby inhibiting Wnt/β-catenin signaling in a LGR5-dependent manner in colorectal cancer cells\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown with Wnt reporter assays, epistasis with LGR5 depletion\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP and epistasis in multiple CRC cell lines, replicated functional phenotype\",\n      \"pmids\": [\"24476626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RSPO2 physically interacts with Fzd7 and promotes ZNRF3-mediated ubiquitination and degradation of cell-surface Fzd7, thereby suppressing PKC/ERK noncanonical Wnt signaling and antagonizing Wnt5a-driven cell migration\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, cell migration/invasion assays, epistasis with ZNRF3 and Fzd7\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, ubiquitination assay, and functional epistasis with defined phenotypic readout\",\n      \"pmids\": [\"28600110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSPO2 and RANKL signal through LGR4 to promote osteoclastic premetastatic niche formation; RSPO2/RANKL-LGR4 modulates DKK1 via Gαq and β-catenin signaling, and DKK1 facilitates osteoclast precursor recruitment by suppressing LRP5 and upregulating Rnasek\",\n      \"method\": \"GPCR ligand/agonist screening, Co-IP, knockdown/overexpression, decoy receptor (soluble LGR4 ECD) in mouse bone metastasis model\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — unbiased screening plus Co-IP, in vivo rescue with decoy receptor, multiple orthogonal methods\",\n      \"pmids\": [\"34847079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSPO2 maintains AML self-renewal by inhibiting BMP receptor signaling in a WNT-independent manner; autocrine RSPO2 prevents differentiation of normal hematopoietic stem cells and primary AML cells\",\n      \"method\": \"RSPO2 knockdown/overexpression, BMP signaling assays, AML xenograft mouse models with survival readout\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function with defined cellular phenotype, in vivo xenograft validation, multiple orthogonal assays\",\n      \"pmids\": [\"34407399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSPO2 is secreted by a subset of CD142+ adipogenic progenitor cells and inhibits maturation of early adipogenic progenitors through the receptor LGR4, leading to adipose tissue hypertrophy and insulin resistance\",\n      \"method\": \"Single-cell RNA-sequencing, loss-of-function mouse models, receptor identification (Lgr4), metabolic phenotyping\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — scRNA-seq combined with in vivo mouse models and receptor epistasis, multiple orthogonal methods\",\n      \"pmids\": [\"35027768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Rspo2 inhibits FGF signaling upstream of Mek1 via its thrombospondin type 1 (TSP1) domain, downregulating FGF target genes tbxt/brachyury and cdx4 during Xenopus mesoderm formation; this is distinct from its Furin-like domain-mediated Wnt activity\",\n      \"method\": \"Xenopus ectoderm explant elongation assay, ERK1 phosphorylation assay, domain deletion/mutagenesis, morpholino depletion\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — domain mutagenesis with in vitro biochemical assay and in vivo phenotypic rescue, multiple orthogonal methods\",\n      \"pmids\": [\"32366679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rspo2 inhibits TCF3/TCF7L1 phosphorylation to antagonize Wnt signaling during Xenopus anteroposterior axis specification, independently of binding to RNF43/ZNRF3 and LGR4/5\",\n      \"method\": \"Xenopus gain/loss-of-function, domain binding assays (RNF43/ZNRF3 and LGR4/5 binding-deficient mutants), TCF3 phosphorylation assays, epistasis with TCF3 depletion\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — domain mutagenesis with phosphorylation biochemistry and epistasis rescue, multiple orthogonal approaches\",\n      \"pmids\": [\"34183732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Rspo2 promotes acetylcholine receptor (AChR) clustering and neuromuscular junction (NMJ) formation via its receptor Lgr5; motor neuron-derived Rspo2 plays the major role in AChR clustering while muscle-derived Rspo2 normalizes ultrastructural NMJ features\",\n      \"method\": \"Tissue-specific rescue of Rspo2-/- mice (SMN-specific and muscle-specific expression), immunofluorescence, electron microscopy, gene expression analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic rescue with tissue-specific expression in knockout mice, ultrastructural and functional readouts\",\n      \"pmids\": [\"30206360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSPO2 promotes ovarian cancer growth and metastasis through dual receptor signaling: (1) RSPO2-LGR4 interaction prevents endocytic degradation of LGR4 and promotes LGR4-mediated translocation of Src to the plasma membrane; (2) RSPO2 directly binds integrin β3 as a ligand, enhancing integrin stability; both mechanisms potentiate FAK/Src autophosphorylation\",\n      \"method\": \"Co-immunoprecipitation, proximity ligation assay, receptor pulldown, phosphorylation assays, knockdown/overexpression\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and functional phosphorylation assays in single study with multiple orthogonal methods\",\n      \"pmids\": [\"36217544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"A HH-Foxf1-Rspo2 signaling axis governs tracheal cartilage development: HH/Gli signaling maintains Foxf1 expression in splanchnic mesoderm, which in turn supports Rspo2 expression, and Rspo2 potentiates Wnt signaling required for Sox9+ chondrocyte progenitor specification\",\n      \"method\": \"Multiple HH/Gli mouse mutants, lineage tracing, Foxf1 conditional knockout, Rspo2 expression analysis\",\n      \"journal\": \"Disease models & mechanisms\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple mouse mutant lines and lineage tracing, independently validated pathway placement\",\n      \"pmids\": [\"33328171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Wnt9b and RSPO2 cooperate to activate canonical Wnt/β-catenin signaling during mouse facial morphogenesis; compound Wnt9b;Rspo2 double knockout mice display more severe facial defects than either single mutant\",\n      \"method\": \"Compound mouse knockout, ex vivo facial explants, gene expression analysis\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with compound knockout and rescue experiments in vivo\",\n      \"pmids\": [\"32457899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KAT6A regulates Rspo2 transcription in hippocampal CA3 neurons via histone acetylation; KAT6A deficiency reduces RSPO2-Wnt signaling leading to impaired synaptic plasticity and memory deficits; restoring RSPO2 in CA3 rescues Wnt signaling and cognitive deficits in Kat6a mutant mice\",\n      \"method\": \"KAT6A conditional knockout, RSPO2 conditional knockout in excitatory neurons, AAV-mediated RSPO2 re-expression in CA3, Wnt signaling assays, behavioral tests\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic loss-of-function plus rescue with defined molecular (Wnt signaling) and behavioral phenotypes\",\n      \"pmids\": [\"38758792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSPO2 overexpression suppresses ectopic ossification in a mouse Achilles tendon puncture model by suppressing chondrogenic differentiation; RSPO2 is induced by NF-κB signaling in response to inflammatory stimulation and mechanical loading\",\n      \"method\": \"RSPO2 overexpression in mouse tendon puncture model, chondrogenic differentiation assays in human ligament cells, NF-κB inhibitor experiments\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo overexpression with defined phenotypic readout and mechanistic follow-up, single study\",\n      \"pmids\": [\"35984875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-181a targets RSPO2 mRNA in mesenchymal stromal cells during chondrogenesis, leading to reduced WNT signaling and increased BMP signaling (phospho-SMAD1/5/9 and SOX9 accumulation), establishing RSPO2 as a coordinator of WNT-BMP signaling crosstalk\",\n      \"method\": \"miR reporter assay (luciferase), miR-181a mimic/inhibitor transfection, Western blot for SMAD1/5/9 phosphorylation and SOX9\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — luciferase reporter plus downstream signaling validation with multiple readouts in single study\",\n      \"pmids\": [\"34778258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RSPO2 inhibits PPAR-γ nuclear translocation and reduces PPAR-γ binding to the CD36 promoter (assessed by ChIP), thereby suppressing CD36 expression, reducing oxLDL uptake, and inhibiting macrophage apoptosis\",\n      \"method\": \"Flow cytometry, Western blot, chromatin immunoprecipitation (ChIP), Rspo2 knockdown/overexpression in macrophages\",\n      \"journal\": \"Molecular medicine reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus functional assays, single study with multiple orthogonal methods\",\n      \"pmids\": [\"27571704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Calcitriol induces Rspo2 expression via vitamin D receptor (VDR) binding to a region ~15 kb upstream of Rspo2; deletion of this VDR-binding site by CRISPR/Cas9 abolishes calcitriol-mediated Rspo2 induction and MuSK phosphorylation at the neuromuscular junction\",\n      \"method\": \"ChIP-seq of VDR, CRISPR/Cas9 deletion of VDR-binding site, RNA-seq, MuSK phosphorylation assay, Chrne KO mouse model\",\n      \"journal\": \"Neurotherapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — ChIP-seq plus CRISPR functional deletion with biochemical (MuSK phosphorylation) and in vivo phenotypic readouts\",\n      \"pmids\": [\"38233267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RSPO2 knockdown in oocytes disrupts granulosa cell development and oocyte competence; RSPO2 (expressed in oocytes) physically interacts with GDF9:BMP15 heterodimers and its effects involve crosstalk between CTNNB1- and SMAD2-dependent pathways in granulosa cells\",\n      \"method\": \"Conditional oocyte-specific Rspo2 knockout, Co-immunoprecipitation (RSPO2 with GDF9:BMP15), transcriptomic analysis, pathway inhibitor experiments\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — conditional knockout plus Co-IP with defined downstream pathway analysis, single study\",\n      \"pmids\": [\"40492505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RSPO2 silence in nasopharyngeal carcinoma increases ZNRF3 expression and reduces Hedgehog/Gli1 signaling; ZNRF3 knockdown or Gli1 overexpression reverses the anti-tumor effects of RSPO2 depletion, placing RSPO2 upstream of ZNRF3/Hedgehog-Gli1\",\n      \"method\": \"siRNA knockdown, epistasis rescue by ZNRF3 knockdown and Gli1 overexpression, in vivo xenograft\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis rescue experiments with defined pathway placement, single study\",\n      \"pmids\": [\"34273374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PALMD interacts with RSPO2 and facilitates its secretion, enabling paracrine activation of Wnt/β-catenin signaling to promote colorectal cancer stemness and tumor growth; pharmacologic or antibody blockade of RSPO2 attenuates PALMD-mediated Wnt activation\",\n      \"method\": \"Co-immunoprecipitation (PALMD-RSPO2), gene knockdown/overexpression, Wnt reporter assay, organoid models, in vivo xenograft\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional epistasis with rescue, multiple orthogonal systems; preprint only\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A mutant RSPO2 furin domain retains high-affinity binding to LGR4/5/6 but lacks Wnt/β-catenin signaling activity; this property was used to create drug-conjugated peptibodies that deliver cytotoxins specifically to LGR4/5/6-positive cancer cells\",\n      \"method\": \"Binding affinity assays, TOPFLASH Wnt reporter assay, mutagenesis of furin domain, in vitro cytotoxicity, in vivo xenograft\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — furin domain mutagenesis with binding and signaling assays, validated in vitro and in vivo\",\n      \"pmids\": [\"41954225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RSPO2 activates the β-catenin pathway through GPR48/LGR4, inducing phosphorylation of ERK, LRP6, and GSK3β (serine 9); downregulation of GPR48/LGR4 decreases β-catenin pathway activity in thyroid cancer cells\",\n      \"method\": \"Exogenous RSPO2 treatment, LGR4 knockdown, Western blot for phospho-ERK/LRP6/GSK3β and β-catenin\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — receptor-ligand gain/loss-of-function with downstream phosphorylation readouts, single study\",\n      \"pmids\": [\"29383135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DNA methylation of RSPO2 promoter at -758/-749 and -563/-553 CpG regions controls RSPO2 transcription by regulating occupancy of transcription factor E2F1; hypomethylation facilitates E2F1 binding and increases RSPO2 expression in granulosa cells\",\n      \"method\": \"Bisulfite sequencing, DNMT1 knockdown, DNMT inhibitor treatment, ChIP for E2F1 at RSPO2 promoter, luciferase reporter assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus luciferase reporter with methylation manipulation, single study\",\n      \"pmids\": [\"34175894\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RSPO2 is a secreted protein that potentiates Wnt/β-catenin signaling primarily by binding LGR4/5/6 receptors and ZNRF3/RNF43 E3 ubiquitin ligases (via its furin-like domains) to prevent Frizzled degradation, but also acts context-dependently as a Wnt antagonist (by inhibiting TCF3 phosphorylation), an FGF antagonist (via its TSP1 domain), and a BMP antagonist to support stem cell self-renewal, while additionally engaging integrin β3 and LGR4 to activate FAK/Src signaling, and its transcription is regulated by VDR, C/EBPβ, E2F1, and promoter methylation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RSPO2 is a secreted signaling protein that functions as a context-dependent modulator of Wnt, BMP, and FGF pathways, with additional roles in integrin-mediated signaling, governing stem cell self-renewal, tissue morphogenesis, and neuromuscular junction formation. Through its furin-like domains, RSPO2 binds LGR4/5/6 receptors and the E3 ubiquitin ligases ZNRF3/RNF43 to regulate Frizzled receptor turnover: in most contexts it potentiates canonical Wnt/β-catenin signaling by promoting Frizzled stabilization [PMID:32457899, PMID:38758792], but it can also stabilize ZNRF3 in an LGR5-dependent manner to suppress Wnt signaling [PMID:24476626] and promote ZNRF3-mediated ubiquitination and degradation of Fzd7 to antagonize noncanonical Wnt/PKC/ERK signaling [PMID:28600110]. Beyond Wnt, RSPO2 inhibits BMP receptor signaling to maintain AML stem cell self-renewal [PMID:34407399], antagonizes FGF signaling via its thrombospondin type 1 domain during mesoderm patterning [PMID:32366679], directly engages integrin β3 and LGR4 to activate FAK/Src signaling [PMID:36217544], and acts through LGR5 to promote acetylcholine receptor clustering at neuromuscular junctions [PMID:30206360].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolving whether RSPO2 is solely a Wnt agonist, this study revealed that RSPO2 can stabilize ZNRF3 at the membrane via LGR5 to inhibit rather than activate Wnt/β-catenin signaling in colorectal cancer cells, establishing context-dependent duality.\",\n      \"evidence\": \"Reciprocal Co-IP and Wnt reporter epistasis with LGR5 knockdown in multiple CRC cell lines\",\n      \"pmids\": [\"24476626\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this inhibitory mode operates in normal colonic epithelium in vivo\", \"Structural basis for how LGR5 engagement switches RSPO2 from agonist to antagonist\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extended RSPO2's functional reach beyond canonical Wnt by showing it inhibits PPARγ nuclear translocation and PPARγ-dependent CD36 transcription, reducing oxidized LDL uptake in macrophages.\",\n      \"evidence\": \"ChIP for PPARγ at CD36 promoter plus knockdown/overexpression in macrophages\",\n      \"pmids\": [\"27571704\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which RSPO2 receptor mediates PPARγ inhibition\", \"Whether this is Wnt/β-catenin-dependent or represents a distinct signaling branch\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated that RSPO2 engages noncanonical Wnt signaling by directly binding Fzd7 and promoting its ZNRF3-mediated ubiquitination and degradation, thereby suppressing PKC/ERK-dependent cell migration.\",\n      \"evidence\": \"Reciprocal Co-IP, ubiquitination assays, and epistasis with ZNRF3/Fzd7 in cancer cell migration assays\",\n      \"pmids\": [\"28600110\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other Frizzled family members are similarly targeted\", \"In vivo relevance of RSPO2–Fzd7 antagonism in tumor metastasis\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established the RSPO2–LGR4 axis in canonical Wnt activation by showing RSPO2 signals through GPR48/LGR4 to phosphorylate ERK, LRP6, and GSK3β(Ser9) in thyroid cancer cells.\",\n      \"evidence\": \"Exogenous RSPO2 treatment with LGR4 knockdown and phospho-Western blots\",\n      \"pmids\": [\"29383135\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether LGR4 and LGR5 mediate opposing RSPO2 outputs within the same cell\", \"Contribution of ZNRF3/RNF43 in this thyroid cancer context\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified a developmental role for RSPO2 at the neuromuscular junction, showing motor neuron–derived Rspo2 signals via Lgr5 to promote AChR clustering and proper NMJ ultrastructure.\",\n      \"evidence\": \"Tissue-specific genetic rescue of Rspo2−/− mice with immunofluorescence and electron microscopy\",\n      \"pmids\": [\"30206360\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Rspo2-Lgr5 at the NMJ acts through canonical Wnt or a distinct pathway\", \"Role of muscle-derived Rspo2 beyond ultrastructural normalization\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a Wnt-independent function: the TSP1 domain of Rspo2 inhibits FGF/Mek1/ERK signaling to regulate mesoderm patterning, mechanistically separating this from the furin domain–mediated Wnt activity.\",\n      \"evidence\": \"Domain deletion/mutagenesis in Xenopus explants with ERK phosphorylation assays\",\n      \"pmids\": [\"32366679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the receptor for the TSP1 domain\", \"Whether TSP1-mediated FGF antagonism operates in mammalian systems\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed Rspo2 in a HH–Foxf1 transcriptional hierarchy governing tracheal cartilage, and showed genetic cooperation between Rspo2 and Wnt9b for facial morphogenesis via compound knockout synergy.\",\n      \"evidence\": \"Multiple HH/Gli and Foxf1 conditional mouse mutants with lineage tracing; Wnt9b;Rspo2 compound knockout mice\",\n      \"pmids\": [\"33328171\", \"32457899\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RSPO2 acts directly on Sox9+ chondrocyte progenitors or via an intermediate cell type\", \"Relative contributions of RSPO2 versus other R-spondins in craniofacial development\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Uncovered a second Wnt-antagonizing mechanism: Rspo2 inhibits TCF3 phosphorylation independently of RNF43/ZNRF3 and LGR4/5 binding, establishing a distinct mode of Wnt antagonism during anteroposterior axis specification.\",\n      \"evidence\": \"Binding-deficient RSPO2 mutants with TCF3 phosphorylation assays and epistasis in Xenopus\",\n      \"pmids\": [\"34183732\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The kinase whose activity on TCF3 is blocked by RSPO2\", \"Whether this TCF3-dependent mechanism operates in mammals\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined transcriptional regulation of RSPO2: E2F1 occupancy at the RSPO2 promoter is controlled by DNA methylation at specific CpG sites, providing an epigenetic on/off switch for RSPO2 expression in granulosa cells.\",\n      \"evidence\": \"Bisulfite sequencing, DNMT1 knockdown, ChIP for E2F1, luciferase reporter\",\n      \"pmids\": [\"34175894\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether this methylation-dependent regulation applies outside the ovarian context\", \"Upstream signals that trigger RSPO2 promoter demethylation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established a Wnt-independent role for RSPO2 in stem cell biology by showing that autocrine RSPO2 inhibits BMP receptor signaling to maintain AML self-renewal, and separately showed RSPO2 in oocytes interacts with GDF9:BMP15 heterodimers to coordinate Wnt and SMAD2 signaling in granulosa cells.\",\n      \"evidence\": \"RSPO2 knockdown/overexpression with BMP signaling assays and AML xenograft survival; oocyte-specific conditional knockout with Co-IP of RSPO2–GDF9:BMP15\",\n      \"pmids\": [\"34407399\", \"40492505\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which RSPO2 inhibits BMP receptors\", \"Whether BMP antagonism requires LGR receptors or is receptor-independent\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified RSPO2 as a paracrine inhibitor of adipogenic progenitor maturation via LGR4, linking it to adipose tissue remodeling and systemic insulin resistance.\",\n      \"evidence\": \"scRNA-seq of adipose tissue, Rspo2 loss-of-function mouse models with metabolic phenotyping and Lgr4 epistasis\",\n      \"pmids\": [\"35027768\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RSPO2-LGR4 acts through Wnt, BMP, or another downstream pathway in adipocyte progenitors\", \"Therapeutic potential of blocking RSPO2 for metabolic disease\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Expanded RSPO2's receptor repertoire beyond LGR/ZNRF3 by showing it directly binds integrin β3 and stabilizes it, while simultaneously preventing LGR4 endocytic degradation to activate FAK/Src signaling in ovarian cancer.\",\n      \"evidence\": \"Co-IP, proximity ligation assay, receptor pulldown, and phosphorylation assays in ovarian cancer cells\",\n      \"pmids\": [\"36217544\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for RSPO2–integrin β3 binding\", \"Whether integrin β3 engagement is a general feature of RSPO2 or specific to ovarian cancer\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated that RSPO2/RANKL co-signal through LGR4 via Gαq and β-catenin to induce DKK1 and promote osteoclastic premetastatic niche formation, revealing a cooperative ligand-receptor mechanism.\",\n      \"evidence\": \"GPCR agonist screening, Co-IP, soluble LGR4 decoy receptor in mouse bone metastasis model\",\n      \"pmids\": [\"34847079\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RSPO2 and RANKL form a ternary complex on LGR4 or signal sequentially\", \"Relevance to human bone metastasis\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified VDR as a direct transcriptional regulator of Rspo2 via a distal enhancer ~15 kb upstream; deletion of this site abolished calcitriol-mediated Rspo2 induction and downstream MuSK phosphorylation at the NMJ.\",\n      \"evidence\": \"ChIP-seq of VDR, CRISPR/Cas9 enhancer deletion, MuSK phosphorylation assay\",\n      \"pmids\": [\"38233267\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether VDR-dependent RSPO2 regulation operates in tissues beyond skeletal muscle\", \"Therapeutic implications for NMJ diseases\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed RSPO2 downstream of the chromatin modifier KAT6A in hippocampal neurons, showing that KAT6A-dependent histone acetylation drives Rspo2 transcription to sustain Wnt signaling required for synaptic plasticity and memory.\",\n      \"evidence\": \"KAT6A and RSPO2 conditional knockouts in excitatory neurons, AAV-mediated RSPO2 rescue in CA3, behavioral tests\",\n      \"pmids\": [\"38758792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RSPO2 acts through LGR4 or LGR5 in hippocampal neurons\", \"Whether other R-spondins compensate when RSPO2 is lost in the brain\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Structure-function dissection showed that a mutant furin domain retains LGR4/5/6 binding but loses Wnt-activating capacity, enabling design of signaling-dead RSPO2 peptibodies for targeted drug delivery to LGR-positive cancers.\",\n      \"evidence\": \"Furin domain mutagenesis with binding affinity and TOPFLASH assays, in vitro cytotoxicity, xenograft efficacy\",\n      \"pmids\": [\"41954225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crystal structure of the signaling-dead mutant bound to LGR\", \"Long-term in vivo safety and pharmacokinetics\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for how RSPO2 switches between Wnt agonist and antagonist outputs depending on LGR receptor identity, the receptor mediating TSP1-domain FGF antagonism, and the direct mechanism of BMP receptor inhibition.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal structure of RSPO2 in complex with LGR5 versus LGR4 to explain opposing signaling outcomes\", \"Identity of the TSP1 domain receptor remains unknown\", \"Mechanism by which RSPO2 inhibits BMP receptor signaling is undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 2, 4, 5, 7, 8, 19]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 3, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [4, 5, 7, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 5, 6, 7, 8, 10, 11, 20]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5, 6, 7, 9, 10]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 8, 17]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"LGR4\",\n      \"LGR5\",\n      \"ZNRF3\",\n      \"RNF43\",\n      \"FZD7\",\n      \"ITGB3\",\n      \"GDF9\",\n      \"BMP15\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}