{"gene":"LGR4","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2011,"finding":"LGR4 binds R-spondins (RSPOs) with high affinity and mediates potentiation of Wnt/β-catenin signaling by enhancing Wnt-induced LRP6 phosphorylation. Neither LGR4 nor LGR5 couples to heterotrimeric G proteins or β-arrestin when stimulated by RSPOs, indicating a unique non-canonical GPCR mechanism.","method":"Binding assays, LRP6 phosphorylation assays, G-protein and β-arrestin coupling assays in mammalian cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (binding, signaling, G-protein coupling), replicated across multiple labs","pmids":["21693646"],"is_preprint":false},{"year":2011,"finding":"R-spondins bind to LGR4 and LGR5 via their Furin domains and promote both Wnt/β-catenin and Wnt/PCP signaling. R-spondin-triggered β-catenin signaling via LGR4 requires Clathrin-mediated (not Caveolin-mediated) endocytosis.","method":"Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos; domain mapping; endocytosis inhibitor experiments","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods, validated in two organism systems, replicated finding of RSPO binding","pmids":["21909076"],"is_preprint":false},{"year":2012,"finding":"LGR4 is the cognate receptor for R-spondin; siRNA depletion of LGR4 completely abolishes RSPO-induced β-catenin signaling. RSPO binds the extracellular domain of LGR4. No coupling of LGR4 to heterotrimeric G proteins was detected in RSPO-treated cells.","method":"Unbiased siRNA screen, overexpression/depletion, binding assays with extracellular domain, G-protein coupling assay, Lgr4-/- crypt culture","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — unbiased screen plus multiple orthogonal validation methods, consistent with independent reports","pmids":["22815884"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of LGR4 ectodomain (ECD) alone and in complex with RSPO1 N-terminal fragment (two furin-like cysteine-rich domains, FU-CRD1 and FU-CRD2) was determined. LGR4-ECD adopts a TLR horseshoe structure and uses its concave surface near the N-termini to bind RSPO1. Both FU-CRD1 and FU-CRD2 contribute to LGR4 binding. Critical RSPO1 residues for biological activity were identified by mutagenesis.","method":"X-ray crystallography of LGR4 ECD–RSPO1 complex; mutagenesis and cellular binding/signaling assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional mutagenesis validation; independently confirmed by another structure paper","pmids":["23756652"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of Lgr4 ectodomain alone and bound to Rspo1 was determined. The receptor adopts an extended horseshoe LRR architecture and binds Rspo1 furin-like repeats via its concave side, providing molecular details of receptor activation.","method":"X-ray crystallography of Lgr4 ECD and Lgr4–Rspo1 complex","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — independent crystal structure determination confirming LGR4-RSPO1 interface; two independent structural studies","pmids":["23891289"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of LGR4–Rspo1 complex determined using hybrid LRR technique. Rspo1 binds in the concave surface of LGR4 through electrostatic and hydrophobic interactions. All Rspo1-binding residues are conserved in LGR4–6, suggesting identical binding surface. LGR4 uses a one-site binding model distinct from the two-step mechanism of LGR1–3.","method":"X-ray crystallography with hybrid LRR fusion protein; structural comparison","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, three independent structural studies converge on same interface","pmids":["25480784"],"is_preprint":false},{"year":2013,"finding":"R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R-spondin-induced LGR4/ZNRF3 complex formation, ZNRF3 membrane clearance, and Wnt signaling activation. LGR4 functions as an engagement receptor while ZNRF3/RNF43 function as effector receptors in a dual receptor model.","method":"Mutagenesis of distinct binding motifs, Co-IP, membrane clearance assays, Wnt signaling assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis plus multiple functional assays; mechanistically defines dual-receptor model","pmids":["24165923"],"is_preprint":false},{"year":2013,"finding":"RSPO-LGR4 recruits the intracellular scaffold protein IQGAP1, which bridges RSPO-LGR4 to the Wnt signalosome through enhanced IQGAP1–DVL interaction following RSPO stimulation. This complex potentiates canonical Wnt/β-catenin signaling via MEK1/2-mediated LRP5/6 phosphorylation and non-canonical signaling through actin dynamics regulation.","method":"Co-IP identification of IQGAP1 as LGR4-interacting protein, pulldown, RSPO stimulation assays, MEK/LRP6 phosphorylation assays, actin dynamics assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP identification plus multiple downstream functional assays in single rigorous study","pmids":["24639526"],"is_preprint":false},{"year":2013,"finding":"Reconstitution of RSPO:LGR4:ZNRF3 ternary complexes was achieved with bacterially produced recombinant proteins. RSPO2 and RSPO3 formed detectable ternary complexes with LGR4 LRR1-14 and ZNRF3 ECD; RSPO4:ZNRF3 complexes were not detected. RSPOs bound LGR4 with nanomolar affinities in order RSPO4 > RSPO2 > RSPO3 > RSPO1.","method":"In vitro reconstitution with recombinant proteins, TR-FRET binding assay, native gel electrophoretic mobility shift assay, cell-based signaling assay","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of ternary complex with multiple orthogonal biophysical methods","pmids":["24050775"],"is_preprint":false},{"year":2016,"finding":"LGR4 is a second receptor for RANKL (in addition to RANK). LGR4 competes with RANK to bind RANKL and suppresses canonical RANK signaling. RANKL binding to LGR4 activates Gαq and GSK3-β signaling, suppressing NFATc1 expression and activity to negatively regulate osteoclastogenesis. Soluble LGR4 ECD binds RANKL and inhibits osteoclast differentiation in vivo.","method":"Binding competition assays, Lgr4 knockout and conditional knockout mice, osteoclast differentiation assays, Gαq/GSK3-β signaling assays, in vivo osteoporosis models with LGR4-ECD treatment","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including KO mice, binding assays, signaling pathway analysis, and in vivo therapeutic validation","pmids":["27064449"],"is_preprint":false},{"year":2020,"finding":"Full-length LGR4 forms a tight constitutive complex with ZNRF3 and RNF43 even without RSPO, whereas LGR5 does not interact with either E3 ligase with or without RSPO. The seven-transmembrane domain of LGR4 confers interaction with E3 ligases (shown by domain-swapping experiments). LGR4 and LGR5 exist as homodimers on the cell surface; LGR5 interacts with FZD and LRP6 to enhance LRP6 phosphorylation.","method":"Coimmunoprecipitation, proximity ligation assay, competition binding, time-resolved FRET, domain-swapping experiments in whole cells","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — four orthogonal methods in one study with rigorous domain-swap controls establishing mechanistic distinction","pmids":["33262293"],"is_preprint":false},{"year":2023,"finding":"LGR4 and RNF43/ZNRF3 form a 2:2 dimer complex that accommodates bivalent RSPO binding with high affinity; LGR5 forms a homodimer that does not complex with E3 ligases and shows similar affinity for monovalent and bivalent RSPO forms. Co-expression of ZNRF3 with LGR4 (but not LGR5) greatly increased binding affinity for monovalent RSPO.","method":"Whole-cell binding assays with monovalent/bivalent RSPO ligands, TR-FRET, co-expression experiments","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple binding assays, single lab, consistent with prior mechanistic studies","pmids":["37402772"],"is_preprint":false},{"year":2013,"finding":"Norrin (Norrie disease gene product, vertebrate ortholog of insect burs/pburs) is a ligand for LGR4 that stimulates Wnt signaling mediated by LGR4 but not by LGR5 or LGR6 in mammalian cells. Binding studies showed norrin interactions with LGR4, 5, and 6. Mutagenesis categorized Norrie disease mutations into subgroups with distinct defects in signaling through three binding partners.","method":"Cell-based Wnt signaling assays, binding studies, site-directed mutagenesis of norrin","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell signaling and binding assays with mutagenesis, single lab","pmids":["23444378"],"is_preprint":false},{"year":2022,"finding":"Nidogen-2 is a novel endogenous ligand of LGR4 (KD ~26.77 nM, measured by surface plasmon resonance). Nidogen-2 binding to LGR4 biased-activates Gαq–PKCα–AMPKα1 signaling to counteract VSMC osteogenic transition and vascular calcification. LGR4 deficiency in VSMCs abolished the protective effect of nidogen-2.","method":"Unbiased membrane protein interactome, Co-IP, flow cytometric binding assay, surface plasmon resonance, G protein signaling assays, VSMC calcification assays, in vivo vascular calcification models with LGR4 KO","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — biophysical binding measurement plus multiple orthogonal mechanistic assays in vitro and in vivo","pmids":["36354004"],"is_preprint":false},{"year":2022,"finding":"RSPO2 and RANKL both signal through LGR4 to modulate the WNT inhibitor DKK1 via Gαq and β-catenin signaling, promoting osteoclastic premetastatic niche formation. Soluble LGR4-ECD acting as a decoy receptor for RSPO2 and RANKL reduced bone metastasis in a mouse model.","method":"Unbiased GPCR ligand/agonist screening, binding assays, signaling pathway analysis (Gαq, β-catenin, DKK1), mouse bone metastasis models with LGR4-ECD treatment","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple assays but single lab; extends known RANKL/LGR4 mechanism to metastatic context","pmids":["34847079"],"is_preprint":false},{"year":2016,"finding":"LGR4/5 LOF in liver disrupts Wnt/β-catenin signaling and hepatic metabolic zonation. The RSPO-LGR4/5-ZNRF3/RNF43 module is a master regulator of Wnt/β-catenin-mediated liver zonation; RSPO1-induced expansion of Wnt signaling gradient requires LGR4/5.","method":"Liver-specific Lgr4/5 LOF mice, RSPO1 protein treatment, ZNRF3/RNF43 LOF mice, functional Wnt reporter assays in liver","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic models with defined molecular readout; establishes pathway hierarchy","pmids":["27088858"],"is_preprint":false},{"year":2014,"finding":"LGR4 potentiates Wnt/β-catenin signaling through IQGAP1 in lung adenocarcinomas with aberrant RSPO3 expression. Knockdown of LGR4 or IQGAP1 reduced tumor growth and metastasis in vivo.","method":"Knockdown of LGR4/IQGAP1 in lung cancer cell lines, in vitro proliferation/migration assays, in vivo xenograft/metastasis models","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo KD with defined pathway, single lab","pmids":["25531322"],"is_preprint":false},{"year":2021,"finding":"LGR4 promotes cancer cell metastasis through a Wnt-independent mechanism involving direct interaction with EGFR. LGR4 blocks EGFR ubiquitination and degradation, resulting in persistent EGFR activation. LGR4 mutants unable to potentiate Wnt signaling still promoted breast cancer migration/invasion in vitro and metastasis in vivo.","method":"Pharmacological and genetic Wnt deactivation, LGR4 mutant overexpression, multiomic screening, Co-IP of LGR4-EGFR, ubiquitination assays, in vitro migration/invasion, in vivo metastasis models","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal approaches including Co-IP, ubiquitination assay, Wnt-independent mutants, and in vivo validation","pmids":["34099494"],"is_preprint":false},{"year":2008,"finding":"Gpr48/LGR4 inactivation impairs definitive erythropoiesis at midgestation through down-regulation of the ATF4 signaling pathway via the cAMP-PKA-CREB pathway. Gpr48-/- fetal livers showed decreased c-Myc and cyclin D1 expression with suppressed proliferation.","method":"Gpr48-/- mice, histological analysis, blood smear assays, real-time PCR for hemoglobin chains and signaling components, proliferation/apoptosis assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with defined molecular pathway, single lab","pmids":["18955481"],"is_preprint":false},{"year":2008,"finding":"GPR48/LGR4 regulates epithelial cell proliferation and migration during eyelid development through activation of the EGFR signaling pathway. Phosphorylation of EGFR was dramatically decreased in Gpr48-/- keratinocytes and developing eyelids, causing reduced F-actin formation, delayed leading-edge extension, and EOB phenotype.","method":"Gpr48-/- mice, histology, scanning EM, scratch assay, Western blot and immunostaining for EGFR phosphorylation","journal":"Investigative ophthalmology & visual science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with in vitro and in vivo readouts, defined signaling pathway, single lab","pmids":["18487371"],"is_preprint":false},{"year":2010,"finding":"GPR48/LGR4 mediates keratinocyte proliferation through HB-EGF-mediated EGFR transactivation. HB-EGF was reduced in Gpr48-/- cell culture medium; addition of HB-EGF restored EGFR, ERK, and STAT3 phosphorylation and cell proliferation. EGFR tyrosine kinase inhibitor AG1478 blocked GPR48-mediated cellular processes.","method":"EGFR inhibitor treatment, conditioned media immunodepletion of EGFR ligands, HB-EGF supplementation rescue, Western blot for EGFR/ERK/STAT3 phosphorylation","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic rescue experiment with inhibitor and ligand supplementation, single lab","pmids":["20732323"],"is_preprint":false},{"year":2013,"finding":"Lgr4 ablation reduces adiposity and promotes white-to-brown fat switch by potentiating brown adipocyte differentiation, partly through Rb1 reduction. Lgr4 homozygous mutant mice show increased energy expenditure and express BAT markers in WAT depots.","method":"Lgr4 homozygous mutant mice, stromal vascular fraction differentiation assays, Rb1 expression analysis, metabolic phenotyping","journal":"Nature cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with mechanistic link to Rb1, single lab","pmids":["24212090"],"is_preprint":false},{"year":2011,"finding":"Lgr4 gene inactivation reduces intestinal epithelial cell proliferation by 50% and Paneth cell terminal differentiation by 80%. LGR4-deficient but not LGR5-deficient crypts die in ex vivo culture with downregulation of stem cell markers and Wnt target genes including Lgr5. Partial rescue with LiCl (GSK3β inhibitor) but not Wnt agonists identifies LGR4 as a permissive factor in the intestinal Wnt pathway.","method":"Lgr4 knockout mice, ex vivo crypt culture, LiCl rescue, quantification of stem cell markers and Wnt targets","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse plus ex vivo rescue experiment with defined pathway position, single lab","pmids":["21508962"],"is_preprint":false},{"year":2013,"finding":"Lgr4 deficiency in macrophages increases CD14 (TLR2/4 co-receptor) expression at the transcriptional level by decreasing CREB binding activity, thereby potentiating TLR2/4-associated cytokine production and increasing susceptibility to septic shock.","method":"Lgr4-/- mice, cytokine production assays, CREB binding assays, CD14 expression analysis in Lgr4-deficient macrophages","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO macrophages with defined CREB-CD14 transcriptional mechanism, single lab","pmids":["23589304"],"is_preprint":false},{"year":2018,"finding":"Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Myeloid-specific Lgr4 knockout mice show reduced M2 tumor-associated macrophages and increased CD8+ T cell infiltration in tumors. Blocking Rspo/Lgr4/Erk/Stat3 signaling with LGR4-ECD or anti-Rspo1 antibody overcame resistance to anti-PD-1 therapy.","method":"Myeloid-specific conditional Lgr4 KO mice, tumor models, ERK/STAT3 signaling analysis, macrophage polarization assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined signaling pathway, multiple tumor models, single lab","pmids":["29967265"],"is_preprint":false},{"year":2020,"finding":"Macrophage-specific Lgr4 knockout mice show improved post-MI cardiac repair with reduced inflammatory macrophages. Lgr4-mediated proinflammatory programming in macrophages occurs via enhancement of CREB-mediated c-Fos, Fosl1, and Fosb transactivation to activate AP-1.","method":"Macrophage-specific Lgr4 KO mice, MI model, transcriptomic profiling, AP-1/CREB activity assays, immune cell quantification","journal":"Circulation research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined AP-1/CREB transcriptional mechanism, single lab with RNA-seq","pmids":["32600176"],"is_preprint":false},{"year":2014,"finding":"Lgr4 regulates corpus luteum maturation through WNT-mediated EGFR-ERK signaling. In Lgr4-/- granulosa-lutein cells, EGFR-ERK signaling activity is attenuated. WNT/β-catenin downstream target MMP9 is reduced, impairing activation of EGF-like factors needed for EGFR-ERK activation and steroidogenesis.","method":"Lgr4-/- mice, primary granulosa cell culture, EGFR-ERK signaling assays, MMP9 inhibitor treatment, HB-EGF rescue","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with mechanistic pathway analysis and rescue experiments, single lab","pmids":["24877628"],"is_preprint":false},{"year":2013,"finding":"LGR4 controls mammary gland development and stem cell repopulation capacity through activation of Sox2 via the Lgr4/Wnt/β-catenin/Lef1 pathway. ChIP analysis showed Sox2 expression is controlled by this pathway; Sox2 overexpression restored mammary regeneration potential of Lgr4-/- stem cells.","method":"Lgr4-/- mice, mammosphere assays, Wnt signaling analysis, ChIP assay for β-catenin/Lef1 at Sox2 promoter, Sox2 overexpression rescue, in vivo mammary regeneration assay","journal":"Stem cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus functional rescue, KO mouse, single lab","pmids":["23712846"],"is_preprint":false},{"year":2013,"finding":"Lgr4 is selectively expressed in peritubular myoid cells (PMCs) of testes, and Lgr4 loss arrests germ cells at meiosis I. Lgr4 in PMCs signals through Wnt/β-catenin to support spermatogenesis; re-activation of Wnt/β-catenin (Apc(min/+) cross or GSK3β inhibitor) partially rescued the Lgr4-/- testis phenotype.","method":"Lgr4-/- mice, Apc(min/+) cross, GSK3β inhibitor treatment, PMC gene expression analysis, meiosis staging","journal":"Development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis rescue, KO mouse, defined pathway, single lab","pmids":["23533175"],"is_preprint":false},{"year":2019,"finding":"The miR-34–LGR4 axis regulates keratinocyte inflammatory response via GSK-3β-induced p65 serine 468 phosphorylation, modulating NF-κB signaling pathway activity. LGR4 was identified as a direct target of miR-34a and miR-34c mediating their proinflammatory function. Lgr4 knockout mice showed impaired wound closure with enhanced inflammation.","method":"miR-34 target validation, Lgr4 KO mice wound model, NF-κB signaling assays including p65 phosphorylation at Ser468, wound healing assays","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse phenotype plus defined signaling mechanism, single lab","pmids":["31376385"],"is_preprint":false},{"year":2013,"finding":"RSPO-LGR4 signaling activates WNT/β-catenin pathway to induce follistatin (Fst) expression in myoblasts, which then antagonizes TGF-β signaling to promote myogenic differentiation. LGR4 depletion severely disrupts myogenic differentiation and abolishes RSPO2 response. Specific TCF/LEF1 binding sites within Fst promoter and intron 1 were required for RSPO2/WNT-induced Fst expression.","method":"LGR4 siRNA depletion in C2C12 myoblasts, RSPO2 stimulation assays, β-catenin activation/inactivation, promoter/ChIP analysis of Fst TCF/LEF1 sites","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter analysis with TCF/LEF1 site mutagenesis plus functional differentiation assays, single lab","pmids":["24344199"],"is_preprint":false},{"year":2015,"finding":"LGR4 (and LGR5) promote formation of long (>80 μm) actin-rich cytoneme-like membrane protrusions through stabilization of nascent filopodia. LGR5-induced cytonemes serve as conduits for β-arrestin-2 and Myosin X transit, linking GPCR signaling to cytoneme-based cell communication.","method":"Overexpression of Lgr4/Lgr5 in cells, live imaging, measurement of filopodial/cytoneme lengths, β-arrestin-2 and Myo10 trafficking assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — overexpression with live imaging and cargo tracking, replicated for both LGR4 and LGR5","pmids":["25653388"],"is_preprint":false},{"year":2020,"finding":"LGR4 deficiency in mice results in delayed puberty with fewer GnRH neurons. LGR4 mutant proteins from delayed puberty patients showed impaired Wnt/β-catenin signaling due to defective protein expression, trafficking, and degradation. Lgr4 knockdown in zebrafish prevented formation and migration of GnRH neurons. Genetic lineage tracing showed strong Lgr4-mediated Wnt/β-catenin signaling activation during GnRH neuron development.","method":"Whole-exome sequencing, LGR4 mutant protein functional assays (expression/trafficking/degradation), Lgr4 KO mice, zebrafish lgr4 knockdown, genetic lineage tracing with Wnt/β-catenin reporter","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple model organisms, lineage tracing, and protein functional assays, single lab","pmids":["32493844"],"is_preprint":false},{"year":2016,"finding":"LGR4 expression in multiple myeloma plasma cells is driven by IL-6/STAT3 signaling, allowing MM cells to hijack osteoblast-derived R-spondins and stabilize Wnt co-receptors via inhibition of RNF43/ZNRF3 E3 ligase activity, dramatically increasing Wnt sensitivity.","method":"IL-6/STAT3 signaling analysis, LGR4 overexpression in MM cells, R-spondin binding assays, Wnt receptor stabilization assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined transcriptional driver (IL-6/STAT3) plus Wnt receptor mechanism, single lab","pmids":["28028233"],"is_preprint":false},{"year":2022,"finding":"In gastric stem cells, Rspo3 signaling via Lgr4 (not Lgr5) drives proliferation of stem cells and induces NF-κB activity. Upon H. pylori exposure, Lgr4-driven NF-κB activation causes expansion of the gland base module and chemokine expression in stem cells, leading to hyperplasia and neutrophil recruitment.","method":"Conditional Lgr4 KO mice, H. pylori infection model, NF-κB reporter assays, conditional Lgr5 KO comparison","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with defined NF-κB signaling mechanism and epistasis between Lgr4 and Lgr5, single lab","pmids":["35767364"],"is_preprint":false},{"year":2014,"finding":"LGR4-dependent Wnt/β-catenin signaling transcriptionally upregulates SLC7A11 (a ferroptosis inhibitor), and LGR4-mAb treatment blocks this pathway, sensitizing chemoresistant colorectal cancer cells to ferroptosis.","method":"Monoclonal antibody (LGR4-mAb) treatment, colorectal cancer organoid biobank, transcriptomic analysis, SLC7A11 expression assays, ferroptosis sensitivity assays","journal":"Nature cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — antibody blockade with defined transcriptional target and mechanistic readout, single lab","pmids":["38291304"],"is_preprint":false},{"year":2020,"finding":"RSPO-LGR4 signaling upregulates key self-renewal genes and is essential for leukemia stem cell (LSC) self-renewal in HOXA9-dependent AML. LGR4 is epigenetically upregulated and cooperates with HOXA9. Blocking RSPO3-LGR4 interaction with anti-RSPO3 antibody impairs self-renewal and induces differentiation in patient-derived xenografts without affecting normal HSCs.","method":"Patient-derived xenograft (PDX) models, anti-RSPO3 antibody (OMP-131R10) treatment, epigenetic analysis, self-renewal assays, differentiation assays","journal":"Cancer cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PDX models with defined epigenetic and self-renewal mechanisms, single lab","pmids":["32559496"],"is_preprint":false},{"year":2021,"finding":"Lgr4 promotes aerobic glycolysis and osteoblast differentiation via canonical Wnt/β-catenin signaling by inducing pyruvate dehydrogenase kinase 1 (pdk1) expression. Lgr4 osteoblast-specific KO mice exhibited decreased bone mass and strength with impaired glycolysis.","method":"Osteoblast-specific Lgr4 KO mice (Lgr4osb-/-), glucose/lactate metabolism assays, Wnt/β-catenin activation rescue, pdk1 expression analysis","journal":"Journal of bone and mineral research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with metabolic and molecular mechanistic readout, single lab","pmids":["33950533"],"is_preprint":false},{"year":2013,"finding":"LGR4 in the hypothalamic arcuate nucleus co-localizes with NPY, POMC, and BDNF neurons. Intracerebroventricular injection of Rspo1 or Rspo3 (ligands acting through LGR4) inhibited food intake, with Rspo1 decreasing NPY and increasing POMC expression. Rspo1 and Rspo3 mRNAs were down-regulated by fasting and up-regulated by insulin.","method":"In situ hybridization, intracerebroventricular injection, food intake measurement, neuropeptide expression assays","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo ligand injection with defined neurochemical readouts, single lab","pmids":["24280058"],"is_preprint":false},{"year":2014,"finding":"LGR4 acts as a link between the peripheral circadian clock and lipid metabolism by regulating circadian expression of Mttp (microsomal triglyceride transfer protein) in hepatocytes, thereby controlling plasma triglyceride rhythms.","method":"Lgr4 mutant mice, measurement of respiratory exchange ratio and plasma lipid rhythms, circadian gene expression analysis in liver and primary hepatocytes","journal":"Journal of molecular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined molecular target (Mttp) in KO mouse, single lab","pmids":["24353284"],"is_preprint":false},{"year":2018,"finding":"LGR4 activation by Rspo1 in hepatocytes reduces TNFα-induced cell death by inhibiting NF-κB nuclear translocation. LGR4 knockdown increased NF-κB-p65 nuclear translocation and caspase-3 levels after acute injury. IKKβ overexpression attenuated Rspo1 protection, and β-catenin disruption reversed protective effects.","method":"Liver-specific Lgr4 KO mice, primary hepatocyte culture, TNFα treatment, NF-κB signaling assays, IKKβ overexpression, β-catenin disruption, HIRI and LPS/D-Gal liver injury models","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with multiple in vitro and in vivo injury models and defined NF-κB mechanism, single lab","pmids":["30406697"],"is_preprint":false},{"year":2020,"finding":"Rspo1/Rspo3-LGR4 signaling inhibits hepatic cholesterol synthesis via the AMPKα-SREBP2 pathway. LGR4 knockdown increased hepatic cholesterol synthesis and decreased AMPKα phosphorylation. AMPKα agonist/antagonist experiments confirmed AMPKα as the downstream mediator of Rspos-LGR4 effects on SREBP2 nuclear translocation.","method":"Lgr4 and Rspo1/3 knockdown mice, AMPKα agonist/antagonist treatment, AMPKα shRNA, SREBP2 nuclear translocation assays, cholesterol synthesis measurements","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological dissection of AMPKα-SREBP2 pathway, single lab","pmids":["32926477"],"is_preprint":false},{"year":2014,"finding":"LGR4/GPR48 inactivation down-regulates histone demethylases Jmjd2a and Fbxl10 through cAMP-CREB signaling pathways in embryonic fibroblasts and urinary/reproductive tissues, contributing to WAGR/AGR syndrome-like defects.","method":"Lgr4-/- mice, gene expression analysis in MEFs and tissues, cAMP-CREB pathway assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with defined epigenetic regulatory target via cAMP-CREB pathway, single lab","pmids":["24519938"],"is_preprint":false},{"year":2006,"finding":"LGR4/GPR48 loss in male mice leads to failed tube elongation and convolution of efferent ducts and epididymis with dramatically reduced cell proliferation. In efferent ducts, LGR4 inactivation decreases estrogen receptor (ESR1) and SLC9A3 expression, causing water reabsorption failure and rete testis dilation.","method":"LGR4 KO mice, histology, cell proliferation assays, gene expression analysis for ESR1 and SLC9A3","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with defined molecular targets (ESR1/SLC9A3), single lab","pmids":["17079737"],"is_preprint":false}],"current_model":"LGR4 (GPR48) is a leucine-rich repeat-containing GPCR that functions as a high-affinity receptor for R-spondins (binding via their furin domains to the concave surface of the LGR4 horseshoe-shaped ECD, as established by crystal structures), and potentiates Wnt/β-catenin signaling by forming constitutive complexes with the E3 ubiquitin ligases ZNRF3/RNF43 through its seven-transmembrane domain (thereby protecting Frizzled/LRP5/6 from degradation) and by recruiting IQGAP1 into the Wnt signalosome to promote MEK1/2-mediated LRP5/6 phosphorylation; additionally, LGR4 acts as a second receptor for RANKL that activates Gαq-GSK3β signaling to suppress NFATc1 and osteoclastogenesis, binds nidogen-2 to activate Gαq-PKCα-AMPKα1 signaling against vascular calcification, and signals independently of Wnt via direct interaction with EGFR to prevent its ubiquitination and degradation, with all these activities broadly required for development, stem cell maintenance, immune regulation, and metabolic homeostasis across multiple tissues."},"narrative":{"mechanistic_narrative":"LGR4 is a leucine-rich-repeat-containing seven-transmembrane receptor that functions as the high-affinity engagement receptor for R-spondins to potentiate Wnt/β-catenin signaling, and serves more broadly as a hub for stem cell maintenance, development, immune regulation, and metabolic homeostasis across many tissues [PMID:21693646, PMID:27088858, PMID:21508962]. R-spondins bind the concave surface of the horseshoe-shaped LGR4 ectodomain through their two furin-like cysteine-rich domains in a one-site binding mode, as resolved by multiple crystal structures [PMID:23756652, PMID:23891289, PMID:25480784]. Rather than coupling to heterotrimeric G proteins or β-arrestin upon R-spondin stimulation, LGR4 acts non-canonically: through its seven-transmembrane domain it forms a constitutive complex with the membrane E3 ubiquitin ligases ZNRF3/RNF43, and R-spondin engagement drives clearance of these ligases to protect Wnt receptors and enhance LRP6 phosphorylation, with LGR4 serving as the \"engagement\" receptor and ZNRF3/RNF43 as the \"effector\" receptors in a dual-receptor model [PMID:21693646, PMID:24165923, PMID:33262293]. Downstream, RSPO-LGR4 recruits the scaffold IQGAP1 to bridge the receptor to the Wnt signalosome and promote MEK1/2-mediated LRP5/6 phosphorylation, also driving actin/cytoneme-based protrusions [PMID:24639526, PMID:25653388]. Beyond R-spondins, LGR4 binds additional ligands—RANKL, where it competes with RANK and activates Gαq-GSK3β signaling to suppress NFATc1 and osteoclastogenesis [PMID:27064449]; nidogen-2, which biased-activates Gαq-PKCα-AMPKα1 signaling to counteract vascular calcification [PMID:36354004]; and norrin, which stimulates LGR4-dependent Wnt signaling [PMID:23444378]. LGR4 also signals independently of Wnt by directly interacting with EGFR to block its ubiquitination and sustain its activation [PMID:34099494]. Through these activities LGR4 governs intestinal, gastric, mammary, and leukemic stem cell self-renewal, liver metabolic zonation, osteoblast/osteoclast balance, macrophage polarization, and reproductive development, and its mutations cause delayed puberty via impaired Wnt/β-catenin signaling in GnRH neurons [PMID:27088858, PMID:21508962, PMID:32493844, PMID:32559496].","teleology":[{"year":2011,"claim":"Established LGR4 as the high-affinity R-spondin receptor that potentiates Wnt/β-catenin signaling through a non-canonical mechanism that does not use classical GPCR effectors, resolving how RSPOs amplify Wnt.","evidence":"Binding, LRP6 phosphorylation, G-protein and β-arrestin coupling assays in mammalian cells and Xenopus, plus domain mapping and siRNA depletion","pmids":["21693646","21909076","22815884"],"confidence":"High","gaps":["Did not resolve the structural basis of the RSPO interface","The intracellular effector translating ligand binding into Wnt potentiation was unidentified"]},{"year":2013,"claim":"Defined the atomic basis of receptor-ligand recognition, showing RSPO furin-like domains dock onto the concave face of the LGR4 horseshoe ectodomain via a one-site binding mode.","evidence":"X-ray crystallography of LGR4 ECD alone and with RSPO1, with mutagenesis validation; confirmed by independent structures","pmids":["23756652","23891289","25480784"],"confidence":"High","gaps":["Structures covered the ectodomain only, not the seven-transmembrane region or full-length receptor","Did not explain how ligand binding signals across the membrane"]},{"year":2013,"claim":"Resolved the downstream signaling machinery, establishing a dual-receptor model where LGR4 engages RSPO and clears the E3 ligases ZNRF3/RNF43, and identifying IQGAP1 as the intracellular scaffold linking LGR4 to the Wnt signalosome.","evidence":"Mutagenesis of distinct binding motifs, Co-IP, membrane clearance assays, in vitro ternary complex reconstitution, and MEK/LRP6 phosphorylation assays","pmids":["24165923","24639526","24050775"],"confidence":"High","gaps":["Stoichiometry of the receptor-ligase complex was not yet defined","Constitutive versus ligand-dependent assembly of the complex was unclear"]},{"year":2020,"claim":"Distinguished LGR4 from LGR5 mechanistically, showing LGR4 forms a constitutive ZNRF3/RNF43 complex via its seven-transmembrane domain as a 2:2 dimer accommodating bivalent RSPO, whereas LGR5 does not bind the E3 ligases.","evidence":"Co-IP, proximity ligation, TR-FRET, domain-swapping, and monovalent/bivalent RSPO binding assays in whole cells","pmids":["33262293","37402772"],"confidence":"Medium","gaps":["No full-length structure of the receptor-ligase dimer","Functional consequences of dimerization for signaling output not fully mapped"]},{"year":2016,"claim":"Expanded LGR4 beyond Wnt by identifying it as a second RANKL receptor that competes with RANK and signals via Gαq-GSK3β to suppress osteoclastogenesis, revealing a G-protein-coupled mode distinct from RSPO signaling.","evidence":"Binding competition, Lgr4 KO and conditional KO mice, osteoclast differentiation, Gαq/GSK3-β signaling assays, and in vivo soluble LGR4-ECD therapy","pmids":["27064449"],"confidence":"High","gaps":["Why RANKL triggers Gαq coupling while RSPO does not was unexplained","Structural basis of RANKL–LGR4 interaction not determined"]},{"year":2022,"claim":"Identified nidogen-2 as a further endogenous ligand activating biased Gαq-PKCα-AMPKα1 signaling, demonstrating that LGR4 integrates multiple ligands into distinct G-protein-dependent outputs in the vasculature.","evidence":"Membrane interactome, Co-IP, SPR, G-protein signaling assays, and in vivo vascular calcification models with LGR4 KO","pmids":["36354004"],"confidence":"High","gaps":["Ligand-binding site on LGR4 for nidogen-2 not structurally defined","How distinct ligands selectively bias different G-protein pathways unresolved"]},{"year":2021,"claim":"Established a Wnt-independent function whereby LGR4 directly binds EGFR and blocks its ubiquitination and degradation, decoupling LGR4's oncogenic activity from its canonical Wnt-potentiating role.","evidence":"Wnt-deactivation experiments, Wnt-incompetent LGR4 mutants, Co-IP, ubiquitination assays, and in vivo metastasis models","pmids":["34099494"],"confidence":"High","gaps":["Whether EGFR stabilization requires a ligand or is constitutive is unclear","Earlier reports of EGFR-pathway effects via HB-EGF transactivation versus direct interaction not reconciled"]},{"year":2020,"claim":"Linked LGR4 to human disease by showing LGR4 mutations cause delayed puberty through impaired Wnt/β-catenin signaling required for GnRH neuron formation and migration.","evidence":"Whole-exome sequencing, LGR4 mutant protein trafficking/degradation assays, Lgr4 KO mice, zebrafish knockdown, and Wnt reporter lineage tracing","pmids":["32493844"],"confidence":"Medium","gaps":["Single-cohort genetic evidence","Direct rescue of the neuronal phenotype by wild-type LGR4 not shown"]},{"year":null,"claim":"How a single receptor selects among R-spondin (non-G-protein), RANKL/nidogen-2 (Gαq-coupled), and EGFR-stabilizing modes from the same seven-transmembrane scaffold remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length structure capturing transmembrane signaling states","Determinants of ligand-biased G-protein coupling versus E3-ligase recruitment are unknown","Mechanism switching between Wnt-dependent and Wnt-independent outputs is uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,9,13]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[3,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,10,17]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[9,10,13]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,7,9,13]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[15,22,32]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[23,24,25]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[37,39,41]}],"complexes":["LGR4–ZNRF3/RNF43 receptor complex","RSPO–LGR4–ZNRF3 ternary complex"],"partners":["RSPO1","RSPO2","RSPO3","ZNRF3","RNF43","IQGAP1","EGFR","NID2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BXB1","full_name":"Leucine-rich repeat-containing G-protein coupled receptor 4","aliases":["G-protein coupled receptor 48"],"length_aa":951,"mass_kda":104.5,"function":"Receptor for R-spondins that potentiates the canonical Wnt signaling pathway and is involved in the formation of various organs. Upon binding to R-spondins (RSPO1, RSPO2, RSPO3 or RSPO4), associates 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. In contrast to classical G-protein coupled receptors, does not activate heterotrimeric G-proteins to transduce the signal. Its function as activator of the Wnt signaling pathway is required for the development of various organs, including liver, kidney, intestine, bone, reproductive tract and eye. May also act as a receptor for norrin (NDP), such results however require additional confirmation in vivo. Required during spermatogenesis to activate the Wnt signaling pathway in peritubular myoid cells. Required for the maintenance of intestinal stem cells and Paneth cell differentiation in postnatal intestinal crypts. Acts as a regulator of bone formation and remodeling. Involved in kidney development; required for maintaining the ureteric bud in an undifferentiated state. Involved in the development of the anterior segment of the eye. Required during erythropoiesis. Also acts as a negative regulator of innate immunity by inhibiting TLR2/TLR4 associated pattern-recognition and pro-inflammatory cytokine production. Plays an important role in regulating the circadian rhythms of plasma lipids, partially through regulating the rhythmic expression of MTTP (By similarity). Required for proper development of GnRH neurons (gonadotropin-releasing hormone expressing neurons) that control the release of reproductive hormones from the pituitary gland (By similarity)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9BXB1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/LGR4","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/LGR4","total_profiled":1310},"omim":[{"mim_id":"620946","title":"UBIQUITIN-SPECIFIC PEPTIDASE 42; USP42","url":"https://www.omim.org/entry/620946"},{"mim_id":"619613","title":"DELAYED PUBERTY, SELF-LIMITED; DPSL","url":"https://www.omim.org/entry/619613"},{"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":"615311","title":"BONE MINERAL DENSITY QUANTITATIVE TRAIT LOCUS 17; BMND17","url":"https://www.omim.org/entry/615311"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Centrosome","reliability":"Approved"},{"location":"Centriolar satellite","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/LGR4"},"hgnc":{"alias_symbol":[],"prev_symbol":["GPR48"]},"alphafold":{"accession":"Q9BXB1","domains":[{"cath_id":"1.20.1070.10","chopping":"540-731_745-819","consensus_level":"high","plddt":84.9439,"start":540,"end":819}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXB1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXB1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BXB1-F1-predicted_aligned_error_v6.png","plddt_mean":78.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=LGR4","jax_strain_url":"https://www.jax.org/strain/search?query=LGR4"},"sequence":{"accession":"Q9BXB1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BXB1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BXB1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BXB1"}},"corpus_meta":[{"pmid":"21693646","id":"PMC_21693646","title":"R-spondins function as 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Neither LGR4 nor LGR5 couples to heterotrimeric G proteins or β-arrestin when stimulated by RSPOs, indicating a unique non-canonical GPCR mechanism.\",\n      \"method\": \"Binding assays, LRP6 phosphorylation assays, G-protein and β-arrestin coupling assays in mammalian cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (binding, signaling, G-protein coupling), replicated across multiple labs\",\n      \"pmids\": [\"21693646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"R-spondins bind to LGR4 and LGR5 via their Furin domains and promote both Wnt/β-catenin and Wnt/PCP signaling. R-spondin-triggered β-catenin signaling via LGR4 requires Clathrin-mediated (not Caveolin-mediated) endocytosis.\",\n      \"method\": \"Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos; domain mapping; endocytosis inhibitor experiments\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods, validated in two organism systems, replicated finding of RSPO binding\",\n      \"pmids\": [\"21909076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"LGR4 is the cognate receptor for R-spondin; siRNA depletion of LGR4 completely abolishes RSPO-induced β-catenin signaling. RSPO binds the extracellular domain of LGR4. No coupling of LGR4 to heterotrimeric G proteins was detected in RSPO-treated cells.\",\n      \"method\": \"Unbiased siRNA screen, overexpression/depletion, binding assays with extracellular domain, G-protein coupling assay, Lgr4-/- crypt culture\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — unbiased screen plus multiple orthogonal validation methods, consistent with independent reports\",\n      \"pmids\": [\"22815884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of LGR4 ectodomain (ECD) alone and in complex with RSPO1 N-terminal fragment (two furin-like cysteine-rich domains, FU-CRD1 and FU-CRD2) was determined. LGR4-ECD adopts a TLR horseshoe structure and uses its concave surface near the N-termini to bind RSPO1. Both FU-CRD1 and FU-CRD2 contribute to LGR4 binding. Critical RSPO1 residues for biological activity were identified by mutagenesis.\",\n      \"method\": \"X-ray crystallography of LGR4 ECD–RSPO1 complex; mutagenesis and cellular binding/signaling assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional mutagenesis validation; independently confirmed by another structure paper\",\n      \"pmids\": [\"23756652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of Lgr4 ectodomain alone and bound to Rspo1 was determined. The receptor adopts an extended horseshoe LRR architecture and binds Rspo1 furin-like repeats via its concave side, providing molecular details of receptor activation.\",\n      \"method\": \"X-ray crystallography of Lgr4 ECD and Lgr4–Rspo1 complex\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — independent crystal structure determination confirming LGR4-RSPO1 interface; two independent structural studies\",\n      \"pmids\": [\"23891289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of LGR4–Rspo1 complex determined using hybrid LRR technique. Rspo1 binds in the concave surface of LGR4 through electrostatic and hydrophobic interactions. All Rspo1-binding residues are conserved in LGR4–6, suggesting identical binding surface. LGR4 uses a one-site binding model distinct from the two-step mechanism of LGR1–3.\",\n      \"method\": \"X-ray crystallography with hybrid LRR fusion protein; structural comparison\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, three independent structural studies converge on same interface\",\n      \"pmids\": [\"25480784\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R-spondin-induced LGR4/ZNRF3 complex formation, ZNRF3 membrane clearance, and Wnt signaling activation. LGR4 functions as an engagement receptor while ZNRF3/RNF43 function as effector receptors in a dual receptor model.\",\n      \"method\": \"Mutagenesis of distinct binding motifs, Co-IP, membrane clearance assays, Wnt signaling assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis plus multiple functional assays; mechanistically defines dual-receptor model\",\n      \"pmids\": [\"24165923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RSPO-LGR4 recruits the intracellular scaffold protein IQGAP1, which bridges RSPO-LGR4 to the Wnt signalosome through enhanced IQGAP1–DVL interaction following RSPO stimulation. This complex potentiates canonical Wnt/β-catenin signaling via MEK1/2-mediated LRP5/6 phosphorylation and non-canonical signaling through actin dynamics regulation.\",\n      \"method\": \"Co-IP identification of IQGAP1 as LGR4-interacting protein, pulldown, RSPO stimulation assays, MEK/LRP6 phosphorylation assays, actin dynamics assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP identification plus multiple downstream functional assays in single rigorous study\",\n      \"pmids\": [\"24639526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Reconstitution of RSPO:LGR4:ZNRF3 ternary complexes was achieved with bacterially produced recombinant proteins. RSPO2 and RSPO3 formed detectable ternary complexes with LGR4 LRR1-14 and ZNRF3 ECD; RSPO4:ZNRF3 complexes were not detected. RSPOs bound LGR4 with nanomolar affinities in order RSPO4 > RSPO2 > RSPO3 > RSPO1.\",\n      \"method\": \"In vitro reconstitution with recombinant proteins, TR-FRET binding assay, native gel electrophoretic mobility shift assay, cell-based signaling assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of ternary complex with multiple orthogonal biophysical methods\",\n      \"pmids\": [\"24050775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LGR4 is a second receptor for RANKL (in addition to RANK). LGR4 competes with RANK to bind RANKL and suppresses canonical RANK signaling. RANKL binding to LGR4 activates Gαq and GSK3-β signaling, suppressing NFATc1 expression and activity to negatively regulate osteoclastogenesis. Soluble LGR4 ECD binds RANKL and inhibits osteoclast differentiation in vivo.\",\n      \"method\": \"Binding competition assays, Lgr4 knockout and conditional knockout mice, osteoclast differentiation assays, Gαq/GSK3-β signaling assays, in vivo osteoporosis models with LGR4-ECD treatment\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including KO mice, binding assays, signaling pathway analysis, and in vivo therapeutic validation\",\n      \"pmids\": [\"27064449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Full-length LGR4 forms a tight constitutive complex with ZNRF3 and RNF43 even without RSPO, whereas LGR5 does not interact with either E3 ligase with or without RSPO. The seven-transmembrane domain of LGR4 confers interaction with E3 ligases (shown by domain-swapping experiments). LGR4 and LGR5 exist as homodimers on the cell surface; LGR5 interacts with FZD and LRP6 to enhance LRP6 phosphorylation.\",\n      \"method\": \"Coimmunoprecipitation, proximity ligation assay, competition binding, time-resolved FRET, domain-swapping experiments in whole cells\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — four orthogonal methods in one study with rigorous domain-swap controls establishing mechanistic distinction\",\n      \"pmids\": [\"33262293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LGR4 and RNF43/ZNRF3 form a 2:2 dimer complex that accommodates bivalent RSPO binding with high affinity; LGR5 forms a homodimer that does not complex with E3 ligases and shows similar affinity for monovalent and bivalent RSPO forms. Co-expression of ZNRF3 with LGR4 (but not LGR5) greatly increased binding affinity for monovalent RSPO.\",\n      \"method\": \"Whole-cell binding assays with monovalent/bivalent RSPO ligands, TR-FRET, co-expression experiments\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple binding assays, single lab, consistent with prior mechanistic studies\",\n      \"pmids\": [\"37402772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Norrin (Norrie disease gene product, vertebrate ortholog of insect burs/pburs) is a ligand for LGR4 that stimulates Wnt signaling mediated by LGR4 but not by LGR5 or LGR6 in mammalian cells. Binding studies showed norrin interactions with LGR4, 5, and 6. Mutagenesis categorized Norrie disease mutations into subgroups with distinct defects in signaling through three binding partners.\",\n      \"method\": \"Cell-based Wnt signaling assays, binding studies, site-directed mutagenesis of norrin\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell signaling and binding assays with mutagenesis, single lab\",\n      \"pmids\": [\"23444378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Nidogen-2 is a novel endogenous ligand of LGR4 (KD ~26.77 nM, measured by surface plasmon resonance). Nidogen-2 binding to LGR4 biased-activates Gαq–PKCα–AMPKα1 signaling to counteract VSMC osteogenic transition and vascular calcification. LGR4 deficiency in VSMCs abolished the protective effect of nidogen-2.\",\n      \"method\": \"Unbiased membrane protein interactome, Co-IP, flow cytometric binding assay, surface plasmon resonance, G protein signaling assays, VSMC calcification assays, in vivo vascular calcification models with LGR4 KO\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — biophysical binding measurement plus multiple orthogonal mechanistic assays in vitro and in vivo\",\n      \"pmids\": [\"36354004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RSPO2 and RANKL both signal through LGR4 to modulate the WNT inhibitor DKK1 via Gαq and β-catenin signaling, promoting osteoclastic premetastatic niche formation. Soluble LGR4-ECD acting as a decoy receptor for RSPO2 and RANKL reduced bone metastasis in a mouse model.\",\n      \"method\": \"Unbiased GPCR ligand/agonist screening, binding assays, signaling pathway analysis (Gαq, β-catenin, DKK1), mouse bone metastasis models with LGR4-ECD treatment\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple assays but single lab; extends known RANKL/LGR4 mechanism to metastatic context\",\n      \"pmids\": [\"34847079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LGR4/5 LOF in liver disrupts Wnt/β-catenin signaling and hepatic metabolic zonation. The RSPO-LGR4/5-ZNRF3/RNF43 module is a master regulator of Wnt/β-catenin-mediated liver zonation; RSPO1-induced expansion of Wnt signaling gradient requires LGR4/5.\",\n      \"method\": \"Liver-specific Lgr4/5 LOF mice, RSPO1 protein treatment, ZNRF3/RNF43 LOF mice, functional Wnt reporter assays in liver\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic models with defined molecular readout; establishes pathway hierarchy\",\n      \"pmids\": [\"27088858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"LGR4 potentiates Wnt/β-catenin signaling through IQGAP1 in lung adenocarcinomas with aberrant RSPO3 expression. Knockdown of LGR4 or IQGAP1 reduced tumor growth and metastasis in vivo.\",\n      \"method\": \"Knockdown of LGR4/IQGAP1 in lung cancer cell lines, in vitro proliferation/migration assays, in vivo xenograft/metastasis models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo KD with defined pathway, single lab\",\n      \"pmids\": [\"25531322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"LGR4 promotes cancer cell metastasis through a Wnt-independent mechanism involving direct interaction with EGFR. LGR4 blocks EGFR ubiquitination and degradation, resulting in persistent EGFR activation. LGR4 mutants unable to potentiate Wnt signaling still promoted breast cancer migration/invasion in vitro and metastasis in vivo.\",\n      \"method\": \"Pharmacological and genetic Wnt deactivation, LGR4 mutant overexpression, multiomic screening, Co-IP of LGR4-EGFR, ubiquitination assays, in vitro migration/invasion, in vivo metastasis models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal approaches including Co-IP, ubiquitination assay, Wnt-independent mutants, and in vivo validation\",\n      \"pmids\": [\"34099494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Gpr48/LGR4 inactivation impairs definitive erythropoiesis at midgestation through down-regulation of the ATF4 signaling pathway via the cAMP-PKA-CREB pathway. Gpr48-/- fetal livers showed decreased c-Myc and cyclin D1 expression with suppressed proliferation.\",\n      \"method\": \"Gpr48-/- mice, histological analysis, blood smear assays, real-time PCR for hemoglobin chains and signaling components, proliferation/apoptosis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with defined molecular pathway, single lab\",\n      \"pmids\": [\"18955481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"GPR48/LGR4 regulates epithelial cell proliferation and migration during eyelid development through activation of the EGFR signaling pathway. Phosphorylation of EGFR was dramatically decreased in Gpr48-/- keratinocytes and developing eyelids, causing reduced F-actin formation, delayed leading-edge extension, and EOB phenotype.\",\n      \"method\": \"Gpr48-/- mice, histology, scanning EM, scratch assay, Western blot and immunostaining for EGFR phosphorylation\",\n      \"journal\": \"Investigative ophthalmology & visual science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with in vitro and in vivo readouts, defined signaling pathway, single lab\",\n      \"pmids\": [\"18487371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"GPR48/LGR4 mediates keratinocyte proliferation through HB-EGF-mediated EGFR transactivation. HB-EGF was reduced in Gpr48-/- cell culture medium; addition of HB-EGF restored EGFR, ERK, and STAT3 phosphorylation and cell proliferation. EGFR tyrosine kinase inhibitor AG1478 blocked GPR48-mediated cellular processes.\",\n      \"method\": \"EGFR inhibitor treatment, conditioned media immunodepletion of EGFR ligands, HB-EGF supplementation rescue, Western blot for EGFR/ERK/STAT3 phosphorylation\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic rescue experiment with inhibitor and ligand supplementation, single lab\",\n      \"pmids\": [\"20732323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Lgr4 ablation reduces adiposity and promotes white-to-brown fat switch by potentiating brown adipocyte differentiation, partly through Rb1 reduction. Lgr4 homozygous mutant mice show increased energy expenditure and express BAT markers in WAT depots.\",\n      \"method\": \"Lgr4 homozygous mutant mice, stromal vascular fraction differentiation assays, Rb1 expression analysis, metabolic phenotyping\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with mechanistic link to Rb1, single lab\",\n      \"pmids\": [\"24212090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Lgr4 gene inactivation reduces intestinal epithelial cell proliferation by 50% and Paneth cell terminal differentiation by 80%. LGR4-deficient but not LGR5-deficient crypts die in ex vivo culture with downregulation of stem cell markers and Wnt target genes including Lgr5. Partial rescue with LiCl (GSK3β inhibitor) but not Wnt agonists identifies LGR4 as a permissive factor in the intestinal Wnt pathway.\",\n      \"method\": \"Lgr4 knockout mice, ex vivo crypt culture, LiCl rescue, quantification of stem cell markers and Wnt targets\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse plus ex vivo rescue experiment with defined pathway position, single lab\",\n      \"pmids\": [\"21508962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Lgr4 deficiency in macrophages increases CD14 (TLR2/4 co-receptor) expression at the transcriptional level by decreasing CREB binding activity, thereby potentiating TLR2/4-associated cytokine production and increasing susceptibility to septic shock.\",\n      \"method\": \"Lgr4-/- mice, cytokine production assays, CREB binding assays, CD14 expression analysis in Lgr4-deficient macrophages\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO macrophages with defined CREB-CD14 transcriptional mechanism, single lab\",\n      \"pmids\": [\"23589304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Myeloid-specific Lgr4 knockout mice show reduced M2 tumor-associated macrophages and increased CD8+ T cell infiltration in tumors. Blocking Rspo/Lgr4/Erk/Stat3 signaling with LGR4-ECD or anti-Rspo1 antibody overcame resistance to anti-PD-1 therapy.\",\n      \"method\": \"Myeloid-specific conditional Lgr4 KO mice, tumor models, ERK/STAT3 signaling analysis, macrophage polarization assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined signaling pathway, multiple tumor models, single lab\",\n      \"pmids\": [\"29967265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Macrophage-specific Lgr4 knockout mice show improved post-MI cardiac repair with reduced inflammatory macrophages. Lgr4-mediated proinflammatory programming in macrophages occurs via enhancement of CREB-mediated c-Fos, Fosl1, and Fosb transactivation to activate AP-1.\",\n      \"method\": \"Macrophage-specific Lgr4 KO mice, MI model, transcriptomic profiling, AP-1/CREB activity assays, immune cell quantification\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined AP-1/CREB transcriptional mechanism, single lab with RNA-seq\",\n      \"pmids\": [\"32600176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Lgr4 regulates corpus luteum maturation through WNT-mediated EGFR-ERK signaling. In Lgr4-/- granulosa-lutein cells, EGFR-ERK signaling activity is attenuated. WNT/β-catenin downstream target MMP9 is reduced, impairing activation of EGF-like factors needed for EGFR-ERK activation and steroidogenesis.\",\n      \"method\": \"Lgr4-/- mice, primary granulosa cell culture, EGFR-ERK signaling assays, MMP9 inhibitor treatment, HB-EGF rescue\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with mechanistic pathway analysis and rescue experiments, single lab\",\n      \"pmids\": [\"24877628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"LGR4 controls mammary gland development and stem cell repopulation capacity through activation of Sox2 via the Lgr4/Wnt/β-catenin/Lef1 pathway. ChIP analysis showed Sox2 expression is controlled by this pathway; Sox2 overexpression restored mammary regeneration potential of Lgr4-/- stem cells.\",\n      \"method\": \"Lgr4-/- mice, mammosphere assays, Wnt signaling analysis, ChIP assay for β-catenin/Lef1 at Sox2 promoter, Sox2 overexpression rescue, in vivo mammary regeneration assay\",\n      \"journal\": \"Stem cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus functional rescue, KO mouse, single lab\",\n      \"pmids\": [\"23712846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Lgr4 is selectively expressed in peritubular myoid cells (PMCs) of testes, and Lgr4 loss arrests germ cells at meiosis I. Lgr4 in PMCs signals through Wnt/β-catenin to support spermatogenesis; re-activation of Wnt/β-catenin (Apc(min/+) cross or GSK3β inhibitor) partially rescued the Lgr4-/- testis phenotype.\",\n      \"method\": \"Lgr4-/- mice, Apc(min/+) cross, GSK3β inhibitor treatment, PMC gene expression analysis, meiosis staging\",\n      \"journal\": \"Development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis rescue, KO mouse, defined pathway, single lab\",\n      \"pmids\": [\"23533175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The miR-34–LGR4 axis regulates keratinocyte inflammatory response via GSK-3β-induced p65 serine 468 phosphorylation, modulating NF-κB signaling pathway activity. LGR4 was identified as a direct target of miR-34a and miR-34c mediating their proinflammatory function. Lgr4 knockout mice showed impaired wound closure with enhanced inflammation.\",\n      \"method\": \"miR-34 target validation, Lgr4 KO mice wound model, NF-κB signaling assays including p65 phosphorylation at Ser468, wound healing assays\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse phenotype plus defined signaling mechanism, single lab\",\n      \"pmids\": [\"31376385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RSPO-LGR4 signaling activates WNT/β-catenin pathway to induce follistatin (Fst) expression in myoblasts, which then antagonizes TGF-β signaling to promote myogenic differentiation. LGR4 depletion severely disrupts myogenic differentiation and abolishes RSPO2 response. Specific TCF/LEF1 binding sites within Fst promoter and intron 1 were required for RSPO2/WNT-induced Fst expression.\",\n      \"method\": \"LGR4 siRNA depletion in C2C12 myoblasts, RSPO2 stimulation assays, β-catenin activation/inactivation, promoter/ChIP analysis of Fst TCF/LEF1 sites\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter analysis with TCF/LEF1 site mutagenesis plus functional differentiation assays, single lab\",\n      \"pmids\": [\"24344199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"LGR4 (and LGR5) promote formation of long (>80 μm) actin-rich cytoneme-like membrane protrusions through stabilization of nascent filopodia. LGR5-induced cytonemes serve as conduits for β-arrestin-2 and Myosin X transit, linking GPCR signaling to cytoneme-based cell communication.\",\n      \"method\": \"Overexpression of Lgr4/Lgr5 in cells, live imaging, measurement of filopodial/cytoneme lengths, β-arrestin-2 and Myo10 trafficking assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — overexpression with live imaging and cargo tracking, replicated for both LGR4 and LGR5\",\n      \"pmids\": [\"25653388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LGR4 deficiency in mice results in delayed puberty with fewer GnRH neurons. LGR4 mutant proteins from delayed puberty patients showed impaired Wnt/β-catenin signaling due to defective protein expression, trafficking, and degradation. Lgr4 knockdown in zebrafish prevented formation and migration of GnRH neurons. Genetic lineage tracing showed strong Lgr4-mediated Wnt/β-catenin signaling activation during GnRH neuron development.\",\n      \"method\": \"Whole-exome sequencing, LGR4 mutant protein functional assays (expression/trafficking/degradation), Lgr4 KO mice, zebrafish lgr4 knockdown, genetic lineage tracing with Wnt/β-catenin reporter\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple model organisms, lineage tracing, and protein functional assays, single lab\",\n      \"pmids\": [\"32493844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LGR4 expression in multiple myeloma plasma cells is driven by IL-6/STAT3 signaling, allowing MM cells to hijack osteoblast-derived R-spondins and stabilize Wnt co-receptors via inhibition of RNF43/ZNRF3 E3 ligase activity, dramatically increasing Wnt sensitivity.\",\n      \"method\": \"IL-6/STAT3 signaling analysis, LGR4 overexpression in MM cells, R-spondin binding assays, Wnt receptor stabilization assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined transcriptional driver (IL-6/STAT3) plus Wnt receptor mechanism, single lab\",\n      \"pmids\": [\"28028233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In gastric stem cells, Rspo3 signaling via Lgr4 (not Lgr5) drives proliferation of stem cells and induces NF-κB activity. Upon H. pylori exposure, Lgr4-driven NF-κB activation causes expansion of the gland base module and chemokine expression in stem cells, leading to hyperplasia and neutrophil recruitment.\",\n      \"method\": \"Conditional Lgr4 KO mice, H. pylori infection model, NF-κB reporter assays, conditional Lgr5 KO comparison\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with defined NF-κB signaling mechanism and epistasis between Lgr4 and Lgr5, single lab\",\n      \"pmids\": [\"35767364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"LGR4-dependent Wnt/β-catenin signaling transcriptionally upregulates SLC7A11 (a ferroptosis inhibitor), and LGR4-mAb treatment blocks this pathway, sensitizing chemoresistant colorectal cancer cells to ferroptosis.\",\n      \"method\": \"Monoclonal antibody (LGR4-mAb) treatment, colorectal cancer organoid biobank, transcriptomic analysis, SLC7A11 expression assays, ferroptosis sensitivity assays\",\n      \"journal\": \"Nature cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — antibody blockade with defined transcriptional target and mechanistic readout, single lab\",\n      \"pmids\": [\"38291304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RSPO-LGR4 signaling upregulates key self-renewal genes and is essential for leukemia stem cell (LSC) self-renewal in HOXA9-dependent AML. LGR4 is epigenetically upregulated and cooperates with HOXA9. Blocking RSPO3-LGR4 interaction with anti-RSPO3 antibody impairs self-renewal and induces differentiation in patient-derived xenografts without affecting normal HSCs.\",\n      \"method\": \"Patient-derived xenograft (PDX) models, anti-RSPO3 antibody (OMP-131R10) treatment, epigenetic analysis, self-renewal assays, differentiation assays\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PDX models with defined epigenetic and self-renewal mechanisms, single lab\",\n      \"pmids\": [\"32559496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Lgr4 promotes aerobic glycolysis and osteoblast differentiation via canonical Wnt/β-catenin signaling by inducing pyruvate dehydrogenase kinase 1 (pdk1) expression. Lgr4 osteoblast-specific KO mice exhibited decreased bone mass and strength with impaired glycolysis.\",\n      \"method\": \"Osteoblast-specific Lgr4 KO mice (Lgr4osb-/-), glucose/lactate metabolism assays, Wnt/β-catenin activation rescue, pdk1 expression analysis\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with metabolic and molecular mechanistic readout, single lab\",\n      \"pmids\": [\"33950533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"LGR4 in the hypothalamic arcuate nucleus co-localizes with NPY, POMC, and BDNF neurons. Intracerebroventricular injection of Rspo1 or Rspo3 (ligands acting through LGR4) inhibited food intake, with Rspo1 decreasing NPY and increasing POMC expression. Rspo1 and Rspo3 mRNAs were down-regulated by fasting and up-regulated by insulin.\",\n      \"method\": \"In situ hybridization, intracerebroventricular injection, food intake measurement, neuropeptide expression assays\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo ligand injection with defined neurochemical readouts, single lab\",\n      \"pmids\": [\"24280058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"LGR4 acts as a link between the peripheral circadian clock and lipid metabolism by regulating circadian expression of Mttp (microsomal triglyceride transfer protein) in hepatocytes, thereby controlling plasma triglyceride rhythms.\",\n      \"method\": \"Lgr4 mutant mice, measurement of respiratory exchange ratio and plasma lipid rhythms, circadian gene expression analysis in liver and primary hepatocytes\",\n      \"journal\": \"Journal of molecular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined molecular target (Mttp) in KO mouse, single lab\",\n      \"pmids\": [\"24353284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"LGR4 activation by Rspo1 in hepatocytes reduces TNFα-induced cell death by inhibiting NF-κB nuclear translocation. LGR4 knockdown increased NF-κB-p65 nuclear translocation and caspase-3 levels after acute injury. IKKβ overexpression attenuated Rspo1 protection, and β-catenin disruption reversed protective effects.\",\n      \"method\": \"Liver-specific Lgr4 KO mice, primary hepatocyte culture, TNFα treatment, NF-κB signaling assays, IKKβ overexpression, β-catenin disruption, HIRI and LPS/D-Gal liver injury models\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with multiple in vitro and in vivo injury models and defined NF-κB mechanism, single lab\",\n      \"pmids\": [\"30406697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Rspo1/Rspo3-LGR4 signaling inhibits hepatic cholesterol synthesis via the AMPKα-SREBP2 pathway. LGR4 knockdown increased hepatic cholesterol synthesis and decreased AMPKα phosphorylation. AMPKα agonist/antagonist experiments confirmed AMPKα as the downstream mediator of Rspos-LGR4 effects on SREBP2 nuclear translocation.\",\n      \"method\": \"Lgr4 and Rspo1/3 knockdown mice, AMPKα agonist/antagonist treatment, AMPKα shRNA, SREBP2 nuclear translocation assays, cholesterol synthesis measurements\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological dissection of AMPKα-SREBP2 pathway, single lab\",\n      \"pmids\": [\"32926477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"LGR4/GPR48 inactivation down-regulates histone demethylases Jmjd2a and Fbxl10 through cAMP-CREB signaling pathways in embryonic fibroblasts and urinary/reproductive tissues, contributing to WAGR/AGR syndrome-like defects.\",\n      \"method\": \"Lgr4-/- mice, gene expression analysis in MEFs and tissues, cAMP-CREB pathway assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with defined epigenetic regulatory target via cAMP-CREB pathway, single lab\",\n      \"pmids\": [\"24519938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"LGR4/GPR48 loss in male mice leads to failed tube elongation and convolution of efferent ducts and epididymis with dramatically reduced cell proliferation. In efferent ducts, LGR4 inactivation decreases estrogen receptor (ESR1) and SLC9A3 expression, causing water reabsorption failure and rete testis dilation.\",\n      \"method\": \"LGR4 KO mice, histology, cell proliferation assays, gene expression analysis for ESR1 and SLC9A3\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with defined molecular targets (ESR1/SLC9A3), single lab\",\n      \"pmids\": [\"17079737\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"LGR4 (GPR48) is a leucine-rich repeat-containing GPCR that functions as a high-affinity receptor for R-spondins (binding via their furin domains to the concave surface of the LGR4 horseshoe-shaped ECD, as established by crystal structures), and potentiates Wnt/β-catenin signaling by forming constitutive complexes with the E3 ubiquitin ligases ZNRF3/RNF43 through its seven-transmembrane domain (thereby protecting Frizzled/LRP5/6 from degradation) and by recruiting IQGAP1 into the Wnt signalosome to promote MEK1/2-mediated LRP5/6 phosphorylation; additionally, LGR4 acts as a second receptor for RANKL that activates Gαq-GSK3β signaling to suppress NFATc1 and osteoclastogenesis, binds nidogen-2 to activate Gαq-PKCα-AMPKα1 signaling against vascular calcification, and signals independently of Wnt via direct interaction with EGFR to prevent its ubiquitination and degradation, with all these activities broadly required for development, stem cell maintenance, immune regulation, and metabolic homeostasis across multiple tissues.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"LGR4 is a leucine-rich-repeat-containing seven-transmembrane receptor that functions as the high-affinity engagement receptor for R-spondins to potentiate Wnt/\\u03b2-catenin signaling, and serves more broadly as a hub for stem cell maintenance, development, immune regulation, and metabolic homeostasis across many tissues [#0, #15, #22]. R-spondins bind the concave surface of the horseshoe-shaped LGR4 ectodomain through their two furin-like cysteine-rich domains in a one-site binding mode, as resolved by multiple crystal structures [#3, #4, #5]. Rather than coupling to heterotrimeric G proteins or \\u03b2-arrestin upon R-spondin stimulation, LGR4 acts non-canonically: through its seven-transmembrane domain it forms a constitutive complex with the membrane E3 ubiquitin ligases ZNRF3/RNF43, and R-spondin engagement drives clearance of these ligases to protect Wnt receptors and enhance LRP6 phosphorylation, with LGR4 serving as the \\\"engagement\\\" receptor and ZNRF3/RNF43 as the \\\"effector\\\" receptors in a dual-receptor model [#0, #6, #10]. Downstream, RSPO-LGR4 recruits the scaffold IQGAP1 to bridge the receptor to the Wnt signalosome and promote MEK1/2-mediated LRP5/6 phosphorylation, also driving actin/cytoneme-based protrusions [#7, #31]. Beyond R-spondins, LGR4 binds additional ligands\\u2014RANKL, where it competes with RANK and activates G\\u03b1q-GSK3\\u03b2 signaling to suppress NFATc1 and osteoclastogenesis [#9]; nidogen-2, which biased-activates G\\u03b1q-PKC\\u03b1-AMPK\\u03b11 signaling to counteract vascular calcification [#13]; and norrin, which stimulates LGR4-dependent Wnt signaling [#12]. LGR4 also signals independently of Wnt by directly interacting with EGFR to block its ubiquitination and sustain its activation [#17]. Through these activities LGR4 governs intestinal, gastric, mammary, and leukemic stem cell self-renewal, liver metabolic zonation, osteoblast/osteoclast balance, macrophage polarization, and reproductive development, and its mutations cause delayed puberty via impaired Wnt/\\u03b2-catenin signaling in GnRH neurons [#15, #22, #32, #36].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established LGR4 as the high-affinity R-spondin receptor that potentiates Wnt/\\u03b2-catenin signaling through a non-canonical mechanism that does not use classical GPCR effectors, resolving how RSPOs amplify Wnt.\",\n      \"evidence\": \"Binding, LRP6 phosphorylation, G-protein and \\u03b2-arrestin coupling assays in mammalian cells and Xenopus, plus domain mapping and siRNA depletion\",\n      \"pmids\": [\"21693646\", \"21909076\", \"22815884\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of the RSPO interface\", \"The intracellular effector translating ligand binding into Wnt potentiation was unidentified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the atomic basis of receptor-ligand recognition, showing RSPO furin-like domains dock onto the concave face of the LGR4 horseshoe ectodomain via a one-site binding mode.\",\n      \"evidence\": \"X-ray crystallography of LGR4 ECD alone and with RSPO1, with mutagenesis validation; confirmed by independent structures\",\n      \"pmids\": [\"23756652\", \"23891289\", \"25480784\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structures covered the ectodomain only, not the seven-transmembrane region or full-length receptor\", \"Did not explain how ligand binding signals across the membrane\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Resolved the downstream signaling machinery, establishing a dual-receptor model where LGR4 engages RSPO and clears the E3 ligases ZNRF3/RNF43, and identifying IQGAP1 as the intracellular scaffold linking LGR4 to the Wnt signalosome.\",\n      \"evidence\": \"Mutagenesis of distinct binding motifs, Co-IP, membrane clearance assays, in vitro ternary complex reconstitution, and MEK/LRP6 phosphorylation assays\",\n      \"pmids\": [\"24165923\", \"24639526\", \"24050775\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the receptor-ligase complex was not yet defined\", \"Constitutive versus ligand-dependent assembly of the complex was unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Distinguished LGR4 from LGR5 mechanistically, showing LGR4 forms a constitutive ZNRF3/RNF43 complex via its seven-transmembrane domain as a 2:2 dimer accommodating bivalent RSPO, whereas LGR5 does not bind the E3 ligases.\",\n      \"evidence\": \"Co-IP, proximity ligation, TR-FRET, domain-swapping, and monovalent/bivalent RSPO binding assays in whole cells\",\n      \"pmids\": [\"33262293\", \"37402772\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length structure of the receptor-ligase dimer\", \"Functional consequences of dimerization for signaling output not fully mapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Expanded LGR4 beyond Wnt by identifying it as a second RANKL receptor that competes with RANK and signals via G\\u03b1q-GSK3\\u03b2 to suppress osteoclastogenesis, revealing a G-protein-coupled mode distinct from RSPO signaling.\",\n      \"evidence\": \"Binding competition, Lgr4 KO and conditional KO mice, osteoclast differentiation, G\\u03b1q/GSK3-\\u03b2 signaling assays, and in vivo soluble LGR4-ECD therapy\",\n      \"pmids\": [\"27064449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why RANKL triggers G\\u03b1q coupling while RSPO does not was unexplained\", \"Structural basis of RANKL\\u2013LGR4 interaction not determined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified nidogen-2 as a further endogenous ligand activating biased G\\u03b1q-PKC\\u03b1-AMPK\\u03b11 signaling, demonstrating that LGR4 integrates multiple ligands into distinct G-protein-dependent outputs in the vasculature.\",\n      \"evidence\": \"Membrane interactome, Co-IP, SPR, G-protein signaling assays, and in vivo vascular calcification models with LGR4 KO\",\n      \"pmids\": [\"36354004\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand-binding site on LGR4 for nidogen-2 not structurally defined\", \"How distinct ligands selectively bias different G-protein pathways unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established a Wnt-independent function whereby LGR4 directly binds EGFR and blocks its ubiquitination and degradation, decoupling LGR4's oncogenic activity from its canonical Wnt-potentiating role.\",\n      \"evidence\": \"Wnt-deactivation experiments, Wnt-incompetent LGR4 mutants, Co-IP, ubiquitination assays, and in vivo metastasis models\",\n      \"pmids\": [\"34099494\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EGFR stabilization requires a ligand or is constitutive is unclear\", \"Earlier reports of EGFR-pathway effects via HB-EGF transactivation versus direct interaction not reconciled\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked LGR4 to human disease by showing LGR4 mutations cause delayed puberty through impaired Wnt/\\u03b2-catenin signaling required for GnRH neuron formation and migration.\",\n      \"evidence\": \"Whole-exome sequencing, LGR4 mutant protein trafficking/degradation assays, Lgr4 KO mice, zebrafish knockdown, and Wnt reporter lineage tracing\",\n      \"pmids\": [\"32493844\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-cohort genetic evidence\", \"Direct rescue of the neuronal phenotype by wild-type LGR4 not shown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single receptor selects among R-spondin (non-G-protein), RANKL/nidogen-2 (G\\u03b1q-coupled), and EGFR-stabilizing modes from the same seven-transmembrane scaffold remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length structure capturing transmembrane signaling states\", \"Determinants of ligand-biased G-protein coupling versus E3-ligase recruitment are unknown\", \"Mechanism switching between Wnt-dependent and Wnt-independent outputs is uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 9, 13]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [3, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 10, 17]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [9, 10, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 7, 9, 13]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [15, 22, 32]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [23, 24, 25]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [37, 39, 41]}\n    ],\n    \"complexes\": [\n      \"LGR4\\u2013ZNRF3/RNF43 receptor complex\",\n      \"RSPO\\u2013LGR4\\u2013ZNRF3 ternary complex\"\n    ],\n    \"partners\": [\n      \"RSPO1\",\n      \"RSPO2\",\n      \"RSPO3\",\n      \"ZNRF3\",\n      \"RNF43\",\n      \"IQGAP1\",\n      \"EGFR\",\n      \"NID2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}