{"gene":"FLRT2","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":1999,"finding":"FLRT1 and FLRT2 are glycosylated type I transmembrane proteins migrating at 90 and 85 kDa respectively, containing 10 leucine-rich repeats, flanking cysteine-rich regions, a fibronectin/collagen-like domain, and an intracellular tail, as demonstrated by expression in SF9 and COS-1 cells.","method":"Heterologous expression in SF9 and COS-1 cells, SDS-PAGE, glycosylation analysis","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein characterization in two cell systems, single lab, structural features confirmed biochemically","pmids":["10644439"],"is_preprint":false},{"year":2011,"finding":"FLRT2 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for Unc5D-positive neurons. FLRT2 binds specifically to Unc5D receptor. During neocortical development, shed FLRT2 ECD repels SVZ neurons (which express Unc5D) from the FLRT2-expressing cortical plate, modulating cortical neuron migration. Deletion of FLRT2 or Unc5D causes premature migration of SVZ-derived neurons toward the cortical plate.","method":"Binding assays, mouse genetics (conditional knockout, overexpression), in vivo cortical neuron migration analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetics (both FLRT2 and Unc5D knockouts show matching phenotypes), binding assays, replicated with gain- and loss-of-function","pmids":["21673655"],"is_preprint":false},{"year":2011,"finding":"FLRT2 is required in the epicardium for heart morphogenesis; Flrt2-null mouse embryos arrest at mid-gestation with cardiac insufficiency, defects in epicardial sheet integrity, and disrupted basement membrane organization. In vitro and in vivo reconstitution assays show FLRT2 and FLRT3 are functionally interchangeable in maintaining cell-cell contacts.","method":"Gene targeting (knockout mouse), in vitro reconstitution, in vivo reconstitution assay","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — knockout with defined phenotype combined with reconstitution assays showing functional interchangeability","pmids":["21350012"],"is_preprint":false},{"year":2011,"finding":"FLRT2 physically interacts with FGFR2 both extracellularly (via FLRT2 leucine-rich repeat domain) and intracellularly (via FLRT2 C-tail domain). FLRT2 knockdown or overexpression causes concomitant decrease or increase in FGFR2 protein, mRNA, and ERK phosphorylation levels, indicating FLRT2 positively regulates FGF signaling in a feedback loop.","method":"Co-immunoprecipitation with embryonic craniofacial tissue lysates, GST pulldown, yeast two-hybrid, stable shRNA knockdown and cDNA overexpression with Western blot and ERK phosphorylation assay","journal":"Journal of dental research","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal methods (Co-IP, GST pulldown, Y2H, functional signaling assays) in single lab","pmids":["21765038"],"is_preprint":false},{"year":2011,"finding":"FLRT2 promotes cellular proliferation and inhibits cell-cell adhesion during early chondrogenesis. Knockdown of FLRT2 in ATDC5 cells slows proliferation, increases N-cadherin expression, and increases PNA-positive aggregates; overexpression has opposite effects. FLRT2 knockdown also reduces cell migration in wound healing assay.","method":"Stable transfection (shRNA knockdown and cDNA overexpression), proliferation assay, lectin PNA staining, N-cadherin Western blot, wound healing assay, Alcian blue staining","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with multiple cellular readouts, single lab","pmids":["21769912"],"is_preprint":false},{"year":2014,"finding":"FLRT2 is present on the cell membrane and in the extracellular matrix of ATDC5 chondroprogenitor cells, where it co-localizes and co-immunoprecipitates with fibronectin. Extracellular FLRT2 accumulation depends on fibronectin fibril formation. Western blot with antibodies to extra- and intracellular domains reveals multiple size bands, suggesting FLRT2 exists in both membrane-bound and shed/cleaved forms.","method":"Immunolocalization, co-immunoprecipitation, fibronectin blocking peptide experiment, Western blot with domain-specific antibodies","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and localization with functional perturbation (blocking peptide), single lab, multiple complementary approaches","pmids":["24585683"],"is_preprint":false},{"year":2017,"finding":"FLRT2 is expressed in endothelial cells of the placental labyrinth. Endothelial cell-specific deletion of FLRT2 in mice causes embryonic lethality with aberrant endothelial alignment and disrupted feto-maternal circulation, mediated through endothelial repulsion via binding to the UNC5B receptor.","method":"Conditional endothelial knockout mouse, embryonic lethality analysis, histological analysis of placental labyrinth, UNC5B binding assay","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with defined molecular mechanism (FLRT2-UNC5B binding) and clear cellular phenotype","pmids":["28576770"],"is_preprint":false},{"year":2017,"finding":"FLRT2 overexpression inhibits breast cancer cell proliferation and migration, while knockdown increases them. FLRT2 overexpression significantly decreases cell adhesion on collagen I-coated plates. Pathway analysis identifies 'cellular growth and proliferation/Cancer' as the top affected pathway.","method":"shRNA knockdown, cDNA overexpression, proliferation assay, migration assay, colony formation assay, adhesion assay, pathway analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional (gain and loss-of-function) with multiple phenotypic readouts, single lab","pmids":["28325946"],"is_preprint":false},{"year":2018,"finding":"FLRT2 haplodeficiency in female mice enhances hippocampal synaptic plasticity and spatial memory recall, accompanied by decreased EAAT2 (glutamate transporter) levels and decreased beta estrogen receptor levels. Male FLRT2+/- mice show deficient memory recall and decreased alpha estrogen receptor levels.","method":"FLRT2+/- knockout mice, in vivo behavioral testing, electrophysiology (LTP), Western blot for EAAT2 and estrogen receptors","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic model with electrophysiology and molecular correlates, single lab, sex-specific effects replicated across multiple assays","pmids":["29487336"],"is_preprint":false},{"year":2019,"finding":"FLRT2 regulates osteoclast multinucleation. FLRT2 expression is induced by RANKL in osteoclast precursors. FLRT2 deficiency reduces hyper-multinucleation, rescued by Unc5B knockdown. FLRT2 promotes Rac1 activation downstream of RANKL; Flrt2-deficient cells show attenuated RANKL-induced Rac1 activation. FLRT2 interferes with Netrin1-Unc5B interaction to modulate multinucleation.","method":"FLRT2-deficient osteoclast differentiation assay, Unc5B RNAi rescue, Netrin1 treatment, Rac1 activation assay","journal":"BMB reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with genetic rescue (Unc5B knockdown) and Rac1 signaling readout, single lab","pmids":["31383250"],"is_preprint":false},{"year":2021,"finding":"FLRT2 and FLRT3 double deletion in mice causes abnormal distribution of cortical interneurons within tangential migratory streams during development, affecting somatostatin+ interneuron laminar positioning postnatally. FLRT2/FLRT3 act non-cell-autonomously as chemorepellents for interneurons in vitro, partly through Unc5B/Unc5D receptors (double Unc5B/Unc5D knockout phenocopies the FLRT double knockout).","method":"FLRT2/FLRT3 double conditional knockout mice, interneuron migration in vitro repulsion assay, Unc5B/Unc5D double knockout genetic epistasis","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — double knockout genetics with in vivo and in vitro phenotype, epistasis with Unc5 receptors, multiple orthogonal approaches","pmids":["34301831"],"is_preprint":false},{"year":2021,"finding":"In the adult hippocampus, FLRT2 expression is confined to CA1 and is partly localized on pre- and post-synapses, as shown by Flrt2-LacZ knock-in reporter mice and subcellular fractionation. After spinal cord injury, FLRT2 is transiently upregulated in reactive astrocytes around lesion sites at 7 days post-injury.","method":"Flrt2-LacZ knock-in mice, X-gal histochemistry, subcellular synaptic fractionation, spinal cord injury model","journal":"Frontiers in molecular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knock-in reporter with fractionation data, direct localization to synaptic compartments, single lab","pmids":["34744626"],"is_preprint":false},{"year":2022,"finding":"FLRT2 forms noncanonical interendothelial adhesions through homophilic binding, safeguarding against oxidative stress. In tumor vasculature, endothelial FLRT2 deletion selectively prunes abnormalized vessels, creating 'oxygen-glucose uncoupling' that suppresses tumor metastasis and increases antitumor effects of immune checkpoint blockers.","method":"Endothelial cell-specific Flrt2 deletion in mice, tumor models, homophilic binding assays, metabolic analysis","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with multiple tumor models, homophilic binding mechanistic data, metabolic phenotyping","pmids":["35104247"],"is_preprint":false},{"year":2022,"finding":"FLRT2 dimerizes in cis (on the same cell) via two Small-X3-Small motifs in its transmembrane helix, which synergize with a third dimerization motif in the extracellular domain. This cis dimerization was demonstrated by molecular dynamics simulations and validated by single particle tracking showing co-diffusion of FLRT2 receptors on live cells.","method":"Molecular dynamics simulations, single particle tracking (SPT) live cell imaging, transmembrane motif mutagenesis","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — MD simulations with experimental validation by SPT and mutagenesis of dimerization motifs, single lab with two orthogonal methods","pmids":["35700726"],"is_preprint":false},{"year":2023,"finding":"In the mouse retina, FLRT2 is expressed by direction-selective (DS) circuit neurons and mediates rejection of inappropriate synaptic partners via transcellular FLRT2-UNC5C/D signaling. UNC5C/D are expressed by non-DS neurons projecting to adjacent IPL sublayers. FLRT2-UNC5 binding eliminates misguided DS dendrites; loss of FLRT2-UNC5 binding allows mistargeted dendrites to elaborate and acquire inappropriate synapses. UNC5s promote dendrite elimination by interfering with FLRT2-mediated adhesion.","method":"In vivo gain- and loss-of-function (FLRT2 knockout, UNC5C misexpression), retinal imaging, synapse analysis","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal gain and loss-of-function in vivo with clear mechanistic model (UNC5 interferes with FLRT2 adhesion), multiple experimental approaches","pmids":["37557174"],"is_preprint":false},{"year":2023,"finding":"FLRT2 elevates ACSL4 expression, increases lipid peroxidation, and subsequently facilitates ferroptosis in bladder cancer cells, thereby inhibiting tumor cell growth, migration, and invasion.","method":"FLRT2 overexpression and silencing in bladder cancer cells, ACSL4 expression assay, lipid peroxidation assay, cell viability/migration/invasion assays","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — bidirectional manipulation with mechanistic pathway (ACSL4/lipid peroxidation/ferroptosis), single lab","pmids":["37480224"],"is_preprint":false},{"year":2024,"finding":"FLRT2 directly associates with integrin subunit beta 4 (ITGB4) and promotes ITGB4 phosphorylation; inhibition of ITGB4 substantially mitigates the induction of endothelial senescence triggered by FLRT2 depletion. FLRT2 prevents endothelial senescence through the mTORC2/AKT/p53 signaling pathway.","method":"Co-immunoprecipitation (FLRT2-ITGB4 interaction), ITGB4 inhibition, FLRT2 knockdown in endothelial cells, Western blot for mTORC2/AKT/p53 pathway, mouse vascular aging model","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding identified by Co-IP, pathway epistasis with ITGB4 inhibition, in vivo mouse aging model with rescue experiment","pmids":["38587072"],"is_preprint":false},{"year":2024,"finding":"FLRT2 interacts with VE-cadherin in endothelial cells of the CNS vasculature; together with the endocytic adaptor Numb, FLRT2 modulates adherens junction morphology. FLRT2 in cortical vessels also regulates crosstalk between adherens and tight junctions, influencing blood-brain barrier development. FLRT2 deletion in endothelial cells specifically impairs retinal vein-driven endothelial proliferation, sprouting, and polarity.","method":"Conditional endothelial FLRT2 deletion (early postnatal), co-immunoprecipitation (FLRT2-VE-cadherin), expansion microscopy for junction morphology, BBB integrity assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with Co-IP interaction data, expansion microscopy validation, multiple vascular phenotypes","pmids":["39609404"],"is_preprint":false},{"year":2024,"finding":"FLRT2 protein is ubiquitinated and degraded by the E3 ubiquitin ligase NEDD4 in NSCLC cells. NEDD4 overexpression abolishes the tumor-suppressive effects of FLRT2 on cancer cell stemness, proliferation, and drug resistance in vitro and in vivo.","method":"Co-immunoprecipitation (NEDD4-FLRT2), ubiquitination assay, NEDD4 overexpression rescue experiment, xenograft mouse model","journal":"Frontiers in pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP confirming binding, functional rescue experiments, in vivo xenograft, single lab","pmids":["39444619"],"is_preprint":false},{"year":2026,"finding":"FLRT2 promotes foam cell formation in macrophages by suppressing ABCA1-mediated cholesterol efflux at the post-transcriptional level. Mechanistically, FLRT2 enhances ABCA1 ubiquitin-proteasome degradation by inhibiting USP22-mediated deubiquitination, defining a FLRT2-USP22-ABCA1 axis. Macrophage-specific Flrt2 deletion reduces atherosclerotic plaque burden in multiple mouse models.","method":"Macrophage-specific Flrt2 conditional knockout mice, adenovirus-mediated FLRT2 overexpression, ABCA1 ubiquitination assay, USP22 interaction/functional assay, cholesterol efflux assay, atherosclerosis mouse models","journal":"Acta pharmacologica Sinica","confidence":"High","confidence_rationale":"Tier 2 / Strong — macrophage-specific knockout with mechanistic dissection of USP22-ABCA1 ubiquitination axis, multiple mouse atherosclerosis models","pmids":["42174129"],"is_preprint":false}],"current_model":"FLRT2 is a shed/membrane-bound leucine-rich repeat transmembrane protein that functions as a repulsive ligand for Unc5 receptors (Unc5B, Unc5C, Unc5D) to control neuronal migration, axon guidance, synaptic partner selection, and osteoclast multinucleation; it also forms homophilic cis-dimers via transmembrane Small-X3-Small motifs and trans-adhesive complexes with VE-cadherin to regulate endothelial organization and vascular development; it positively regulates FGF/ERK signaling through physical interaction with FGFR2, prevents endothelial senescence by binding ITGB4 and activating mTORC2/AKT, and in macrophages promotes ABCA1 degradation by inhibiting USP22 deubiquitination, while its own stability is regulated by NEDD4-mediated ubiquitination."},"narrative":{"mechanistic_narrative":"FLRT2 is a glycosylated type I transmembrane protein with ten leucine-rich repeats and a fibronectin/collagen-like domain that functions as a shed repulsive ligand and adhesion molecule governing neuronal migration, vascular organization, and synaptic partner selection [PMID:10644439, PMID:21673655, PMID:28576770]. Its ectodomain is cleaved from cells and binds the Unc5 family of receptors (Unc5B, Unc5C, Unc5D) to deliver repulsive guidance: shed FLRT2 repels Unc5D-positive neurons from the cortical plate, controls cortical interneuron laminar positioning together with FLRT3, and directs rejection of inappropriate synaptic partners in the retina, where Unc5 binding antagonizes FLRT2-mediated adhesion to eliminate misguided dendrites [PMID:21673655, PMID:34301831, PMID:37557174]. In the vasculature, FLRT2 acts in endothelial cells to organize feto-maternal circulation through UNC5B-mediated repulsion and forms noncanonical homophilic interendothelial adhesions—stabilized by cis-dimerization through transmembrane Small-X3-Small motifs—that protect against oxidative stress; it also interacts with VE-cadherin and the adaptor Numb to remodel adherens junctions and shape blood-brain barrier development [PMID:28576770, PMID:35104247, PMID:35700726, PMID:39609404]. FLRT2 positively regulates FGF/ERK signaling through direct extracellular and intracellular interaction with FGFR2 [PMID:21765038], prevents endothelial senescence by associating with ITGB4 and engaging the mTORC2/AKT/p53 pathway [PMID:38587072], and promotes osteoclast multinucleation by driving RANKL-induced Rac1 activation [PMID:31383250]. FLRT2 acts as a tumor suppressor in several epithelial cancers, restraining proliferation and migration [PMID:28325946], and in bladder cancer it elevates ACSL4 to drive ferroptosis [PMID:37480224]; its protein levels are controlled by NEDD4-mediated ubiquitination and degradation [PMID:39444619]. In macrophages, FLRT2 suppresses ABCA1-dependent cholesterol efflux by inhibiting USP22-mediated deubiquitination of ABCA1, promoting foam cell formation and atherosclerotic plaque burden [PMID:42174129].","teleology":[{"year":1999,"claim":"Established FLRT2 as a discrete protein, defining the domain architecture that would later explain its dual roles as a ligand and adhesion molecule.","evidence":"Heterologous expression in SF9 and COS-1 cells with SDS-PAGE and glycosylation analysis","pmids":["10644439"],"confidence":"Medium","gaps":["No function assigned at this stage","No binding partners identified","Cleavage/shedding not yet demonstrated"]},{"year":2011,"claim":"Showed FLRT2 ectodomains are shed and act as a repulsive ligand for Unc5D, answering how FLRT2 guides neuronal migration non-cell-autonomously.","evidence":"Binding assays and reciprocal mouse genetics (FLRT2 and Unc5D knockouts) with cortical migration analysis","pmids":["21673655"],"confidence":"High","gaps":["Protease responsible for shedding not identified","Downstream signaling in Unc5D neurons not dissected"]},{"year":2011,"claim":"Defined an essential developmental requirement for FLRT2 in epicardial integrity and basement membrane organization, with FLRT3 functional interchangeability.","evidence":"Knockout mouse phenotyping plus in vitro and in vivo reconstitution assays","pmids":["21350012"],"confidence":"High","gaps":["Molecular receptor/partner mediating epicardial adhesion not defined here","Relationship to Unc5 repulsion unclear"]},{"year":2011,"claim":"Linked FLRT2 to growth factor signaling by showing direct extracellular and intracellular interaction with FGFR2 and positive feedback on ERK activation.","evidence":"Co-IP from craniofacial tissue, GST pulldown, yeast two-hybrid, knockdown/overexpression with ERK phosphorylation readout","pmids":["21765038"],"confidence":"High","gaps":["Structural basis of FGFR2 binding not resolved","Whether this operates in cis or via shed ectodomain unclear"]},{"year":2011,"claim":"Demonstrated FLRT2 controls proliferation versus adhesion balance in chondroprogenitors, inversely regulating N-cadherin.","evidence":"shRNA/overexpression in ATDC5 cells with proliferation, PNA staining, N-cadherin Western blot and wound healing assays","pmids":["21769912"],"confidence":"Medium","gaps":["Mechanism linking FLRT2 to N-cadherin not defined","Receptor not identified"]},{"year":2014,"claim":"Placed FLRT2 in the extracellular matrix in association with fibronectin, supporting both membrane-bound and shed/cleaved pools.","evidence":"Immunolocalization, Co-IP, fibronectin blocking peptide and domain-specific Western blots in ATDC5 cells","pmids":["24585683"],"confidence":"Medium","gaps":["Functional consequence of fibronectin association not established","Cleavage mechanism not defined"]},{"year":2017,"claim":"Extended FLRT2 repulsion to the vasculature, showing endothelial FLRT2-UNC5B signaling organizes placental endothelial alignment and is essential for embryonic circulation.","evidence":"Endothelial-specific conditional knockout mouse with histology and UNC5B binding assay","pmids":["28576770"],"confidence":"High","gaps":["Intracellular signaling downstream of endothelial repulsion not defined"]},{"year":2017,"claim":"Identified FLRT2 as a tumor suppressor in breast cancer, restraining proliferation, migration, and collagen adhesion.","evidence":"Bidirectional knockdown/overexpression with proliferation, migration, colony, and adhesion assays plus pathway analysis","pmids":["28325946"],"confidence":"Medium","gaps":["Molecular mediator of tumor suppression not identified","In vivo validation absent in this study"]},{"year":2018,"claim":"Revealed sex-specific roles for FLRT2 in hippocampal synaptic plasticity and memory, with estrogen receptor and glutamate transporter correlates.","evidence":"FLRT2+/- mice with behavioral testing, LTP electrophysiology, and Western blots for EAAT2 and estrogen receptors","pmids":["29487336"],"confidence":"Medium","gaps":["Causal link between FLRT2 and EAAT2/estrogen receptor changes not established","Receptor mediating synaptic effect unknown"]},{"year":2019,"claim":"Showed FLRT2 promotes osteoclast multinucleation downstream of RANKL by activating Rac1 and modulating Netrin1-Unc5B interaction.","evidence":"FLRT2-deficient osteoclast differentiation with Unc5B RNAi rescue, Netrin1 treatment, and Rac1 activation assay","pmids":["31383250"],"confidence":"Medium","gaps":["Direct link from Unc5B to Rac1 not fully resolved","In vivo bone phenotype not established here"]},{"year":2021,"claim":"Demonstrated FLRT2/FLRT3 act as chemorepellents controlling cortical interneuron migration and laminar positioning via Unc5B/Unc5D.","evidence":"FLRT2/FLRT3 double conditional knockout with in vitro repulsion assay and Unc5B/Unc5D double knockout epistasis","pmids":["34301831"],"confidence":"High","gaps":["Relative contributions of FLRT2 versus FLRT3 not separated","Receptor-bearing cell signaling not dissected"]},{"year":2021,"claim":"Localized adult FLRT2 to CA1 pre- and post-synapses and showed injury-induced upregulation in reactive astrocytes.","evidence":"Flrt2-LacZ knock-in reporter mice, X-gal histochemistry, synaptic fractionation, and spinal cord injury model","pmids":["34744626"],"confidence":"Medium","gaps":["Synaptic function of CA1 FLRT2 not directly tested","Role of injury-induced astrocytic FLRT2 unknown"]},{"year":2022,"claim":"Established that FLRT2 forms noncanonical homophilic interendothelial adhesions protecting against oxidative stress, with therapeutic relevance to tumor vessel pruning.","evidence":"Endothelial-specific Flrt2 deletion in tumor models with homophilic binding and metabolic analyses","pmids":["35104247"],"confidence":"High","gaps":["Molecular basis coupling homophilic adhesion to oxidative stress protection not fully defined"]},{"year":2022,"claim":"Defined the structural mechanism of FLRT2 cis-dimerization through transmembrane Small-X3-Small motifs synergizing with an extracellular motif.","evidence":"Molecular dynamics simulations validated by single particle tracking and transmembrane motif mutagenesis","pmids":["35700726"],"confidence":"High","gaps":["Functional consequence of cis-dimerization for ligand/adhesion activity not directly tested"]},{"year":2023,"claim":"Showed FLRT2-UNC5C/D transcellular signaling mediates synaptic partner rejection in the retina, with UNC5 antagonizing FLRT2 adhesion to eliminate misguided dendrites.","evidence":"In vivo FLRT2 knockout and UNC5C misexpression with retinal imaging and synapse analysis","pmids":["37557174"],"confidence":"High","gaps":["Intracellular effectors of dendrite elimination not identified"]},{"year":2023,"claim":"Connected FLRT2 tumor suppression in bladder cancer to ferroptosis via ACSL4 elevation and lipid peroxidation.","evidence":"FLRT2 overexpression/silencing with ACSL4 expression, lipid peroxidation, and viability/migration/invasion assays","pmids":["37480224"],"confidence":"Medium","gaps":["Mechanism linking FLRT2 to ACSL4 regulation not defined","Receptor dependence not tested"]},{"year":2024,"claim":"Identified FLRT2-ITGB4 association and mTORC2/AKT/p53 signaling as the pathway by which FLRT2 prevents endothelial senescence.","evidence":"Co-IP, ITGB4 inhibition, FLRT2 knockdown in endothelial cells, and a mouse vascular aging model with rescue","pmids":["38587072"],"confidence":"High","gaps":["How ITGB4 phosphorylation activates mTORC2 not resolved"]},{"year":2024,"claim":"Revealed FLRT2 interaction with VE-cadherin and Numb shapes adherens/tight junction crosstalk and blood-brain barrier development.","evidence":"Early postnatal endothelial Flrt2 deletion with Co-IP, expansion microscopy, and BBB integrity assays","pmids":["39609404"],"confidence":"High","gaps":["Mechanism of Numb-dependent junction remodeling not fully dissected"]},{"year":2024,"claim":"Established that NEDD4 ubiquitinates and degrades FLRT2, defining post-translational control of FLRT2 tumor-suppressive activity.","evidence":"Co-IP, ubiquitination assay, NEDD4 overexpression rescue, and xenograft model in NSCLC","pmids":["39444619"],"confidence":"Medium","gaps":["Ubiquitination site on FLRT2 not mapped","Upstream regulation of NEDD4 not addressed"]},{"year":2026,"claim":"Defined a FLRT2-USP22-ABCA1 axis whereby FLRT2 suppresses cholesterol efflux to promote macrophage foam cell formation and atherosclerosis.","evidence":"Macrophage-specific Flrt2 knockout, ABCA1 ubiquitination and USP22 functional assays, cholesterol efflux assays, and atherosclerosis mouse models","pmids":["42174129"],"confidence":"High","gaps":["How membrane FLRT2 communicates with cytoplasmic USP22/ABCA1 machinery not resolved"]},{"year":null,"claim":"How FLRT2's distinct activities—Unc5-mediated repulsion, homophilic and VE-cadherin adhesion, FGFR2/ITGB4 signaling, and intracellular ubiquitination control of ABCA1—are integrated within a single cell and which protease governs ectodomain shedding remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Sheddase identity unknown","No unified structural model of ligand versus adhesion states","Tissue-specific selection among partners undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[1,6,14]},{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[12,17]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,16,19]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,5,13]},{"term_id":"GO:0031012","term_label":"extracellular matrix","supporting_discovery_ids":[5]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[1,5]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,2,10,14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,16]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[2,5]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[18,19]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[7,15,19]}],"complexes":[],"partners":["UNC5B","UNC5C","UNC5D","FGFR2","ITGB4","VE-CADHERIN","NEDD4","FLRT3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43155","full_name":"Leucine-rich repeat transmembrane protein FLRT2","aliases":["Fibronectin-like domain-containing leucine-rich transmembrane protein 2"],"length_aa":660,"mass_kda":74.0,"function":"Functions in cell-cell adhesion, cell migration and axon guidance. Mediates cell-cell adhesion via its interactions with ADGRL3 and probably also other latrophilins that are expressed at the surface of adjacent cells. May play a role in the migration of cortical neurons during brain development via its interaction with UNC5D. Mediates axon growth cone collapse and plays a repulsive role in neuron guidance via its interaction with UNC5D, and possibly also other UNC-5 family members. Plays a role in fibroblast growth factor-mediated signaling cascades. Required for normal organization of the cardiac basement membrane during embryogenesis, and for normal embryonic epicardium and heart morphogenesis","subcellular_location":"Cell membrane; Endoplasmic reticulum membrane; Cell junction, focal adhesion; Secreted, extracellular space, extracellular matrix; Microsome membrane; Secreted; Synapse, synaptosome","url":"https://www.uniprot.org/uniprotkb/O43155/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FLRT2","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/FLRT2","total_profiled":1310},"omim":[{"mim_id":"616417","title":"ADHESION G PROTEIN-COUPLED RECEPTOR L3; ADGRL3","url":"https://www.omim.org/entry/616417"},{"mim_id":"604808","title":"FIBRONECTIN-LIKE DOMAIN-CONTAINING LEUCINE-RICH TRANSMEMBRANE PROTEIN 3; FLRT3","url":"https://www.omim.org/entry/604808"},{"mim_id":"604807","title":"FIBRONECTIN-LIKE DOMAIN-CONTAINING LEUCINE-RICH TRANSMEMBRANE PROTEIN 2; FLRT2","url":"https://www.omim.org/entry/604807"},{"mim_id":"604806","title":"FIBRONECTIN-LIKE DOMAIN-CONTAINING LEUCINE-RICH TRANSMEMBRANE PROTEIN 1; FLRT1","url":"https://www.omim.org/entry/604806"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"ovary","ntpm":38.6}],"url":"https://www.proteinatlas.org/search/FLRT2"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O43155","domains":[{"cath_id":"3.80.10.10","chopping":"36-171","consensus_level":"medium","plddt":96.1697,"start":36,"end":171},{"cath_id":"2.60.40.10","chopping":"426-515","consensus_level":"high","plddt":80.2139,"start":426,"end":515}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43155","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43155-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43155-F1-predicted_aligned_error_v6.png","plddt_mean":74.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FLRT2","jax_strain_url":"https://www.jax.org/strain/search?query=FLRT2"},"sequence":{"accession":"O43155","fasta_url":"https://rest.uniprot.org/uniprotkb/O43155.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43155/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43155"}},"corpus_meta":[{"pmid":"21673655","id":"PMC_21673655","title":"FLRT2 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Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/33193897","citation_count":14,"is_preprint":false},{"pmid":"29487336","id":"PMC_29487336","title":"Enhanced synaptic plasticity and spatial memory in female but not male FLRT2-haplodeficient mice.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29487336","citation_count":13,"is_preprint":false},{"pmid":"24585683","id":"PMC_24585683","title":"FLRT2 interacts with fibronectin in the ATDC5 chondroprogenitor cells.","date":"2014","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/24585683","citation_count":13,"is_preprint":false},{"pmid":"35700726","id":"PMC_35700726","title":"The guidance and adhesion protein FLRT2 dimerizes in cis via dual small-X3-small transmembrane motifs.","date":"2022","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/35700726","citation_count":11,"is_preprint":false},{"pmid":"38587072","id":"PMC_38587072","title":"FLRT2 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signaling","date":"2024-10-03","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.01.616077","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16469,"output_tokens":5282,"usd":0.064319,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13630,"output_tokens":5600,"usd":0.104075,"stage2_stop_reason":"end_turn"},"total_usd":0.168394,"stage1_batch_id":"msgbatch_01AsR7g8dGTEiEw9U83bJeWn","stage2_batch_id":"msgbatch_01YV5ZyxtFNUZEjxRcpd9jzb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"FLRT1 and FLRT2 are glycosylated type I transmembrane proteins migrating at 90 and 85 kDa respectively, containing 10 leucine-rich repeats, flanking cysteine-rich regions, a fibronectin/collagen-like domain, and an intracellular tail, as demonstrated by expression in SF9 and COS-1 cells.\",\n      \"method\": \"Heterologous expression in SF9 and COS-1 cells, SDS-PAGE, glycosylation analysis\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein characterization in two cell systems, single lab, structural features confirmed biochemically\",\n      \"pmids\": [\"10644439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"FLRT2 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for Unc5D-positive neurons. FLRT2 binds specifically to Unc5D receptor. During neocortical development, shed FLRT2 ECD repels SVZ neurons (which express Unc5D) from the FLRT2-expressing cortical plate, modulating cortical neuron migration. Deletion of FLRT2 or Unc5D causes premature migration of SVZ-derived neurons toward the cortical plate.\",\n      \"method\": \"Binding assays, mouse genetics (conditional knockout, overexpression), in vivo cortical neuron migration analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetics (both FLRT2 and Unc5D knockouts show matching phenotypes), binding assays, replicated with gain- and loss-of-function\",\n      \"pmids\": [\"21673655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"FLRT2 is required in the epicardium for heart morphogenesis; Flrt2-null mouse embryos arrest at mid-gestation with cardiac insufficiency, defects in epicardial sheet integrity, and disrupted basement membrane organization. In vitro and in vivo reconstitution assays show FLRT2 and FLRT3 are functionally interchangeable in maintaining cell-cell contacts.\",\n      \"method\": \"Gene targeting (knockout mouse), in vitro reconstitution, in vivo reconstitution assay\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — knockout with defined phenotype combined with reconstitution assays showing functional interchangeability\",\n      \"pmids\": [\"21350012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"FLRT2 physically interacts with FGFR2 both extracellularly (via FLRT2 leucine-rich repeat domain) and intracellularly (via FLRT2 C-tail domain). FLRT2 knockdown or overexpression causes concomitant decrease or increase in FGFR2 protein, mRNA, and ERK phosphorylation levels, indicating FLRT2 positively regulates FGF signaling in a feedback loop.\",\n      \"method\": \"Co-immunoprecipitation with embryonic craniofacial tissue lysates, GST pulldown, yeast two-hybrid, stable shRNA knockdown and cDNA overexpression with Western blot and ERK phosphorylation assay\",\n      \"journal\": \"Journal of dental research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal methods (Co-IP, GST pulldown, Y2H, functional signaling assays) in single lab\",\n      \"pmids\": [\"21765038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"FLRT2 promotes cellular proliferation and inhibits cell-cell adhesion during early chondrogenesis. Knockdown of FLRT2 in ATDC5 cells slows proliferation, increases N-cadherin expression, and increases PNA-positive aggregates; overexpression has opposite effects. FLRT2 knockdown also reduces cell migration in wound healing assay.\",\n      \"method\": \"Stable transfection (shRNA knockdown and cDNA overexpression), proliferation assay, lectin PNA staining, N-cadherin Western blot, wound healing assay, Alcian blue staining\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with multiple cellular readouts, single lab\",\n      \"pmids\": [\"21769912\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"FLRT2 is present on the cell membrane and in the extracellular matrix of ATDC5 chondroprogenitor cells, where it co-localizes and co-immunoprecipitates with fibronectin. Extracellular FLRT2 accumulation depends on fibronectin fibril formation. Western blot with antibodies to extra- and intracellular domains reveals multiple size bands, suggesting FLRT2 exists in both membrane-bound and shed/cleaved forms.\",\n      \"method\": \"Immunolocalization, co-immunoprecipitation, fibronectin blocking peptide experiment, Western blot with domain-specific antibodies\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and localization with functional perturbation (blocking peptide), single lab, multiple complementary approaches\",\n      \"pmids\": [\"24585683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FLRT2 is expressed in endothelial cells of the placental labyrinth. Endothelial cell-specific deletion of FLRT2 in mice causes embryonic lethality with aberrant endothelial alignment and disrupted feto-maternal circulation, mediated through endothelial repulsion via binding to the UNC5B receptor.\",\n      \"method\": \"Conditional endothelial knockout mouse, embryonic lethality analysis, histological analysis of placental labyrinth, UNC5B binding assay\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with defined molecular mechanism (FLRT2-UNC5B binding) and clear cellular phenotype\",\n      \"pmids\": [\"28576770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"FLRT2 overexpression inhibits breast cancer cell proliferation and migration, while knockdown increases them. FLRT2 overexpression significantly decreases cell adhesion on collagen I-coated plates. Pathway analysis identifies 'cellular growth and proliferation/Cancer' as the top affected pathway.\",\n      \"method\": \"shRNA knockdown, cDNA overexpression, proliferation assay, migration assay, colony formation assay, adhesion assay, pathway analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional (gain and loss-of-function) with multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"28325946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"FLRT2 haplodeficiency in female mice enhances hippocampal synaptic plasticity and spatial memory recall, accompanied by decreased EAAT2 (glutamate transporter) levels and decreased beta estrogen receptor levels. Male FLRT2+/- mice show deficient memory recall and decreased alpha estrogen receptor levels.\",\n      \"method\": \"FLRT2+/- knockout mice, in vivo behavioral testing, electrophysiology (LTP), Western blot for EAAT2 and estrogen receptors\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic model with electrophysiology and molecular correlates, single lab, sex-specific effects replicated across multiple assays\",\n      \"pmids\": [\"29487336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FLRT2 regulates osteoclast multinucleation. FLRT2 expression is induced by RANKL in osteoclast precursors. FLRT2 deficiency reduces hyper-multinucleation, rescued by Unc5B knockdown. FLRT2 promotes Rac1 activation downstream of RANKL; Flrt2-deficient cells show attenuated RANKL-induced Rac1 activation. FLRT2 interferes with Netrin1-Unc5B interaction to modulate multinucleation.\",\n      \"method\": \"FLRT2-deficient osteoclast differentiation assay, Unc5B RNAi rescue, Netrin1 treatment, Rac1 activation assay\",\n      \"journal\": \"BMB reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with genetic rescue (Unc5B knockdown) and Rac1 signaling readout, single lab\",\n      \"pmids\": [\"31383250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FLRT2 and FLRT3 double deletion in mice causes abnormal distribution of cortical interneurons within tangential migratory streams during development, affecting somatostatin+ interneuron laminar positioning postnatally. FLRT2/FLRT3 act non-cell-autonomously as chemorepellents for interneurons in vitro, partly through Unc5B/Unc5D receptors (double Unc5B/Unc5D knockout phenocopies the FLRT double knockout).\",\n      \"method\": \"FLRT2/FLRT3 double conditional knockout mice, interneuron migration in vitro repulsion assay, Unc5B/Unc5D double knockout genetic epistasis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — double knockout genetics with in vivo and in vitro phenotype, epistasis with Unc5 receptors, multiple orthogonal approaches\",\n      \"pmids\": [\"34301831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In the adult hippocampus, FLRT2 expression is confined to CA1 and is partly localized on pre- and post-synapses, as shown by Flrt2-LacZ knock-in reporter mice and subcellular fractionation. After spinal cord injury, FLRT2 is transiently upregulated in reactive astrocytes around lesion sites at 7 days post-injury.\",\n      \"method\": \"Flrt2-LacZ knock-in mice, X-gal histochemistry, subcellular synaptic fractionation, spinal cord injury model\",\n      \"journal\": \"Frontiers in molecular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knock-in reporter with fractionation data, direct localization to synaptic compartments, single lab\",\n      \"pmids\": [\"34744626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FLRT2 forms noncanonical interendothelial adhesions through homophilic binding, safeguarding against oxidative stress. In tumor vasculature, endothelial FLRT2 deletion selectively prunes abnormalized vessels, creating 'oxygen-glucose uncoupling' that suppresses tumor metastasis and increases antitumor effects of immune checkpoint blockers.\",\n      \"method\": \"Endothelial cell-specific Flrt2 deletion in mice, tumor models, homophilic binding assays, metabolic analysis\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with multiple tumor models, homophilic binding mechanistic data, metabolic phenotyping\",\n      \"pmids\": [\"35104247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FLRT2 dimerizes in cis (on the same cell) via two Small-X3-Small motifs in its transmembrane helix, which synergize with a third dimerization motif in the extracellular domain. This cis dimerization was demonstrated by molecular dynamics simulations and validated by single particle tracking showing co-diffusion of FLRT2 receptors on live cells.\",\n      \"method\": \"Molecular dynamics simulations, single particle tracking (SPT) live cell imaging, transmembrane motif mutagenesis\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — MD simulations with experimental validation by SPT and mutagenesis of dimerization motifs, single lab with two orthogonal methods\",\n      \"pmids\": [\"35700726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In the mouse retina, FLRT2 is expressed by direction-selective (DS) circuit neurons and mediates rejection of inappropriate synaptic partners via transcellular FLRT2-UNC5C/D signaling. UNC5C/D are expressed by non-DS neurons projecting to adjacent IPL sublayers. FLRT2-UNC5 binding eliminates misguided DS dendrites; loss of FLRT2-UNC5 binding allows mistargeted dendrites to elaborate and acquire inappropriate synapses. UNC5s promote dendrite elimination by interfering with FLRT2-mediated adhesion.\",\n      \"method\": \"In vivo gain- and loss-of-function (FLRT2 knockout, UNC5C misexpression), retinal imaging, synapse analysis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal gain and loss-of-function in vivo with clear mechanistic model (UNC5 interferes with FLRT2 adhesion), multiple experimental approaches\",\n      \"pmids\": [\"37557174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FLRT2 elevates ACSL4 expression, increases lipid peroxidation, and subsequently facilitates ferroptosis in bladder cancer cells, thereby inhibiting tumor cell growth, migration, and invasion.\",\n      \"method\": \"FLRT2 overexpression and silencing in bladder cancer cells, ACSL4 expression assay, lipid peroxidation assay, cell viability/migration/invasion assays\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — bidirectional manipulation with mechanistic pathway (ACSL4/lipid peroxidation/ferroptosis), single lab\",\n      \"pmids\": [\"37480224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FLRT2 directly associates with integrin subunit beta 4 (ITGB4) and promotes ITGB4 phosphorylation; inhibition of ITGB4 substantially mitigates the induction of endothelial senescence triggered by FLRT2 depletion. FLRT2 prevents endothelial senescence through the mTORC2/AKT/p53 signaling pathway.\",\n      \"method\": \"Co-immunoprecipitation (FLRT2-ITGB4 interaction), ITGB4 inhibition, FLRT2 knockdown in endothelial cells, Western blot for mTORC2/AKT/p53 pathway, mouse vascular aging model\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding identified by Co-IP, pathway epistasis with ITGB4 inhibition, in vivo mouse aging model with rescue experiment\",\n      \"pmids\": [\"38587072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FLRT2 interacts with VE-cadherin in endothelial cells of the CNS vasculature; together with the endocytic adaptor Numb, FLRT2 modulates adherens junction morphology. FLRT2 in cortical vessels also regulates crosstalk between adherens and tight junctions, influencing blood-brain barrier development. FLRT2 deletion in endothelial cells specifically impairs retinal vein-driven endothelial proliferation, sprouting, and polarity.\",\n      \"method\": \"Conditional endothelial FLRT2 deletion (early postnatal), co-immunoprecipitation (FLRT2-VE-cadherin), expansion microscopy for junction morphology, BBB integrity assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with Co-IP interaction data, expansion microscopy validation, multiple vascular phenotypes\",\n      \"pmids\": [\"39609404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FLRT2 protein is ubiquitinated and degraded by the E3 ubiquitin ligase NEDD4 in NSCLC cells. NEDD4 overexpression abolishes the tumor-suppressive effects of FLRT2 on cancer cell stemness, proliferation, and drug resistance in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation (NEDD4-FLRT2), ubiquitination assay, NEDD4 overexpression rescue experiment, xenograft mouse model\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP confirming binding, functional rescue experiments, in vivo xenograft, single lab\",\n      \"pmids\": [\"39444619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"FLRT2 promotes foam cell formation in macrophages by suppressing ABCA1-mediated cholesterol efflux at the post-transcriptional level. Mechanistically, FLRT2 enhances ABCA1 ubiquitin-proteasome degradation by inhibiting USP22-mediated deubiquitination, defining a FLRT2-USP22-ABCA1 axis. Macrophage-specific Flrt2 deletion reduces atherosclerotic plaque burden in multiple mouse models.\",\n      \"method\": \"Macrophage-specific Flrt2 conditional knockout mice, adenovirus-mediated FLRT2 overexpression, ABCA1 ubiquitination assay, USP22 interaction/functional assay, cholesterol efflux assay, atherosclerosis mouse models\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — macrophage-specific knockout with mechanistic dissection of USP22-ABCA1 ubiquitination axis, multiple mouse atherosclerosis models\",\n      \"pmids\": [\"42174129\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"FLRT2 is a shed/membrane-bound leucine-rich repeat transmembrane protein that functions as a repulsive ligand for Unc5 receptors (Unc5B, Unc5C, Unc5D) to control neuronal migration, axon guidance, synaptic partner selection, and osteoclast multinucleation; it also forms homophilic cis-dimers via transmembrane Small-X3-Small motifs and trans-adhesive complexes with VE-cadherin to regulate endothelial organization and vascular development; it positively regulates FGF/ERK signaling through physical interaction with FGFR2, prevents endothelial senescence by binding ITGB4 and activating mTORC2/AKT, and in macrophages promotes ABCA1 degradation by inhibiting USP22 deubiquitination, while its own stability is regulated by NEDD4-mediated ubiquitination.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FLRT2 is a glycosylated type I transmembrane protein with ten leucine-rich repeats and a fibronectin/collagen-like domain that functions as a shed repulsive ligand and adhesion molecule governing neuronal migration, vascular organization, and synaptic partner selection [#0, #1, #6]. Its ectodomain is cleaved from cells and binds the Unc5 family of receptors (Unc5B, Unc5C, Unc5D) to deliver repulsive guidance: shed FLRT2 repels Unc5D-positive neurons from the cortical plate, controls cortical interneuron laminar positioning together with FLRT3, and directs rejection of inappropriate synaptic partners in the retina, where Unc5 binding antagonizes FLRT2-mediated adhesion to eliminate misguided dendrites [#1, #10, #14]. In the vasculature, FLRT2 acts in endothelial cells to organize feto-maternal circulation through UNC5B-mediated repulsion and forms noncanonical homophilic interendothelial adhesions—stabilized by cis-dimerization through transmembrane Small-X3-Small motifs—that protect against oxidative stress; it also interacts with VE-cadherin and the adaptor Numb to remodel adherens junctions and shape blood-brain barrier development [#6, #12, #13, #17]. FLRT2 positively regulates FGF/ERK signaling through direct extracellular and intracellular interaction with FGFR2 [#3], prevents endothelial senescence by associating with ITGB4 and engaging the mTORC2/AKT/p53 pathway [#16], and promotes osteoclast multinucleation by driving RANKL-induced Rac1 activation [#9]. FLRT2 acts as a tumor suppressor in several epithelial cancers, restraining proliferation and migration [#7], and in bladder cancer it elevates ACSL4 to drive ferroptosis [#15]; its protein levels are controlled by NEDD4-mediated ubiquitination and degradation [#18]. In macrophages, FLRT2 suppresses ABCA1-dependent cholesterol efflux by inhibiting USP22-mediated deubiquitination of ABCA1, promoting foam cell formation and atherosclerotic plaque burden [#19].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established FLRT2 as a discrete protein, defining the domain architecture that would later explain its dual roles as a ligand and adhesion molecule.\",\n      \"evidence\": \"Heterologous expression in SF9 and COS-1 cells with SDS-PAGE and glycosylation analysis\",\n      \"pmids\": [\"10644439\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No function assigned at this stage\", \"No binding partners identified\", \"Cleavage/shedding not yet demonstrated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed FLRT2 ectodomains are shed and act as a repulsive ligand for Unc5D, answering how FLRT2 guides neuronal migration non-cell-autonomously.\",\n      \"evidence\": \"Binding assays and reciprocal mouse genetics (FLRT2 and Unc5D knockouts) with cortical migration analysis\",\n      \"pmids\": [\"21673655\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Protease responsible for shedding not identified\", \"Downstream signaling in Unc5D neurons not dissected\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined an essential developmental requirement for FLRT2 in epicardial integrity and basement membrane organization, with FLRT3 functional interchangeability.\",\n      \"evidence\": \"Knockout mouse phenotyping plus in vitro and in vivo reconstitution assays\",\n      \"pmids\": [\"21350012\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular receptor/partner mediating epicardial adhesion not defined here\", \"Relationship to Unc5 repulsion unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Linked FLRT2 to growth factor signaling by showing direct extracellular and intracellular interaction with FGFR2 and positive feedback on ERK activation.\",\n      \"evidence\": \"Co-IP from craniofacial tissue, GST pulldown, yeast two-hybrid, knockdown/overexpression with ERK phosphorylation readout\",\n      \"pmids\": [\"21765038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of FGFR2 binding not resolved\", \"Whether this operates in cis or via shed ectodomain unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated FLRT2 controls proliferation versus adhesion balance in chondroprogenitors, inversely regulating N-cadherin.\",\n      \"evidence\": \"shRNA/overexpression in ATDC5 cells with proliferation, PNA staining, N-cadherin Western blot and wound healing assays\",\n      \"pmids\": [\"21769912\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking FLRT2 to N-cadherin not defined\", \"Receptor not identified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placed FLRT2 in the extracellular matrix in association with fibronectin, supporting both membrane-bound and shed/cleaved pools.\",\n      \"evidence\": \"Immunolocalization, Co-IP, fibronectin blocking peptide and domain-specific Western blots in ATDC5 cells\",\n      \"pmids\": [\"24585683\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of fibronectin association not established\", \"Cleavage mechanism not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended FLRT2 repulsion to the vasculature, showing endothelial FLRT2-UNC5B signaling organizes placental endothelial alignment and is essential for embryonic circulation.\",\n      \"evidence\": \"Endothelial-specific conditional knockout mouse with histology and UNC5B binding assay\",\n      \"pmids\": [\"28576770\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Intracellular signaling downstream of endothelial repulsion not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified FLRT2 as a tumor suppressor in breast cancer, restraining proliferation, migration, and collagen adhesion.\",\n      \"evidence\": \"Bidirectional knockdown/overexpression with proliferation, migration, colony, and adhesion assays plus pathway analysis\",\n      \"pmids\": [\"28325946\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mediator of tumor suppression not identified\", \"In vivo validation absent in this study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealed sex-specific roles for FLRT2 in hippocampal synaptic plasticity and memory, with estrogen receptor and glutamate transporter correlates.\",\n      \"evidence\": \"FLRT2+/- mice with behavioral testing, LTP electrophysiology, and Western blots for EAAT2 and estrogen receptors\",\n      \"pmids\": [\"29487336\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal link between FLRT2 and EAAT2/estrogen receptor changes not established\", \"Receptor mediating synaptic effect unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed FLRT2 promotes osteoclast multinucleation downstream of RANKL by activating Rac1 and modulating Netrin1-Unc5B interaction.\",\n      \"evidence\": \"FLRT2-deficient osteoclast differentiation with Unc5B RNAi rescue, Netrin1 treatment, and Rac1 activation assay\",\n      \"pmids\": [\"31383250\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link from Unc5B to Rac1 not fully resolved\", \"In vivo bone phenotype not established here\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated FLRT2/FLRT3 act as chemorepellents controlling cortical interneuron migration and laminar positioning via Unc5B/Unc5D.\",\n      \"evidence\": \"FLRT2/FLRT3 double conditional knockout with in vitro repulsion assay and Unc5B/Unc5D double knockout epistasis\",\n      \"pmids\": [\"34301831\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of FLRT2 versus FLRT3 not separated\", \"Receptor-bearing cell signaling not dissected\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Localized adult FLRT2 to CA1 pre- and post-synapses and showed injury-induced upregulation in reactive astrocytes.\",\n      \"evidence\": \"Flrt2-LacZ knock-in reporter mice, X-gal histochemistry, synaptic fractionation, and spinal cord injury model\",\n      \"pmids\": [\"34744626\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Synaptic function of CA1 FLRT2 not directly tested\", \"Role of injury-induced astrocytic FLRT2 unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established that FLRT2 forms noncanonical homophilic interendothelial adhesions protecting against oxidative stress, with therapeutic relevance to tumor vessel pruning.\",\n      \"evidence\": \"Endothelial-specific Flrt2 deletion in tumor models with homophilic binding and metabolic analyses\",\n      \"pmids\": [\"35104247\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis coupling homophilic adhesion to oxidative stress protection not fully defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined the structural mechanism of FLRT2 cis-dimerization through transmembrane Small-X3-Small motifs synergizing with an extracellular motif.\",\n      \"evidence\": \"Molecular dynamics simulations validated by single particle tracking and transmembrane motif mutagenesis\",\n      \"pmids\": [\"35700726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of cis-dimerization for ligand/adhesion activity not directly tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showed FLRT2-UNC5C/D transcellular signaling mediates synaptic partner rejection in the retina, with UNC5 antagonizing FLRT2 adhesion to eliminate misguided dendrites.\",\n      \"evidence\": \"In vivo FLRT2 knockout and UNC5C misexpression with retinal imaging and synapse analysis\",\n      \"pmids\": [\"37557174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Intracellular effectors of dendrite elimination not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected FLRT2 tumor suppression in bladder cancer to ferroptosis via ACSL4 elevation and lipid peroxidation.\",\n      \"evidence\": \"FLRT2 overexpression/silencing with ACSL4 expression, lipid peroxidation, and viability/migration/invasion assays\",\n      \"pmids\": [\"37480224\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking FLRT2 to ACSL4 regulation not defined\", \"Receptor dependence not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified FLRT2-ITGB4 association and mTORC2/AKT/p53 signaling as the pathway by which FLRT2 prevents endothelial senescence.\",\n      \"evidence\": \"Co-IP, ITGB4 inhibition, FLRT2 knockdown in endothelial cells, and a mouse vascular aging model with rescue\",\n      \"pmids\": [\"38587072\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ITGB4 phosphorylation activates mTORC2 not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed FLRT2 interaction with VE-cadherin and Numb shapes adherens/tight junction crosstalk and blood-brain barrier development.\",\n      \"evidence\": \"Early postnatal endothelial Flrt2 deletion with Co-IP, expansion microscopy, and BBB integrity assays\",\n      \"pmids\": [\"39609404\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of Numb-dependent junction remodeling not fully dissected\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established that NEDD4 ubiquitinates and degrades FLRT2, defining post-translational control of FLRT2 tumor-suppressive activity.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, NEDD4 overexpression rescue, and xenograft model in NSCLC\",\n      \"pmids\": [\"39444619\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination site on FLRT2 not mapped\", \"Upstream regulation of NEDD4 not addressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined a FLRT2-USP22-ABCA1 axis whereby FLRT2 suppresses cholesterol efflux to promote macrophage foam cell formation and atherosclerosis.\",\n      \"evidence\": \"Macrophage-specific Flrt2 knockout, ABCA1 ubiquitination and USP22 functional assays, cholesterol efflux assays, and atherosclerosis mouse models\",\n      \"pmids\": [\"42174129\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How membrane FLRT2 communicates with cytoplasmic USP22/ABCA1 machinery not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How FLRT2's distinct activities—Unc5-mediated repulsion, homophilic and VE-cadherin adhesion, FGFR2/ITGB4 signaling, and intracellular ubiquitination control of ABCA1—are integrated within a single cell and which protease governs ectodomain shedding remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Sheddase identity unknown\", \"No unified structural model of ligand versus adhesion states\", \"Tissue-specific selection among partners undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [1, 6, 14]},\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [12, 17]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 16, 19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 5, 13]},\n      {\"term_id\": \"GO:0031012\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [1, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 2, 10, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 16]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [18, 19]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [7, 15, 19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"UNC5B\", \"UNC5C\", \"UNC5D\", \"FGFR2\", \"ITGB4\", \"VE-cadherin\", \"NEDD4\", \"FLRT3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}