{"gene":"IGSF5","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2003,"finding":"JAM4 (IGSF5) directly binds MAGI-1 (but not ZO-1) through its carboxyl terminus, and when coexpressed in COS-7 cells, JAM4 and MAGI-1 interact and form clusters at cell contacts. JAM4 mediates calcium-independent homophilic adhesion in L cells and recruits MAGI-1, ZO-1, and occludin to JAM4-based cell contacts. MAGI-1 strengthens JAM4-mediated cell adhesion and barrier sealing effects in CHO cell monolayers.","method":"Biochemical in vitro binding assays, coexpression in COS-7 cells, cell adhesion assays in L cells, permeability assays in CHO monolayers","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal biochemical binding assays, multiple cell-based functional assays, replicated across multiple cell systems in a single rigorous study","pmids":["12773569"],"is_preprint":false},{"year":2003,"finding":"JAM4 localization to tight junctions depends on the first Ig-loop, not the MAGI-1-interacting region. Both Ig-loops are necessary for homophilic trans interactions, while cis interactions depend only on the first Ig-loop. JAM4 determines the subcellular localization of MAGI-1 in MDCK cells.","method":"Deletion/domain mutant analysis in MDCK cells, immunofluorescence localization, cell adhesion assays","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutagenesis with localization readout, single lab, multiple constructs tested","pmids":["12940823"],"is_preprint":false},{"year":2005,"finding":"MAGI-1 forms a tripartite complex with nephrin and JAM4 in vitro, and MAGI-1 is localized at the glomerular slit diaphragm where it interacts with both nephrin and JAM4 in vivo. JAM4 is also distributed on apical membranes of podocytes beyond the slit diaphragm.","method":"Yeast two-hybrid screening, in vitro biochemical pulldown for tripartite complex, immunoelectron microscopy","journal":"Laboratory investigation; a journal of technical methods and pathology","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid, in vitro complex reconstitution, and immunoelectron microscopy in same study; consistent with prior work","pmids":["16155592"],"is_preprint":false},{"year":2004,"finding":"JAM4 overexpression in MDCK cells enhances HGF-induced branching and scattering. JAM4 activates Rac GTPase (demonstrated by pull-down with CRIB domain of PAK and protrusion formation suppressed by dominant-negative Rac in COS-7 cells), suggesting JAM4 augments HGF-mediated Rac activation.","method":"JAM4-overexpressing MDCK cells treated with HGF; COS-7 cell morphology assay; dominant-negative Rac construct; GST-CRIB pulldown assay for active Rac","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical Rac activation assay combined with dominant-negative epistasis and cell morphology readout, single lab","pmids":["15330858"],"is_preprint":false},{"year":2006,"finding":"LNX1 (LNXp70 isoform) directly binds the C-terminal PDZ-binding motif of JAM4 via its second PDZ domain, forming a tripartite complex with JAM4 and Numb. LNX1 facilitates endocytosis of JAM4, and this endocytosis requires Numb (shown by dominant-negative constructs and RNAi). LNX1 is involved in TGF-β-induced redistribution of JAM4 in mammary epithelial cells.","method":"Yeast two-hybrid screening, immunoprecipitation from kidney lysates, in vitro biochemical binding assays, dominant-negative constructs, RNA interference, endocytosis assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid, in vivo co-IP from tissue, in vitro tripartite complex, RNAi and dominant-negative epistasis; multiple orthogonal methods in single study","pmids":["16832352"],"is_preprint":false},{"year":2006,"finding":"JAM4 protein is localized to the plasma membrane of male germ cells and is expressed in stem cell and progenitor cell populations in male germ cell and hematopoietic lineages. JAM4-deficient mice show no obvious histological abnormalities in testes, liver, or kidney, and hematopoietic stem cell numbers are normal, suggesting functional redundancy with other cell adhesion molecules.","method":"Signal sequence trap cloning, immunolocalization, JAM4-knockout mouse generation and phenotypic analysis (histology, flow cytometry)","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with defined cellular phenotype readout; negative phenotype is informative for redundancy; single lab","pmids":["16982697"],"is_preprint":false},{"year":2005,"finding":"In injured podocytes (PAN nephropathy model), JAM4 expression and subcellular localization are altered — JAM4 is focally increased at the apical membrane of podocytes with effaced foot processes, while its colocalization with MAGI-1 is disrupted. JAM4 remains colocalized with ezrin at the apical membrane in PAN nephropathy.","method":"Immunomicroscopy and immunoelectron microscopy in rat proteinuric models; selective detergent extraction to assess subcellular compartmentalization","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by immunoelectron microscopy with biochemical fractionation in disease model, single lab","pmids":["16118391"],"is_preprint":false},{"year":2021,"finding":"Surface plasmon resonance of recombinant extracellular domains shows that heterotypic interactions among JAM family members (including JAM4) can be greatly favored over homotypic interactions, and JAM family members have unique tertiary structures despite similar secondary structures.","method":"Recombinant protein expression (MBP fusion), surface plasmon resonance (SPR), structural characterization","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro SPR binding assay with recombinant proteins; single lab, single study","pmids":["33801758"],"is_preprint":false}],"current_model":"JAM4/IGSF5 is an immunoglobulin superfamily cell adhesion molecule at tight junctions that mediates calcium-independent homophilic adhesion via its two extracellular Ig-loops; the first Ig-loop directs its localization to tight junctions and drives cis interactions, while both loops are required for trans homophilic adhesion. JAM4 binds MAGI-1 (via its C-terminal PDZ-binding motif) and together they regulate paracellular permeability and form part of the glomerular slit diaphragm complex with nephrin. JAM4 endocytosis is regulated by LNX1-Numb scaffold, and JAM4 activates Rac GTPase to enhance HGF-mediated branching and scattering of epithelial cells."},"narrative":{"mechanistic_narrative":"IGSF5 (JAM4) is an immunoglobulin-superfamily cell adhesion molecule that organizes epithelial junctions and the glomerular slit diaphragm through its two extracellular Ig-loops and a C-terminal PDZ-binding motif [PMID:12773569, PMID:16155592]. It mediates calcium-independent homophilic adhesion and recruits the junctional scaffolds MAGI-1, ZO-1, and occludin to sites of cell contact, with MAGI-1 binding through the JAM4 carboxyl terminus and reinforcing adhesion and paracellular barrier sealing [PMID:12773569]. The two Ig-loops are functionally divided: the first Ig-loop directs tight-junction localization and supports cis interactions, while both loops are required for trans homophilic adhesion, and JAM4 in turn dictates the subcellular distribution of MAGI-1 [PMID:12940823]. In the kidney, JAM4 forms a tripartite complex with MAGI-1 and nephrin at the glomerular slit diaphragm, a localization that is disrupted in proteinuric injury where JAM4 redistributes apically and loses MAGI-1 colocalization [PMID:16155592, PMID:16118391]. Beyond static adhesion, JAM4 signals to promote epithelial remodeling: it activates Rac GTPase to augment HGF-induced branching and scattering [PMID:15330858], and its surface levels are controlled by an LNX1–Numb scaffold that drives JAM4 endocytosis, contributing to TGF-β-induced receptor redistribution [PMID:16832352]. Knockout mice lack overt histological defects in tissues where JAM4 is expressed, indicating functional redundancy with other adhesion molecules [PMID:16982697].","teleology":[{"year":2003,"claim":"Establishing that JAM4 is an adhesion molecule that nucleates a junctional scaffold answered whether it does more than mediate contact — it physically organizes the tight-junction protein network.","evidence":"In vitro binding assays, COS-7 coexpression, L-cell adhesion assays, and CHO monolayer permeability assays","pmids":["12773569"],"confidence":"High","gaps":["Does not define the structural basis of MAGI-1 binding","Physiological tissue context of the adhesion not addressed in this study"]},{"year":2003,"claim":"Domain dissection answered which parts of JAM4 carry out localization versus adhesion, separating tight-junction targeting and cis interactions (first Ig-loop) from trans adhesion (both loops).","evidence":"Deletion/domain mutant analysis with immunofluorescence and adhesion assays in MDCK cells","pmids":["12940823"],"confidence":"Medium","gaps":["No structural model of the Ig-loops","Single-lab domain mapping without orthogonal validation"]},{"year":2004,"claim":"Linking JAM4 to Rac activation answered whether it has a signaling role beyond adhesion, showing it augments HGF-driven epithelial branching and scattering.","evidence":"JAM4-overexpressing MDCK cells with HGF, GST-CRIB pulldown for active Rac, and dominant-negative Rac in COS-7 morphology assays","pmids":["15330858"],"confidence":"Medium","gaps":["Mechanism coupling JAM4 to Rac GEFs unidentified","Relevance to endogenous JAM4 levels not established"]},{"year":2005,"claim":"Placing JAM4 in a nephrin–MAGI-1 complex answered where it acts in vivo, localizing it to the glomerular slit diaphragm.","evidence":"Yeast two-hybrid, in vitro tripartite complex reconstitution, and immunoelectron microscopy","pmids":["16155592"],"confidence":"High","gaps":["Functional consequence of the tripartite complex for filtration not tested","Stoichiometry and direct vs bridged interactions unresolved"]},{"year":2005,"claim":"Examining injured podocytes answered whether JAM4 localization is dynamic in disease, revealing apical redistribution and loss of MAGI-1 colocalization in proteinuria.","evidence":"Immunoelectron microscopy and detergent-extraction fractionation in rat PAN nephropathy","pmids":["16118391"],"confidence":"Medium","gaps":["Causal role of JAM4 redistribution in foot-process effacement unknown","Correlative disease-model observation"]},{"year":2006,"claim":"Identifying the LNX1–Numb scaffold answered how JAM4 surface levels are controlled, defining an endocytic regulatory mechanism tied to TGF-β signaling.","evidence":"Yeast two-hybrid, co-IP from kidney lysates, in vitro tripartite binding, RNAi and dominant-negative endocytosis assays","pmids":["16832352"],"confidence":"High","gaps":["Trigger linking TGF-β to LNX1-Numb recruitment unresolved","In vivo consequence of JAM4 endocytosis not tested"]},{"year":2006,"claim":"Knockout mice answered whether JAM4 is individually required in its expressing tissues, showing no overt defect and implying redundancy.","evidence":"Signal sequence trap cloning, immunolocalization, and phenotypic analysis of JAM4-knockout mice (histology, flow cytometry)","pmids":["16982697"],"confidence":"Medium","gaps":["Redundant adhesion molecules not identified","Subtle functional or stress-dependent phenotypes not probed"]},{"year":2021,"claim":"Quantitative binding measurements answered whether JAM4 prefers homotypic or heterotypic partners, showing heterotypic JAM-family interactions can dominate.","evidence":"Surface plasmon resonance of recombinant MBP-fused extracellular domains","pmids":["33801758"],"confidence":"Medium","gaps":["Cellular relevance of heterotypic JAM4 interactions untested","Specific JAM4 heterotypic partner affinities not detailed in context"]},{"year":null,"claim":"Whether JAM4 adhesion, Rac signaling, and slit-diaphragm assembly are integrated into a single regulatable pathway, and what physiological process specifically requires JAM4, remains open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated in vivo loss-of-function phenotype defined","Redundant partners and disease relevance unestablished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098631","term_label":"cell adhesion mediator activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,2,4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,5,6]}],"pathway":[],"complexes":["JAM4-MAGI-1-nephrin slit diaphragm complex","JAM4-LNX1-Numb endocytic complex"],"partners":["MAGI1","NPHS1","LNX1","NUMB","ZO-1","OCLN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NSI5","full_name":"Immunoglobulin superfamily member 5","aliases":["Junctional adhesion molecule 4","JAM-4"],"length_aa":407,"mass_kda":44.6,"function":"Provides, together with MAGI1, an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells. Mediates calcium-independent homophilic cell adhesion. In testis, it may function as a cell adhesion molecule rather than a tight-junction protein. It may participate in the adhesion between spermatogonia-spermatogonia, spermatogonia-Sertoli cells, and Sertoli cells-Sertoli cells (By similarity)","subcellular_location":"Apical cell membrane; Cell junction, tight junction","url":"https://www.uniprot.org/uniprotkb/Q9NSI5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/IGSF5","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/IGSF5","total_profiled":1310},"omim":[{"mim_id":"610638","title":"IMMUNOGLOBULIN SUPERFAMILY, MEMBER 5; IGSF5","url":"https://www.omim.org/entry/610638"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Focal adhesion sites","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in some","driving_tissues":[],"url":"https://www.proteinatlas.org/search/IGSF5"},"hgnc":{"alias_symbol":["JAM4"],"prev_symbol":[]},"alphafold":{"accession":"Q9NSI5","domains":[{"cath_id":"2.60.40.10","chopping":"42-140","consensus_level":"high","plddt":91.4542,"start":42,"end":140},{"cath_id":"2.60.40.10","chopping":"144-236","consensus_level":"high","plddt":88.7318,"start":144,"end":236}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NSI5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NSI5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NSI5-F1-predicted_aligned_error_v6.png","plddt_mean":65.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=IGSF5","jax_strain_url":"https://www.jax.org/strain/search?query=IGSF5"},"sequence":{"accession":"Q9NSI5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NSI5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NSI5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NSI5"}},"corpus_meta":[{"pmid":"12773569","id":"PMC_12773569","title":"JAM4, a junctional cell adhesion molecule interacting with a tight junction protein, MAGI-1.","date":"2003","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12773569","citation_count":142,"is_preprint":false},{"pmid":"24667924","id":"PMC_24667924","title":"JAM-related proteins in mucosal homeostasis and inflammation.","date":"2014","source":"Seminars in immunopathology","url":"https://pubmed.ncbi.nlm.nih.gov/24667924","citation_count":88,"is_preprint":false},{"pmid":"16155592","id":"PMC_16155592","title":"MAGI-1 is a component of the glomerular slit diaphragm that is tightly associated with nephrin.","date":"2005","source":"Laboratory investigation; a journal of technical methods and pathology","url":"https://pubmed.ncbi.nlm.nih.gov/16155592","citation_count":55,"is_preprint":false},{"pmid":"16832352","id":"PMC_16832352","title":"Ligand-of-Numb protein X is an endocytic scaffold for junctional adhesion molecule 4.","date":"2006","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/16832352","citation_count":36,"is_preprint":false},{"pmid":"24376456","id":"PMC_24376456","title":"Gene-alcohol interactions identify several novel blood pressure loci including a promising locus near SLC16A9.","date":"2013","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24376456","citation_count":32,"is_preprint":false},{"pmid":"20562105","id":"PMC_20562105","title":"Up-regulation of the homophilic adhesion molecule sidekick-1 in podocytes contributes to glomerulosclerosis.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20562105","citation_count":29,"is_preprint":false},{"pmid":"16982697","id":"PMC_16982697","title":"A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages.","date":"2006","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16982697","citation_count":23,"is_preprint":false},{"pmid":"39129336","id":"PMC_39129336","title":"The MIND diet, brain transcriptomic alterations, and dementia.","date":"2024","source":"Alzheimer's & dementia : the journal of the Alzheimer's Association","url":"https://pubmed.ncbi.nlm.nih.gov/39129336","citation_count":19,"is_preprint":false},{"pmid":"15200235","id":"PMC_15200235","title":"A high proportion of chromosome 21 promoter polymorphisms influence transcriptional activity.","date":"2004","source":"Gene expression","url":"https://pubmed.ncbi.nlm.nih.gov/15200235","citation_count":17,"is_preprint":false},{"pmid":"12940823","id":"PMC_12940823","title":"Roles of immunoglobulin-like loops of junctional cell adhesion molecule 4; involvement in the subcellular localization and the cell adhesion.","date":"2003","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/12940823","citation_count":15,"is_preprint":false},{"pmid":"34453080","id":"PMC_34453080","title":"Dietary supplementation of β-conglycinin, with or without sodium butyrate on the growth, immune response and intestinal health of hybrid grouper.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/34453080","citation_count":11,"is_preprint":false},{"pmid":"15330858","id":"PMC_15330858","title":"JAM4 enhances hepatocyte growth factor-mediated branching and scattering of Madin-Darby canine kidney cells.","date":"2004","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/15330858","citation_count":9,"is_preprint":false},{"pmid":"33801758","id":"PMC_33801758","title":"Molecular Characterization of the Extracellular Domain of Human Junctional Adhesion Proteins.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33801758","citation_count":9,"is_preprint":false},{"pmid":"24084763","id":"PMC_24084763","title":"Germline variants and advanced colorectal adenomas: adenoma prevention with celecoxib trial genome-wide association study.","date":"2013","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/24084763","citation_count":9,"is_preprint":false},{"pmid":"16118391","id":"PMC_16118391","title":"Altered expression of junctional adhesion molecule 4 in injured podocytes.","date":"2005","source":"American journal of physiology. 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JAM4 mediates calcium-independent homophilic adhesion in L cells and recruits MAGI-1, ZO-1, and occludin to JAM4-based cell contacts. MAGI-1 strengthens JAM4-mediated cell adhesion and barrier sealing effects in CHO cell monolayers.\",\n      \"method\": \"Biochemical in vitro binding assays, coexpression in COS-7 cells, cell adhesion assays in L cells, permeability assays in CHO monolayers\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal biochemical binding assays, multiple cell-based functional assays, replicated across multiple cell systems in a single rigorous study\",\n      \"pmids\": [\"12773569\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"JAM4 localization to tight junctions depends on the first Ig-loop, not the MAGI-1-interacting region. Both Ig-loops are necessary for homophilic trans interactions, while cis interactions depend only on the first Ig-loop. JAM4 determines the subcellular localization of MAGI-1 in MDCK cells.\",\n      \"method\": \"Deletion/domain mutant analysis in MDCK cells, immunofluorescence localization, cell adhesion assays\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutagenesis with localization readout, single lab, multiple constructs tested\",\n      \"pmids\": [\"12940823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"MAGI-1 forms a tripartite complex with nephrin and JAM4 in vitro, and MAGI-1 is localized at the glomerular slit diaphragm where it interacts with both nephrin and JAM4 in vivo. JAM4 is also distributed on apical membranes of podocytes beyond the slit diaphragm.\",\n      \"method\": \"Yeast two-hybrid screening, in vitro biochemical pulldown for tripartite complex, immunoelectron microscopy\",\n      \"journal\": \"Laboratory investigation; a journal of technical methods and pathology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid, in vitro complex reconstitution, and immunoelectron microscopy in same study; consistent with prior work\",\n      \"pmids\": [\"16155592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"JAM4 overexpression in MDCK cells enhances HGF-induced branching and scattering. JAM4 activates Rac GTPase (demonstrated by pull-down with CRIB domain of PAK and protrusion formation suppressed by dominant-negative Rac in COS-7 cells), suggesting JAM4 augments HGF-mediated Rac activation.\",\n      \"method\": \"JAM4-overexpressing MDCK cells treated with HGF; COS-7 cell morphology assay; dominant-negative Rac construct; GST-CRIB pulldown assay for active Rac\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical Rac activation assay combined with dominant-negative epistasis and cell morphology readout, single lab\",\n      \"pmids\": [\"15330858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"LNX1 (LNXp70 isoform) directly binds the C-terminal PDZ-binding motif of JAM4 via its second PDZ domain, forming a tripartite complex with JAM4 and Numb. LNX1 facilitates endocytosis of JAM4, and this endocytosis requires Numb (shown by dominant-negative constructs and RNAi). LNX1 is involved in TGF-β-induced redistribution of JAM4 in mammary epithelial cells.\",\n      \"method\": \"Yeast two-hybrid screening, immunoprecipitation from kidney lysates, in vitro biochemical binding assays, dominant-negative constructs, RNA interference, endocytosis assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid, in vivo co-IP from tissue, in vitro tripartite complex, RNAi and dominant-negative epistasis; multiple orthogonal methods in single study\",\n      \"pmids\": [\"16832352\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"JAM4 protein is localized to the plasma membrane of male germ cells and is expressed in stem cell and progenitor cell populations in male germ cell and hematopoietic lineages. JAM4-deficient mice show no obvious histological abnormalities in testes, liver, or kidney, and hematopoietic stem cell numbers are normal, suggesting functional redundancy with other cell adhesion molecules.\",\n      \"method\": \"Signal sequence trap cloning, immunolocalization, JAM4-knockout mouse generation and phenotypic analysis (histology, flow cytometry)\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with defined cellular phenotype readout; negative phenotype is informative for redundancy; single lab\",\n      \"pmids\": [\"16982697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In injured podocytes (PAN nephropathy model), JAM4 expression and subcellular localization are altered — JAM4 is focally increased at the apical membrane of podocytes with effaced foot processes, while its colocalization with MAGI-1 is disrupted. JAM4 remains colocalized with ezrin at the apical membrane in PAN nephropathy.\",\n      \"method\": \"Immunomicroscopy and immunoelectron microscopy in rat proteinuric models; selective detergent extraction to assess subcellular compartmentalization\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by immunoelectron microscopy with biochemical fractionation in disease model, single lab\",\n      \"pmids\": [\"16118391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Surface plasmon resonance of recombinant extracellular domains shows that heterotypic interactions among JAM family members (including JAM4) can be greatly favored over homotypic interactions, and JAM family members have unique tertiary structures despite similar secondary structures.\",\n      \"method\": \"Recombinant protein expression (MBP fusion), surface plasmon resonance (SPR), structural characterization\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro SPR binding assay with recombinant proteins; single lab, single study\",\n      \"pmids\": [\"33801758\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"JAM4/IGSF5 is an immunoglobulin superfamily cell adhesion molecule at tight junctions that mediates calcium-independent homophilic adhesion via its two extracellular Ig-loops; the first Ig-loop directs its localization to tight junctions and drives cis interactions, while both loops are required for trans homophilic adhesion. JAM4 binds MAGI-1 (via its C-terminal PDZ-binding motif) and together they regulate paracellular permeability and form part of the glomerular slit diaphragm complex with nephrin. JAM4 endocytosis is regulated by LNX1-Numb scaffold, and JAM4 activates Rac GTPase to enhance HGF-mediated branching and scattering of epithelial cells.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"IGSF5 (JAM4) is an immunoglobulin-superfamily cell adhesion molecule that organizes epithelial junctions and the glomerular slit diaphragm through its two extracellular Ig-loops and a C-terminal PDZ-binding motif [#0, #2]. It mediates calcium-independent homophilic adhesion and recruits the junctional scaffolds MAGI-1, ZO-1, and occludin to sites of cell contact, with MAGI-1 binding through the JAM4 carboxyl terminus and reinforcing adhesion and paracellular barrier sealing [#0]. The two Ig-loops are functionally divided: the first Ig-loop directs tight-junction localization and supports cis interactions, while both loops are required for trans homophilic adhesion, and JAM4 in turn dictates the subcellular distribution of MAGI-1 [#1]. In the kidney, JAM4 forms a tripartite complex with MAGI-1 and nephrin at the glomerular slit diaphragm, a localization that is disrupted in proteinuric injury where JAM4 redistributes apically and loses MAGI-1 colocalization [#2, #6]. Beyond static adhesion, JAM4 signals to promote epithelial remodeling: it activates Rac GTPase to augment HGF-induced branching and scattering [#3], and its surface levels are controlled by an LNX1–Numb scaffold that drives JAM4 endocytosis, contributing to TGF-β-induced receptor redistribution [#4]. Knockout mice lack overt histological defects in tissues where JAM4 is expressed, indicating functional redundancy with other adhesion molecules [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing that JAM4 is an adhesion molecule that nucleates a junctional scaffold answered whether it does more than mediate contact — it physically organizes the tight-junction protein network.\",\n      \"evidence\": \"In vitro binding assays, COS-7 coexpression, L-cell adhesion assays, and CHO monolayer permeability assays\",\n      \"pmids\": [\"12773569\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define the structural basis of MAGI-1 binding\", \"Physiological tissue context of the adhesion not addressed in this study\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Domain dissection answered which parts of JAM4 carry out localization versus adhesion, separating tight-junction targeting and cis interactions (first Ig-loop) from trans adhesion (both loops).\",\n      \"evidence\": \"Deletion/domain mutant analysis with immunofluorescence and adhesion assays in MDCK cells\",\n      \"pmids\": [\"12940823\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the Ig-loops\", \"Single-lab domain mapping without orthogonal validation\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Linking JAM4 to Rac activation answered whether it has a signaling role beyond adhesion, showing it augments HGF-driven epithelial branching and scattering.\",\n      \"evidence\": \"JAM4-overexpressing MDCK cells with HGF, GST-CRIB pulldown for active Rac, and dominant-negative Rac in COS-7 morphology assays\",\n      \"pmids\": [\"15330858\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism coupling JAM4 to Rac GEFs unidentified\", \"Relevance to endogenous JAM4 levels not established\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Placing JAM4 in a nephrin–MAGI-1 complex answered where it acts in vivo, localizing it to the glomerular slit diaphragm.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro tripartite complex reconstitution, and immunoelectron microscopy\",\n      \"pmids\": [\"16155592\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the tripartite complex for filtration not tested\", \"Stoichiometry and direct vs bridged interactions unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Examining injured podocytes answered whether JAM4 localization is dynamic in disease, revealing apical redistribution and loss of MAGI-1 colocalization in proteinuria.\",\n      \"evidence\": \"Immunoelectron microscopy and detergent-extraction fractionation in rat PAN nephropathy\",\n      \"pmids\": [\"16118391\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal role of JAM4 redistribution in foot-process effacement unknown\", \"Correlative disease-model observation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identifying the LNX1–Numb scaffold answered how JAM4 surface levels are controlled, defining an endocytic regulatory mechanism tied to TGF-β signaling.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP from kidney lysates, in vitro tripartite binding, RNAi and dominant-negative endocytosis assays\",\n      \"pmids\": [\"16832352\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger linking TGF-β to LNX1-Numb recruitment unresolved\", \"In vivo consequence of JAM4 endocytosis not tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Knockout mice answered whether JAM4 is individually required in its expressing tissues, showing no overt defect and implying redundancy.\",\n      \"evidence\": \"Signal sequence trap cloning, immunolocalization, and phenotypic analysis of JAM4-knockout mice (histology, flow cytometry)\",\n      \"pmids\": [\"16982697\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Redundant adhesion molecules not identified\", \"Subtle functional or stress-dependent phenotypes not probed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Quantitative binding measurements answered whether JAM4 prefers homotypic or heterotypic partners, showing heterotypic JAM-family interactions can dominate.\",\n      \"evidence\": \"Surface plasmon resonance of recombinant MBP-fused extracellular domains\",\n      \"pmids\": [\"33801758\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular relevance of heterotypic JAM4 interactions untested\", \"Specific JAM4 heterotypic partner affinities not detailed in context\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether JAM4 adhesion, Rac signaling, and slit-diaphragm assembly are integrated into a single regulatable pathway, and what physiological process specifically requires JAM4, remains open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated in vivo loss-of-function phenotype defined\", \"Redundant partners and disease relevance unestablished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098631\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 5, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005911\", \"supporting_discovery_ids\": []}\n    ],\n    \"complexes\": [\"JAM4-MAGI-1-nephrin slit diaphragm complex\", \"JAM4-LNX1-Numb endocytic complex\"],\n    \"partners\": [\"MAGI1\", \"NPHS1\", \"LNX1\", \"NUMB\", \"ZO-1\", \"OCLN\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}