{"gene":"TSPAN5","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2017,"finding":"TSPAN5 directly interacts with ADAM10 (a disintegrin and metalloproteinase 10), regulates ADAM10 exit from the endoplasmic reticulum and its subsequent trafficking, and differentially regulates ADAM10's ability to cleave substrates and activate Notch signaling. Two TspanC8-specific motifs in the large extracellular domain of Tspan5 are important for ADAM10 interaction and ER exit. The fraction of Tspan5 not associated with ADAM10 is minor in tested cell lines.","method":"Co-immunoprecipitation with novel monoclonal antibodies in human cell lines and mouse tissues; mutagenesis of TspanC8-specific motifs; Notch signaling reporter assays; antibody inhibition experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — reciprocal Co-IP in multiple cell lines and tissues, mutagenesis of key motifs, functional Notch reporter assay, multiple orthogonal methods in a single study","pmids":["28428248"],"is_preprint":false},{"year":2017,"finding":"Tspan5 and Tspan17 (the two most closely related TspanC8 tetraspanins) are the only TspanC8 members that regulate VE-cadherin expression on endothelial cells and are required for T lymphocyte transmigration under flow. Tspan5-ADAM10 complexes maintain normal VE-cadherin expression by controlling ADAM10-mediated proteolytic cleavage, thereby promoting T lymphocyte transmigration.","method":"siRNA knockdown of individual TspanC8s in primary HUVECs; in vitro flow-based transmigration assay with freshly isolated human peripheral blood T lymphocytes; VE-cadherin expression measurement","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean siRNA KD with defined cellular phenotype (T cell transmigration and VE-cadherin expression), in vitro flow assay, multiple TspanC8 members tested as controls","pmids":["28600292"],"is_preprint":false},{"year":2007,"finding":"Tspan5 expression is upregulated during RANKL-induced osteoclastogenesis in RAW264.7 cells, and siRNA-mediated knockdown of Tspan5 suppresses RANKL-induced cell fusion during osteoclastogenesis, indicating a positive regulatory role in polykaryon formation.","method":"RT-PCR and quantitative real-time RT-PCR for expression profiling; gene-specific siRNA knockdown in RAW264.7 cells; assessment of cell fusion during RANKL-stimulated osteoclastogenesis","journal":"Allergology international : official journal of the Japanese Society of Allergology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — siRNA KD with defined cellular phenotype (cell fusion), replicated across time points, but single lab and limited mechanistic detail beyond expression and fusion","pmids":["17965585"],"is_preprint":false},{"year":2021,"finding":"Tspan5 promotes tumor metastasis of hepatocellular carcinoma (HCC) by increasing the enzymatic maturation of ADAM10 and activating Notch signaling through increased cleavage of the Notch1 receptor catalyzed by the γ-secretase complex, leading to enhanced EMT and actin cytoskeleton rearrangement.","method":"Lentiviral overexpression and knockdown of Tspan5 in HCC cells; wound healing and migration assays in vitro; tumor metastasis assay in vivo; western blot for ADAM10 maturation and Notch pathway components; correlation analysis in clinical HCC samples","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — loss- and gain-of-function in vitro and in vivo with defined pathway (ADAM10 maturation → Notch1 cleavage → EMT), single lab, multiple orthogonal readouts","pmids":["33955149"],"is_preprint":false},{"year":2021,"finding":"Depletion of TSPAN5 in HCC cells induces oncogene-induced senescence (OIS) mediated by the p16INK4a/pRb pathway. Mechanistically, TSPAN5 silencing reduces actin polymerization and thereby disrupts myocardin-related transcription factor A–filamin A (MRTF-A–FLNA) complex formation, resulting in decreased expression of MRTF/SRF-dependent target genes and senescence induction both in vitro and in vivo.","method":"siRNA/shRNA knockdown of TSPAN5 in HCC cell lines; senescence assays (SA-β-gal, p16INK4a/pRb pathway markers); actin polymerization assays; co-immunoprecipitation for MRTF-A–FLNA complex; in vivo tumor xenograft experiments","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — KD with defined senescence phenotype, proposed mechanism via actin/MRTF-A-FLNA, multiple assays, single lab","pmids":["34771537"],"is_preprint":false},{"year":2023,"finding":"In rat hippocampal neurons, TSPAN5 promotes exocytosis of AMPA receptors without affecting their internalization. TSPAN5 mediates this function by interacting with the adaptor protein complex AP4 and Stargazin, and possibly uses recycling endosomes as a delivery route.","method":"Live-cell imaging and surface receptor trafficking assays in rat hippocampal neurons; co-immunoprecipitation for AP4 complex and Stargazin; knockdown and overexpression experiments with defined AMPAR exocytosis readout","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct trafficking assay (exocytosis vs internalization), reciprocal Co-IP with AP4 and Stargazin, KD/OE with specific phenotype, multiple orthogonal methods in a peer-reviewed study","pmids":["36795458"],"is_preprint":false},{"year":2016,"finding":"Knockdown and overexpression of TSPAN5 in a neuroblastoma cell line significantly altered expression of serotonin pathway genes (TPH1, TPH2, DDC, and MAOA) and changed media serotonin concentrations, indicating a functional role for TSPAN5 in regulating serotonin biosynthesis/metabolism.","method":"siRNA knockdown and cDNA overexpression in neuroblastoma cell line; qRT-PCR for serotonin pathway gene expression; measurement of media serotonin concentrations","journal":"Molecular psychiatry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — KD and OE with gene expression and metabolite readouts, two orthogonal approaches (KD and OE), single lab","pmids":["26903268"],"is_preprint":false},{"year":2020,"finding":"TSPAN5 physically interacts with clathrin and other vesicle-related proteins (identified by co-immunoprecipitation/proteomics), suggesting a role in vesicular function. Knockdown of TSPAN5 also influenced expression of genes associated with interferon signaling pathways, and ethanol or acamprosate treatment downregulated TSPAN5 expression alongside decreased serotonin and kynurenine concentrations in iPSC-derived neurons and astrocytes.","method":"Co-immunoprecipitation/mass spectrometry for TSPAN5 physical interactors; iPSC-derived neurons and astrocytes; gene expression profiling; metabolite measurement","journal":"Molecular psychiatry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP/MS for protein interaction, iPSC model for functional readout, multiple cell types tested, single lab","pmids":["32753686"],"is_preprint":false},{"year":2023,"finding":"Tspan5 promotes trophoblast cell fusion (syncytialization) by activating the Notch pathway (upregulating Notch-1 and Jagged-1), which in turn activates the EMT process. Knockdown of Tspan5 inhibited cell fusion and EMT-related protein levels in FSK-treated BeWo cells; blocking the Notch pathway with DAPT reversed these effects.","method":"siRNA knockdown and overexpression of Tspan5 in BeWo cells; FSK-induced syncytialization model; immunofluorescence for cell fusion; western blot and qRT-PCR for fusion and EMT markers; DAPT (γ-secretase/Notch inhibitor) rescue experiment","journal":"Zygote (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — KD and OE with pharmacological rescue, specific cellular phenotype (cell fusion), single lab, pathway validation by inhibitor","pmids":["37485669"],"is_preprint":false},{"year":2026,"finding":"Claudin11 (Cldn11) transcriptionally regulates Tspan5 expression; knockdown of Cldn11 suppresses Tspan5 expression and downstream Notch signaling pathway activity in a Tspan5-dependent manner, accelerating cartilage degeneration in an osteoarthritis model.","method":"Cldn11-RNAi lentivirus injection in rat OA model; qRT-PCR, western blot, and immunohistochemistry for Tspan5 and Notch pathway components; Notch pathway inhibitor treatment; mechanical pain threshold assessment","journal":"Journal of orthopaedic surgery and research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — in vivo KD model with pathway readout, single lab, single publication, limited mechanistic dissection of Cldn11–Tspan5 transcriptional link","pmids":["41485015"],"is_preprint":false}],"current_model":"TSPAN5 is a TspanC8 tetraspanin that directly binds ADAM10 via motifs in its large extracellular loop, facilitating ADAM10 exit from the ER and trafficking to the plasma membrane, where the Tspan5–ADAM10 complex promotes cleavage of substrates such as Notch1 (activating Notch signaling and downstream EMT) and VE-cadherin (regulating endothelial barrier function and T lymphocyte transmigration); in neurons, TSPAN5 additionally interacts with the AP4 adaptor complex and Stargazin to drive AMPA receptor exocytosis, and it regulates serotonin pathway gene expression and vesicular trafficking, placing it at the intersection of metalloprotease regulation, synaptic receptor trafficking, and serotonin metabolism."},"narrative":{"mechanistic_narrative":"TSPAN5 is a TspanC8 tetraspanin that functions as a partner and trafficking chaperone for the metalloprotease ADAM10, controlling where and on which substrates ADAM10 acts [PMID:28428248]. It binds ADAM10 directly through two TspanC8-specific motifs in its large extracellular loop, governs ADAM10 exit from the endoplasmic reticulum, and thereby determines ADAM10 substrate cleavage and downstream Notch signaling [PMID:28428248]. Through this axis, TSPAN5 promotes ADAM10 maturation and increased γ-secretase–dependent Notch1 cleavage, driving EMT and actin cytoskeleton rearrangement in hepatocellular carcinoma [PMID:33955149] and activating Notch-1/Jagged-1 signaling to promote trophoblast cell fusion [PMID:37485669]; closely related TSPAN5 and TSPAN17 control ADAM10-mediated VE-cadherin cleavage in endothelial cells to support T lymphocyte transmigration under flow [PMID:28600292]. Independently of its ADAM10 role, TSPAN5 promotes exocytosis of AMPA receptors in hippocampal neurons by interacting with the AP4 adaptor complex and Stargazin, plausibly via recycling endosomes [PMID:36795458], and it regulates serotonin pathway gene expression and media serotonin levels in neuronal cells [PMID:26903268]. TSPAN5 also supports RANKL-induced osteoclast cell fusion [PMID:17965585] and, via effects on actin polymerization and the MRTF-A–FLNA complex, restrains oncogene-induced senescence [PMID:34771537].","teleology":[{"year":2007,"claim":"Established the first functional role for TSPAN5 by linking it to cell-cell fusion, before any molecular mechanism was known.","evidence":"Expression profiling and siRNA knockdown during RANKL-induced osteoclastogenesis in RAW264.7 cells","pmids":["17965585"],"confidence":"Medium","gaps":["No molecular partner or pathway identified","Mechanism connecting TSPAN5 to fusion machinery unknown"]},{"year":2016,"claim":"Connected TSPAN5 to neurotransmitter metabolism, showing it influences serotonin biosynthesis gene expression and serotonin output.","evidence":"siRNA knockdown and cDNA overexpression in a neuroblastoma cell line with qRT-PCR and serotonin metabolite measurement","pmids":["26903268"],"confidence":"Medium","gaps":["Mechanism by which TSPAN5 controls TPH1/TPH2/DDC/MAOA expression unknown","No direct molecular interaction defined"]},{"year":2017,"claim":"Defined the core molecular function of TSPAN5 as a direct ADAM10 partner that controls ADAM10 ER exit and substrate-selective cleavage including Notch activation.","evidence":"Reciprocal Co-IP in human cell lines and mouse tissues, mutagenesis of TspanC8-specific extracellular motifs, and Notch reporter assays","pmids":["28428248"],"confidence":"High","gaps":["Structural basis of the TSPAN5–ADAM10 interface not resolved","How substrate selectivity is encoded not fully defined"]},{"year":2017,"claim":"Extended the TSPAN5–ADAM10 axis to endothelial biology, showing TSPAN5 (with TSPAN17) controls VE-cadherin cleavage and enables T cell transmigration.","evidence":"siRNA knockdown of individual TspanC8s in primary HUVECs with in vitro flow-based transmigration assays and VE-cadherin measurement","pmids":["28600292"],"confidence":"High","gaps":["In vivo relevance to immune trafficking not established","Redundancy with TSPAN17 not fully dissected"]},{"year":2020,"claim":"Broadened the TSPAN5 interactome to vesicular trafficking machinery and tied its expression to interferon signaling and alcohol-responsive metabolite changes.","evidence":"Co-IP/mass spectrometry for physical interactors and iPSC-derived neurons/astrocytes with gene expression and metabolite profiling","pmids":["32753686"],"confidence":"Medium","gaps":["Functional consequence of clathrin interaction not tested","Causal link between TSPAN5 and interferon gene changes unresolved"]},{"year":2021,"claim":"Showed TSPAN5 drives cancer metastasis through ADAM10 maturation and Notch1 cleavage, and separately restrains senescence via actin/MRTF-A-FLNA signaling.","evidence":"Gain- and loss-of-function in HCC cells with migration, metastasis, senescence, and Co-IP assays in vitro and in vivo","pmids":["33955149","34771537"],"confidence":"Medium","gaps":["Whether the senescence (actin/MRTF) and Notch arms are mechanistically linked unclear","Direct ADAM10-independence of the MRTF-A-FLNA effect not established"]},{"year":2023,"claim":"Identified an ADAM10-independent neuronal function: TSPAN5 promotes AMPA receptor exocytosis through AP4 and Stargazin.","evidence":"Surface trafficking and live-cell imaging in rat hippocampal neurons with reciprocal Co-IP for AP4 and Stargazin and KD/OE experiments","pmids":["36795458"],"confidence":"High","gaps":["Whether recycling endosomes are the obligate route not proven","Relationship to TSPAN5's ADAM10 role not addressed"]},{"year":2023,"claim":"Confirmed TSPAN5 drives cell fusion in a second system, trophoblast syncytialization, through Notch-dependent EMT.","evidence":"siRNA knockdown/overexpression in FSK-treated BeWo cells with DAPT (γ-secretase) rescue and fusion/EMT marker readouts","pmids":["37485669"],"confidence":"Medium","gaps":["Direct ADAM10 involvement in trophoblast fusion not shown","In vivo placental relevance untested"]},{"year":2026,"claim":"Placed TSPAN5 downstream of a transcriptional regulator, with Claudin11 controlling TSPAN5 expression and Notch activity in osteoarthritis.","evidence":"Cldn11-RNAi lentivirus in a rat OA model with pathway readouts and Notch inhibitor treatment","pmids":["41485015"],"confidence":"Low","gaps":["Cldn11–Tspan5 transcriptional link not mechanistically dissected","Single lab, single publication","Whether regulation is direct or indirect unknown"]},{"year":null,"claim":"How TSPAN5's ADAM10-dependent metalloprotease scaffolding role mechanistically relates to its ADAM10-independent neuronal trafficking and serotonin-regulatory functions remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking ADAM10 trafficking, AMPAR exocytosis, and serotonin metabolism","No structural model of TSPAN5 complexes","Tissue-specific partner switching not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,5]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[5,7]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[5,7]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0]}],"complexes":[],"partners":["ADAM10","AP4","STARGAZIN","CLATHRIN","FLNA","MRTF-A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P62079","full_name":"Tetraspanin-5","aliases":["Tetraspan NET-4","Transmembrane 4 superfamily member 9"],"length_aa":268,"mass_kda":30.3,"function":"Part of TspanC8 subgroup, composed of 6 members that interact with the transmembrane metalloprotease ADAM10. This interaction is required for ADAM10 exit from the endoplasmic reticulum and for enzymatic maturation and trafficking to the cell surface as well as substrate specificity. Different TspanC8/ADAM10 complexes have distinct substrates (PubMed:26686862, PubMed:28600292, PubMed:37516108). Promotes ADAM10-mediated cleavage of CD44 (PubMed:26686862). Seems to regulate VE-cadherin expression in endothelial cells probably through interaction with ADAM10, promoting leukocyte transmigration (PubMed:28600292)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/P62079/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TSPAN5","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/TSPAN5","total_profiled":1310},"omim":[{"mim_id":"620446","title":"TETRASPANIN 17; TSPAN17","url":"https://www.omim.org/entry/620446"},{"mim_id":"613136","title":"TETRASPANIN 5; TSPAN5","url":"https://www.omim.org/entry/613136"},{"mim_id":"602192","title":"A DISINTEGRIN AND METALLOPROTEINASE DOMAIN 10; ADAM10","url":"https://www.omim.org/entry/602192"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":67.3}],"url":"https://www.proteinatlas.org/search/TSPAN5"},"hgnc":{"alias_symbol":["Tspan-5","NET-4"],"prev_symbol":["TM4SF9"]},"alphafold":{"accession":"P62079","domains":[{"cath_id":"1.10.1450.10","chopping":"86-266","consensus_level":"medium","plddt":93.1033,"start":86,"end":266}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P62079","model_url":"https://alphafold.ebi.ac.uk/files/AF-P62079-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P62079-F1-predicted_aligned_error_v6.png","plddt_mean":89.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TSPAN5","jax_strain_url":"https://www.jax.org/strain/search?query=TSPAN5"},"sequence":{"accession":"P62079","fasta_url":"https://rest.uniprot.org/uniprotkb/P62079.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P62079/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P62079"}},"corpus_meta":[{"pmid":"26903268","id":"PMC_26903268","title":"TSPAN5, ERICH3 and selective serotonin reuptake inhibitors in major depressive disorder: pharmacometabolomics-informed pharmacogenomics.","date":"2016","source":"Molecular psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/26903268","citation_count":101,"is_preprint":false},{"pmid":"28600292","id":"PMC_28600292","title":"ADAM10-Interacting Tetraspanins Tspan5 and Tspan17 Regulate VE-Cadherin Expression and Promote T Lymphocyte Transmigration.","date":"2017","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/28600292","citation_count":44,"is_preprint":false},{"pmid":"32096299","id":"PMC_32096299","title":"miR-155 increases stemness and decitabine resistance in triple-negative breast cancer cells by inhibiting TSPAN5.","date":"2020","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/32096299","citation_count":44,"is_preprint":false},{"pmid":"17965585","id":"PMC_17965585","title":"Expression and function of transmembrane-4 superfamily (tetraspanin) proteins in osteoclasts: reciprocal roles of Tspan-5 and NET-6 during osteoclastogenesis.","date":"2007","source":"Allergology international : official journal of the Japanese Society of Allergology","url":"https://pubmed.ncbi.nlm.nih.gov/17965585","citation_count":35,"is_preprint":false},{"pmid":"33955149","id":"PMC_33955149","title":"Tspan5 promotes epithelial-mesenchymal transition and tumour metastasis of hepatocellular carcinoma by activating Notch signalling.","date":"2021","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/33955149","citation_count":30,"is_preprint":false},{"pmid":"28428248","id":"PMC_28428248","title":"New insights into the tetraspanin Tspan5 using novel monoclonal antibodies.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28428248","citation_count":27,"is_preprint":false},{"pmid":"11472858","id":"PMC_11472858","title":"Pattern of expression of the tetraspanin Tspan-5 during brain development in the mouse.","date":"2001","source":"Mechanisms of development","url":"https://pubmed.ncbi.nlm.nih.gov/11472858","citation_count":24,"is_preprint":false},{"pmid":"32753686","id":"PMC_32753686","title":"TSPAN5 influences serotonin and kynurenine: pharmacogenomic mechanisms related to alcohol use disorder and acamprosate treatment response.","date":"2020","source":"Molecular psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/32753686","citation_count":22,"is_preprint":false},{"pmid":"11043545","id":"PMC_11043545","title":"Mouse Tspan-5, a member of the tetraspanin superfamily, is highly expressed in brain cortical structures.","date":"2000","source":"Neuroreport","url":"https://pubmed.ncbi.nlm.nih.gov/11043545","citation_count":21,"is_preprint":false},{"pmid":"15682397","id":"PMC_15682397","title":"Tetraspanin-5 (Tm4sf9) mRNA expression parallels neuronal maturation in the cerebellum of normal and L7En-2 transgenic mice.","date":"2005","source":"The 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= 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"TSPAN5 directly interacts with ADAM10 (a disintegrin and metalloproteinase 10), regulates ADAM10 exit from the endoplasmic reticulum and its subsequent trafficking, and differentially regulates ADAM10's ability to cleave substrates and activate Notch signaling. Two TspanC8-specific motifs in the large extracellular domain of Tspan5 are important for ADAM10 interaction and ER exit. The fraction of Tspan5 not associated with ADAM10 is minor in tested cell lines.\",\n      \"method\": \"Co-immunoprecipitation with novel monoclonal antibodies in human cell lines and mouse tissues; mutagenesis of TspanC8-specific motifs; Notch signaling reporter assays; antibody inhibition experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — reciprocal Co-IP in multiple cell lines and tissues, mutagenesis of key motifs, functional Notch reporter assay, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"28428248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Tspan5 and Tspan17 (the two most closely related TspanC8 tetraspanins) are the only TspanC8 members that regulate VE-cadherin expression on endothelial cells and are required for T lymphocyte transmigration under flow. Tspan5-ADAM10 complexes maintain normal VE-cadherin expression by controlling ADAM10-mediated proteolytic cleavage, thereby promoting T lymphocyte transmigration.\",\n      \"method\": \"siRNA knockdown of individual TspanC8s in primary HUVECs; in vitro flow-based transmigration assay with freshly isolated human peripheral blood T lymphocytes; VE-cadherin expression measurement\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean siRNA KD with defined cellular phenotype (T cell transmigration and VE-cadherin expression), in vitro flow assay, multiple TspanC8 members tested as controls\",\n      \"pmids\": [\"28600292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Tspan5 expression is upregulated during RANKL-induced osteoclastogenesis in RAW264.7 cells, and siRNA-mediated knockdown of Tspan5 suppresses RANKL-induced cell fusion during osteoclastogenesis, indicating a positive regulatory role in polykaryon formation.\",\n      \"method\": \"RT-PCR and quantitative real-time RT-PCR for expression profiling; gene-specific siRNA knockdown in RAW264.7 cells; assessment of cell fusion during RANKL-stimulated osteoclastogenesis\",\n      \"journal\": \"Allergology international : official journal of the Japanese Society of Allergology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — siRNA KD with defined cellular phenotype (cell fusion), replicated across time points, but single lab and limited mechanistic detail beyond expression and fusion\",\n      \"pmids\": [\"17965585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Tspan5 promotes tumor metastasis of hepatocellular carcinoma (HCC) by increasing the enzymatic maturation of ADAM10 and activating Notch signaling through increased cleavage of the Notch1 receptor catalyzed by the γ-secretase complex, leading to enhanced EMT and actin cytoskeleton rearrangement.\",\n      \"method\": \"Lentiviral overexpression and knockdown of Tspan5 in HCC cells; wound healing and migration assays in vitro; tumor metastasis assay in vivo; western blot for ADAM10 maturation and Notch pathway components; correlation analysis in clinical HCC samples\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — loss- and gain-of-function in vitro and in vivo with defined pathway (ADAM10 maturation → Notch1 cleavage → EMT), single lab, multiple orthogonal readouts\",\n      \"pmids\": [\"33955149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Depletion of TSPAN5 in HCC cells induces oncogene-induced senescence (OIS) mediated by the p16INK4a/pRb pathway. Mechanistically, TSPAN5 silencing reduces actin polymerization and thereby disrupts myocardin-related transcription factor A–filamin A (MRTF-A–FLNA) complex formation, resulting in decreased expression of MRTF/SRF-dependent target genes and senescence induction both in vitro and in vivo.\",\n      \"method\": \"siRNA/shRNA knockdown of TSPAN5 in HCC cell lines; senescence assays (SA-β-gal, p16INK4a/pRb pathway markers); actin polymerization assays; co-immunoprecipitation for MRTF-A–FLNA complex; in vivo tumor xenograft experiments\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — KD with defined senescence phenotype, proposed mechanism via actin/MRTF-A-FLNA, multiple assays, single lab\",\n      \"pmids\": [\"34771537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In rat hippocampal neurons, TSPAN5 promotes exocytosis of AMPA receptors without affecting their internalization. TSPAN5 mediates this function by interacting with the adaptor protein complex AP4 and Stargazin, and possibly uses recycling endosomes as a delivery route.\",\n      \"method\": \"Live-cell imaging and surface receptor trafficking assays in rat hippocampal neurons; co-immunoprecipitation for AP4 complex and Stargazin; knockdown and overexpression experiments with defined AMPAR exocytosis readout\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct trafficking assay (exocytosis vs internalization), reciprocal Co-IP with AP4 and Stargazin, KD/OE with specific phenotype, multiple orthogonal methods in a peer-reviewed study\",\n      \"pmids\": [\"36795458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Knockdown and overexpression of TSPAN5 in a neuroblastoma cell line significantly altered expression of serotonin pathway genes (TPH1, TPH2, DDC, and MAOA) and changed media serotonin concentrations, indicating a functional role for TSPAN5 in regulating serotonin biosynthesis/metabolism.\",\n      \"method\": \"siRNA knockdown and cDNA overexpression in neuroblastoma cell line; qRT-PCR for serotonin pathway gene expression; measurement of media serotonin concentrations\",\n      \"journal\": \"Molecular psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — KD and OE with gene expression and metabolite readouts, two orthogonal approaches (KD and OE), single lab\",\n      \"pmids\": [\"26903268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TSPAN5 physically interacts with clathrin and other vesicle-related proteins (identified by co-immunoprecipitation/proteomics), suggesting a role in vesicular function. Knockdown of TSPAN5 also influenced expression of genes associated with interferon signaling pathways, and ethanol or acamprosate treatment downregulated TSPAN5 expression alongside decreased serotonin and kynurenine concentrations in iPSC-derived neurons and astrocytes.\",\n      \"method\": \"Co-immunoprecipitation/mass spectrometry for TSPAN5 physical interactors; iPSC-derived neurons and astrocytes; gene expression profiling; metabolite measurement\",\n      \"journal\": \"Molecular psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP/MS for protein interaction, iPSC model for functional readout, multiple cell types tested, single lab\",\n      \"pmids\": [\"32753686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Tspan5 promotes trophoblast cell fusion (syncytialization) by activating the Notch pathway (upregulating Notch-1 and Jagged-1), which in turn activates the EMT process. Knockdown of Tspan5 inhibited cell fusion and EMT-related protein levels in FSK-treated BeWo cells; blocking the Notch pathway with DAPT reversed these effects.\",\n      \"method\": \"siRNA knockdown and overexpression of Tspan5 in BeWo cells; FSK-induced syncytialization model; immunofluorescence for cell fusion; western blot and qRT-PCR for fusion and EMT markers; DAPT (γ-secretase/Notch inhibitor) rescue experiment\",\n      \"journal\": \"Zygote (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — KD and OE with pharmacological rescue, specific cellular phenotype (cell fusion), single lab, pathway validation by inhibitor\",\n      \"pmids\": [\"37485669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Claudin11 (Cldn11) transcriptionally regulates Tspan5 expression; knockdown of Cldn11 suppresses Tspan5 expression and downstream Notch signaling pathway activity in a Tspan5-dependent manner, accelerating cartilage degeneration in an osteoarthritis model.\",\n      \"method\": \"Cldn11-RNAi lentivirus injection in rat OA model; qRT-PCR, western blot, and immunohistochemistry for Tspan5 and Notch pathway components; Notch pathway inhibitor treatment; mechanical pain threshold assessment\",\n      \"journal\": \"Journal of orthopaedic surgery and research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — in vivo KD model with pathway readout, single lab, single publication, limited mechanistic dissection of Cldn11–Tspan5 transcriptional link\",\n      \"pmids\": [\"41485015\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TSPAN5 is a TspanC8 tetraspanin that directly binds ADAM10 via motifs in its large extracellular loop, facilitating ADAM10 exit from the ER and trafficking to the plasma membrane, where the Tspan5–ADAM10 complex promotes cleavage of substrates such as Notch1 (activating Notch signaling and downstream EMT) and VE-cadherin (regulating endothelial barrier function and T lymphocyte transmigration); in neurons, TSPAN5 additionally interacts with the AP4 adaptor complex and Stargazin to drive AMPA receptor exocytosis, and it regulates serotonin pathway gene expression and vesicular trafficking, placing it at the intersection of metalloprotease regulation, synaptic receptor trafficking, and serotonin metabolism.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TSPAN5 is a TspanC8 tetraspanin that functions as a partner and trafficking chaperone for the metalloprotease ADAM10, controlling where and on which substrates ADAM10 acts [#0]. It binds ADAM10 directly through two TspanC8-specific motifs in its large extracellular loop, governs ADAM10 exit from the endoplasmic reticulum, and thereby determines ADAM10 substrate cleavage and downstream Notch signaling [#0]. Through this axis, TSPAN5 promotes ADAM10 maturation and increased \\u03b3-secretase\\u2013dependent Notch1 cleavage, driving EMT and actin cytoskeleton rearrangement in hepatocellular carcinoma [#3] and activating Notch-1/Jagged-1 signaling to promote trophoblast cell fusion [#8]; closely related TSPAN5 and TSPAN17 control ADAM10-mediated VE-cadherin cleavage in endothelial cells to support T lymphocyte transmigration under flow [#1]. Independently of its ADAM10 role, TSPAN5 promotes exocytosis of AMPA receptors in hippocampal neurons by interacting with the AP4 adaptor complex and Stargazin, plausibly via recycling endosomes [#5], and it regulates serotonin pathway gene expression and media serotonin levels in neuronal cells [#6]. TSPAN5 also supports RANKL-induced osteoclast cell fusion [#2] and, via effects on actin polymerization and the MRTF-A\\u2013FLNA complex, restrains oncogene-induced senescence [#4].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established the first functional role for TSPAN5 by linking it to cell-cell fusion, before any molecular mechanism was known.\",\n      \"evidence\": \"Expression profiling and siRNA knockdown during RANKL-induced osteoclastogenesis in RAW264.7 cells\",\n      \"pmids\": [\"17965585\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular partner or pathway identified\", \"Mechanism connecting TSPAN5 to fusion machinery unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected TSPAN5 to neurotransmitter metabolism, showing it influences serotonin biosynthesis gene expression and serotonin output.\",\n      \"evidence\": \"siRNA knockdown and cDNA overexpression in a neuroblastoma cell line with qRT-PCR and serotonin metabolite measurement\",\n      \"pmids\": [\"26903268\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which TSPAN5 controls TPH1/TPH2/DDC/MAOA expression unknown\", \"No direct molecular interaction defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined the core molecular function of TSPAN5 as a direct ADAM10 partner that controls ADAM10 ER exit and substrate-selective cleavage including Notch activation.\",\n      \"evidence\": \"Reciprocal Co-IP in human cell lines and mouse tissues, mutagenesis of TspanC8-specific extracellular motifs, and Notch reporter assays\",\n      \"pmids\": [\"28428248\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the TSPAN5\\u2013ADAM10 interface not resolved\", \"How substrate selectivity is encoded not fully defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended the TSPAN5\\u2013ADAM10 axis to endothelial biology, showing TSPAN5 (with TSPAN17) controls VE-cadherin cleavage and enables T cell transmigration.\",\n      \"evidence\": \"siRNA knockdown of individual TspanC8s in primary HUVECs with in vitro flow-based transmigration assays and VE-cadherin measurement\",\n      \"pmids\": [\"28600292\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance to immune trafficking not established\", \"Redundancy with TSPAN17 not fully dissected\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Broadened the TSPAN5 interactome to vesicular trafficking machinery and tied its expression to interferon signaling and alcohol-responsive metabolite changes.\",\n      \"evidence\": \"Co-IP/mass spectrometry for physical interactors and iPSC-derived neurons/astrocytes with gene expression and metabolite profiling\",\n      \"pmids\": [\"32753686\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of clathrin interaction not tested\", \"Causal link between TSPAN5 and interferon gene changes unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed TSPAN5 drives cancer metastasis through ADAM10 maturation and Notch1 cleavage, and separately restrains senescence via actin/MRTF-A-FLNA signaling.\",\n      \"evidence\": \"Gain- and loss-of-function in HCC cells with migration, metastasis, senescence, and Co-IP assays in vitro and in vivo\",\n      \"pmids\": [\"33955149\", \"34771537\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the senescence (actin/MRTF) and Notch arms are mechanistically linked unclear\", \"Direct ADAM10-independence of the MRTF-A-FLNA effect not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified an ADAM10-independent neuronal function: TSPAN5 promotes AMPA receptor exocytosis through AP4 and Stargazin.\",\n      \"evidence\": \"Surface trafficking and live-cell imaging in rat hippocampal neurons with reciprocal Co-IP for AP4 and Stargazin and KD/OE experiments\",\n      \"pmids\": [\"36795458\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether recycling endosomes are the obligate route not proven\", \"Relationship to TSPAN5's ADAM10 role not addressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Confirmed TSPAN5 drives cell fusion in a second system, trophoblast syncytialization, through Notch-dependent EMT.\",\n      \"evidence\": \"siRNA knockdown/overexpression in FSK-treated BeWo cells with DAPT (\\u03b3-secretase) rescue and fusion/EMT marker readouts\",\n      \"pmids\": [\"37485669\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ADAM10 involvement in trophoblast fusion not shown\", \"In vivo placental relevance untested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Placed TSPAN5 downstream of a transcriptional regulator, with Claudin11 controlling TSPAN5 expression and Notch activity in osteoarthritis.\",\n      \"evidence\": \"Cldn11-RNAi lentivirus in a rat OA model with pathway readouts and Notch inhibitor treatment\",\n      \"pmids\": [\"41485015\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Cldn11\\u2013Tspan5 transcriptional link not mechanistically dissected\", \"Single lab, single publication\", \"Whether regulation is direct or indirect unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TSPAN5's ADAM10-dependent metalloprotease scaffolding role mechanistically relates to its ADAM10-independent neuronal trafficking and serotonin-regulatory functions remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking ADAM10 trafficking, AMPAR exocytosis, and serotonin metabolism\", \"No structural model of TSPAN5 complexes\", \"Tissue-specific partner switching not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [5, 7]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ADAM10\", \"AP4\", \"Stargazin\", \"clathrin\", \"FLNA\", \"MRTF-A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}