{"gene":"TSPAN4","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":1997,"finding":"NAG-2 (TSPAN4) co-immunoprecipitates with other TM4SF proteins (CD9 and CD81) and integrins (alpha3beta1 and alpha6beta1) in non-stringent detergent conditions, and co-localizes with CD81 on the surface of spread HT1080 cells, demonstrating its incorporation into specific TM4SF·TM4SF and TM4SF-integrin complexes.","method":"Co-immunoprecipitation, two-color immunofluorescence, expression cloning","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and co-localization, single lab, two orthogonal methods","pmids":["9360996"],"is_preprint":false},{"year":1999,"finding":"Under digitonin conditions that disrupt tetraspan-tetraspan interactions, NAG-2 (TSPAN4) does not form stable complexes with integrins, in contrast to CD81 and CD151, suggesting NAG-2 interacts with integrins indirectly through CD81 or CD151 rather than by direct binding.","method":"Immunoprecipitation in digitonin detergent from multiple cell lines","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic detergent-disruption Co-IP across multiple cell lines, single lab, well-controlled negative result for direct interaction","pmids":["10229664"],"is_preprint":false},{"year":2021,"finding":"TSPAN4 physically interacts with the histamine H4 receptor (H4R), as confirmed by bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation in transfected cells. Histamine stimulation reduced the TSPAN4-H4R interaction, but TSPAN4 did not affect H4R-mediated G protein signaling.","method":"Split-ubiquitin membrane yeast two-hybrid, BRET, BiFC, co-immunoprecipitation","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — three orthogonal methods confirming interaction, single lab","pmids":["34439793"],"is_preprint":false},{"year":2022,"finding":"TGF-β1 increases TSPAN4 expression via the Smad2/3 signaling pathway in retinal pigmented epithelium (RPE) cells, leading to migrasome production. Migrasomes can be internalized by RPE cells and increase their migration and proliferation. TSPAN4-inhibited RPE cells show reduced ability to initiate experimental proliferative vitreoretinopathy (PVR).","method":"Ex vivo and in vitro RPE models, siRNA knockdown, electron microscopy, in vivo experimental PVR model","journal":"Journal of nanobiotechnology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple models (ex vivo, in vitro, in vivo) with loss-of-function, but single lab and mechanistic pathway placement is signaling upstream (Smad2/3)","pmids":["36494806"],"is_preprint":false},{"year":2024,"finding":"TSPAN4 interacts with EGFR and regulates its protein stability in glioblastoma cells. TSPAN4 knockdown disrupts the TSPAN4-EGFR interaction, destabilizes EGFR expression, and inactivates downstream MEK/ERK, STAT3, and AKT signaling pathways, inhibiting GBM cell proliferation and invasion in vitro and tumorigenicity in vivo.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression, western blot, in vitro proliferation/invasion assays, in vivo xenograft","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional loss-of-function with defined pathway readout, single lab","pmids":["39108703"],"is_preprint":false},{"year":2025,"finding":"TSPAN4 interacts with and influences the expression and localization of tropomyosin-1 (TPM1) in vascular smooth muscle cells, affecting cytoskeletal organization and driving phenotypic switching from contractile to synthetic phenotype. TSPAN4 deficiency in mice attenuated neointimal formation following carotid artery ligation.","method":"Co-immunoprecipitation, western blot, EdU assays, Transwell assays, TSPAN4-deficient mouse carotid ligation model","journal":"Clinical science (London, England : 1979)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP identifying binding partner plus in vivo knockout phenotype, single lab","pmids":["41004162"],"is_preprint":false},{"year":2024,"finding":"The extracellular loop 2 (EC2/LEL) of TSPAN4 contributes to its curvature sensitivity and curvature-induced lateral interactions. Deletion of EC2 had the most significant impact on TSPAN4 enrichment in tubular membranes and its interactions, as determined by micropipette aspiration with membrane tube pulling from giant plasma membrane vesicles.","method":"Micropipette aspiration, optical tweezers, confocal microscopy of giant plasma membrane vesicles expressing TSPAN4-GFP truncation mutants","journal":"Biophysical reports","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — reconstitution-like biophysical assay with domain deletion mutagenesis, single lab","pmids":["38562622"],"is_preprint":false},{"year":2026,"finding":"TSPAN4 is palmitoylated at six juxtamembrane cysteines, with DHHC6 and PPT1 identified as the main writer and eraser enzymes, respectively. This palmitoylation is critical for TSPAN4 clustering and cholesterol recruitment, enabling tetraspan-enriched microdomain (TEM) to macrodomain (TEMA) assembly required for migrasome formation. Palmitoylation-deficient TSPAN4 acts as a dominant negative, suppressing migrasome formation in cultured cells and zebrafish embryos, disrupting left-right asymmetry and organ morphogenesis.","method":"Palmitoylation assays, mutagenesis of cysteines, DHHC6/PPT1 knockout/knockdown, live cell imaging, zebrafish morphology assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — identification of writer (DHHC6) and eraser (PPT1) enzymes with mutagenesis, multiple orthogonal methods, functional validation in cells and in vivo zebrafish","pmids":["41984101"],"is_preprint":false},{"year":2026,"finding":"TSPAN4 physically associates with PD-L1 on melanoma cell surfaces (colocalizing on migrasomes and retraction fibers) and negatively regulates PD-L1 protein levels by enhancing its degradation and restricting its lateral mobility at the plasma membrane. Loss of TSPAN4 stabilizes PD-L1, promotes its interaction with CMTM6, and increases PD-L1 surface availability for PD-1 binding, leading to more efficient immune checkpoint engagement.","method":"Cell surface proximity biotinylation coupled with mass spectrometry, co-immunoprecipitation, TSPAN4 knockdown, PD-1/PD-L1 binding assays, T cell functional assays","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity biotinylation MS discovery confirmed by Co-IP plus functional knockdown assays, single lab, two orthogonal methods","pmids":["41525200"],"is_preprint":false},{"year":2012,"finding":"TSPAN4 (as NAG-2/TM4SF7) is expressed in Xenopus laevis in migrating cranial neural crest cells, somites, developing eye, brain, and otic vesicles, suggesting a role in regulation of migration during development, consistent with its expression in cells with high migratory potential.","method":"Quantitative real-time PCR and in situ hybridization during Xenopus laevis embryonic development","journal":"Gene expression patterns : GEP","confidence":"Low","confidence_rationale":"Tier 4 / Weak — expression profiling only during development, no functional experiment performed on TSPAN4 specifically","pmids":["22940433"],"is_preprint":false},{"year":2024,"finding":"In migrating macrophages, TSPAN4 is a component of tetraspanin-enriched microdomains (TEMs) on retraction fibers and migrasomes. SCIMP (like TSPAN4) modulates retraction fiber and migrasome formation. Transmembrane TNF is delivered via SNARE-mediated carriers to the surfaces of retraction fibers and migrasomes, identifying retraction fibers as cytokine secretion sites.","method":"In vitro and in vivo macrophage imaging (mouse and zebrafish), TSPAN4 and SCIMP expression modulation, live cell microscopy","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, imaging-based localization with partial functional follow-up, single lab","pmids":[],"is_preprint":true}],"current_model":"TSPAN4 is a tetraspanin membrane protein that localizes to tetraspanin-enriched microdomains (TEMs), retraction fibers, and migrasomes; its palmitoylation at juxtamembrane cysteines (written by DHHC6, erased by PPT1) drives cholesterol recruitment and TEM-to-macrodomain assembly required for migrasome formation and function, while its large extracellular loop 2 governs membrane curvature sensing; TSPAN4 forms protein complexes with integrins (indirectly via CD81/CD151), the histamine H4 receptor, EGFR (stabilizing it and activating downstream MEK/ERK, STAT3, and AKT), tropomyosin-1 (regulating cytoskeletal organization and VSMC phenotypic switching), and PD-L1 (negatively regulating its stability and surface availability)."},"narrative":{"mechanistic_narrative":"TSPAN4 is a tetraspanin that organizes specialized plasma-membrane microdomains and drives the biogenesis of migration-associated membrane structures, including retraction fibers and migrasomes [PMID:41984101]. It is incorporated into tetraspanin-enriched microdomains through lateral associations with other tetraspanins (CD9, CD81) and, via these partners, indirectly couples to integrins (alpha3beta1, alpha6beta1) rather than binding them directly [PMID:9360996, PMID:10229664]. Palmitoylation at six juxtamembrane cysteines—installed by DHHC6 and removed by PPT1—is critical for TSPAN4 clustering and cholesterol recruitment, enabling the conversion of tetraspanin-enriched microdomains into the macrodomains required for migrasome formation; palmitoylation-deficient TSPAN4 acts as a dominant negative that suppresses migrasome biogenesis and disrupts left-right asymmetry and organ morphogenesis in zebrafish [PMID:41984101]. Its large extracellular loop 2 confers membrane-curvature sensitivity and governs curvature-induced lateral interactions that enrich TSPAN4 in tubular membranes [PMID:38562622]. Through these microdomains TSPAN4 modulates the surface fate of partner proteins: it associates with PD-L1 on melanoma cells and limits PD-L1 stability, lateral mobility, and surface availability for PD-1 engagement [PMID:41525200]. TSPAN4 also engages signaling and cytoskeletal partners in disease contexts, stabilizing EGFR and sustaining MEK/ERK, STAT3, and AKT signaling to promote glioblastoma proliferation and invasion [PMID:39108703], and binding tropomyosin-1 to drive vascular smooth muscle cell phenotypic switching and neointimal formation [PMID:41004162]. TGF-beta1 induces TSPAN4 expression via Smad2/3 to promote migrasome production in retinal pigmented epithelium during proliferative vitreoretinopathy [PMID:36494806].","teleology":[{"year":1997,"claim":"Established that TSPAN4 is not an isolated membrane protein but a participant in the tetraspanin web, defining its membership in TM4SF-TM4SF and TM4SF-integrin complexes.","evidence":"Co-immunoprecipitation and two-color immunofluorescence in HT1080 cells showing association with CD9, CD81, and integrins alpha3beta1/alpha6beta1","pmids":["9360996"],"confidence":"Medium","gaps":["Did not distinguish direct from indirect binding","No functional consequence of the complexes tested"]},{"year":1999,"claim":"Resolved the topology of TSPAN4-integrin association, showing the integrin link is indirect and bridged by other tetraspanins.","evidence":"Digitonin detergent immunoprecipitation across multiple cell lines disrupting tetraspan-tetraspan but not direct interactions","pmids":["10229664"],"confidence":"Medium","gaps":["Which tetraspanin (CD81 vs CD151) provides the primary bridge not resolved","No structural basis for the lateral interaction"]},{"year":2012,"claim":"Linked TSPAN4 expression to migratory cell populations during development, hinting at a role in cell migration before any mechanism was known.","evidence":"qRT-PCR and in situ hybridization in Xenopus laevis embryos","pmids":["22940433"],"confidence":"Low","gaps":["Expression correlation only, no functional perturbation of TSPAN4","No molecular mechanism addressed"]},{"year":2021,"claim":"Identified a GPCR partner, showing TSPAN4 can physically engage the histamine H4 receptor in a ligand-sensitive manner.","evidence":"Split-ubiquitin yeast two-hybrid, BRET, BiFC, and Co-IP in transfected cells","pmids":["34439793"],"confidence":"Medium","gaps":["No effect on H4R G protein signaling, so functional purpose unclear","Interaction shown only in overexpression systems"]},{"year":2022,"claim":"Placed TSPAN4 downstream of TGF-beta1/Smad2/3 signaling as an inducible driver of migrasome production with pathological consequence.","evidence":"RPE cell models, siRNA knockdown, electron microscopy, and an in vivo proliferative vitreoretinopathy model","pmids":["36494806"],"confidence":"Medium","gaps":["Mechanism of migrasome internalization not defined","Direct transcriptional regulation of TSPAN4 by Smad not shown"]},{"year":2024,"claim":"Connected TSPAN4 to oncogenic receptor signaling by showing it stabilizes EGFR and sustains downstream proliferative/invasive pathways.","evidence":"Co-IP, siRNA knockdown, western blot, in vitro proliferation/invasion assays, and xenografts in glioblastoma","pmids":["39108703"],"confidence":"Medium","gaps":["Mechanism of EGFR stabilization (degradation vs trafficking) not defined","Direct vs tetraspanin-bridged EGFR contact not resolved"]},{"year":2024,"claim":"Provided a biophysical basis for TSPAN4 enrichment in curved membranes, assigning curvature sensing to extracellular loop 2.","evidence":"Micropipette aspiration and membrane tube pulling from giant plasma membrane vesicles with EC2 deletion mutants","pmids":["38562622"],"confidence":"Medium","gaps":["Structural mechanism by which EC2 senses curvature unknown","Link between curvature sensing and full migrasome assembly not directly tested"]},{"year":2026,"claim":"Defined the post-translational switch controlling TSPAN4 microdomain assembly, identifying palmitoylation writer/eraser enzymes and cholesterol recruitment as the trigger for TEM-to-macrodomain conversion and migrasome formation.","evidence":"Palmitoylation and cysteine mutagenesis, DHHC6/PPT1 knockout/knockdown, live imaging, and zebrafish morphogenesis assays","pmids":["41984101"],"confidence":"High","gaps":["Stoichiometry and dynamics of palmitoylation cycling in vivo not quantified","How cholesterol is recruited mechanistically not fully resolved"]},{"year":2026,"claim":"Revealed an immune-regulatory role at the membrane, showing TSPAN4 restrains PD-L1 surface availability and checkpoint engagement.","evidence":"Surface proximity biotinylation-MS, Co-IP, TSPAN4 knockdown, and PD-1/PD-L1 binding and T cell assays in melanoma","pmids":["41525200"],"confidence":"Medium","gaps":["Mechanism by which TSPAN4 enhances PD-L1 degradation not defined","Interplay between TSPAN4 and CMTM6 at the molecular level unresolved"]},{"year":null,"claim":"How TSPAN4's diverse partner-specific roles (EGFR, PD-L1, TPM1, H4R) integrate with its core migrasome-organizing function under a single mechanistic framework remains open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking microdomain assembly to partner-protein fate","Structural data on TSPAN4 complexes lacking","Whether partner regulation is migrasome-dependent untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[7,6]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,8]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,7,8,10]}],"pathway":[],"complexes":["tetraspanin-enriched microdomain"],"partners":["CD81","CD9","EGFR","TPM1","PD-L1","HRH4","ITGA3","ITGA6"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O14817","full_name":"Tetraspanin-4","aliases":["Novel antigen 2","NAG-2","Transmembrane 4 superfamily member 7"],"length_aa":238,"mass_kda":26.1,"function":"Structural component of specialized membrane microdomains known as tetraspanin-enriched microdomains (TERMs), which act as platforms for receptor clustering and signaling. Plays an essential role in migrasome formation and migration on retracting fibers at the rear end of migrating cells (PubMed:31371828). Migrasomes are cellular organelles that form as large vesicle-like structures on retraction fibers of migrating cells (PubMed:31371828). Mechanistically, acts as a membrane curvature sensor and participates in stabilizing the migrasome structure in a late stage of biogenesis (PubMed:36252000, PubMed:36823145). May also play a regulatory role for the histamine H4 receptor/HRH4 without affecting histamine binding to HRH4 or signaling (PubMed:34439793)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/O14817/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TSPAN4","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TSPAN4","total_profiled":1310},"omim":[{"mim_id":"602644","title":"TETRASPANIN 4; TSPAN4","url":"https://www.omim.org/entry/602644"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TSPAN4"},"hgnc":{"alias_symbol":["NAG-2","TSPAN-4","TETRASPAN"],"prev_symbol":["TM4SF7"]},"alphafold":{"accession":"O14817","domains":[{"cath_id":"-","chopping":"2-139_205-238","consensus_level":"high","plddt":88.8467,"start":2,"end":238}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O14817","model_url":"https://alphafold.ebi.ac.uk/files/AF-O14817-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O14817-F1-predicted_aligned_error_v6.png","plddt_mean":89.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TSPAN4","jax_strain_url":"https://www.jax.org/strain/search?query=TSPAN4"},"sequence":{"accession":"O14817","fasta_url":"https://rest.uniprot.org/uniprotkb/O14817.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O14817/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O14817"}},"corpus_meta":[{"pmid":"9685355","id":"PMC_9685355","title":"Selective 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Histamine stimulation reduced the TSPAN4-H4R interaction, but TSPAN4 did not affect H4R-mediated G protein signaling.\",\n      \"method\": \"Split-ubiquitin membrane yeast two-hybrid, BRET, BiFC, co-immunoprecipitation\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — three orthogonal methods confirming interaction, single lab\",\n      \"pmids\": [\"34439793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TGF-β1 increases TSPAN4 expression via the Smad2/3 signaling pathway in retinal pigmented epithelium (RPE) cells, leading to migrasome production. Migrasomes can be internalized by RPE cells and increase their migration and proliferation. TSPAN4-inhibited RPE cells show reduced ability to initiate experimental proliferative vitreoretinopathy (PVR).\",\n      \"method\": \"Ex vivo and in vitro RPE models, siRNA knockdown, electron microscopy, in vivo experimental PVR model\",\n      \"journal\": \"Journal of nanobiotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple models (ex vivo, in vitro, in vivo) with loss-of-function, but single lab and mechanistic pathway placement is signaling upstream (Smad2/3)\",\n      \"pmids\": [\"36494806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TSPAN4 interacts with EGFR and regulates its protein stability in glioblastoma cells. TSPAN4 knockdown disrupts the TSPAN4-EGFR interaction, destabilizes EGFR expression, and inactivates downstream MEK/ERK, STAT3, and AKT signaling pathways, inhibiting GBM cell proliferation and invasion in vitro and tumorigenicity in vivo.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression, western blot, in vitro proliferation/invasion assays, in vivo xenograft\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional loss-of-function with defined pathway readout, single lab\",\n      \"pmids\": [\"39108703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TSPAN4 interacts with and influences the expression and localization of tropomyosin-1 (TPM1) in vascular smooth muscle cells, affecting cytoskeletal organization and driving phenotypic switching from contractile to synthetic phenotype. TSPAN4 deficiency in mice attenuated neointimal formation following carotid artery ligation.\",\n      \"method\": \"Co-immunoprecipitation, western blot, EdU assays, Transwell assays, TSPAN4-deficient mouse carotid ligation model\",\n      \"journal\": \"Clinical science (London, England : 1979)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP identifying binding partner plus in vivo knockout phenotype, single lab\",\n      \"pmids\": [\"41004162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The extracellular loop 2 (EC2/LEL) of TSPAN4 contributes to its curvature sensitivity and curvature-induced lateral interactions. Deletion of EC2 had the most significant impact on TSPAN4 enrichment in tubular membranes and its interactions, as determined by micropipette aspiration with membrane tube pulling from giant plasma membrane vesicles.\",\n      \"method\": \"Micropipette aspiration, optical tweezers, confocal microscopy of giant plasma membrane vesicles expressing TSPAN4-GFP truncation mutants\",\n      \"journal\": \"Biophysical reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution-like biophysical assay with domain deletion mutagenesis, single lab\",\n      \"pmids\": [\"38562622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TSPAN4 is palmitoylated at six juxtamembrane cysteines, with DHHC6 and PPT1 identified as the main writer and eraser enzymes, respectively. This palmitoylation is critical for TSPAN4 clustering and cholesterol recruitment, enabling tetraspan-enriched microdomain (TEM) to macrodomain (TEMA) assembly required for migrasome formation. Palmitoylation-deficient TSPAN4 acts as a dominant negative, suppressing migrasome formation in cultured cells and zebrafish embryos, disrupting left-right asymmetry and organ morphogenesis.\",\n      \"method\": \"Palmitoylation assays, mutagenesis of cysteines, DHHC6/PPT1 knockout/knockdown, live cell imaging, zebrafish morphology assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — identification of writer (DHHC6) and eraser (PPT1) enzymes with mutagenesis, multiple orthogonal methods, functional validation in cells and in vivo zebrafish\",\n      \"pmids\": [\"41984101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TSPAN4 physically associates with PD-L1 on melanoma cell surfaces (colocalizing on migrasomes and retraction fibers) and negatively regulates PD-L1 protein levels by enhancing its degradation and restricting its lateral mobility at the plasma membrane. Loss of TSPAN4 stabilizes PD-L1, promotes its interaction with CMTM6, and increases PD-L1 surface availability for PD-1 binding, leading to more efficient immune checkpoint engagement.\",\n      \"method\": \"Cell surface proximity biotinylation coupled with mass spectrometry, co-immunoprecipitation, TSPAN4 knockdown, PD-1/PD-L1 binding assays, T cell functional assays\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity biotinylation MS discovery confirmed by Co-IP plus functional knockdown assays, single lab, two orthogonal methods\",\n      \"pmids\": [\"41525200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TSPAN4 (as NAG-2/TM4SF7) is expressed in Xenopus laevis in migrating cranial neural crest cells, somites, developing eye, brain, and otic vesicles, suggesting a role in regulation of migration during development, consistent with its expression in cells with high migratory potential.\",\n      \"method\": \"Quantitative real-time PCR and in situ hybridization during Xenopus laevis embryonic development\",\n      \"journal\": \"Gene expression patterns : GEP\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — expression profiling only during development, no functional experiment performed on TSPAN4 specifically\",\n      \"pmids\": [\"22940433\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In migrating macrophages, TSPAN4 is a component of tetraspanin-enriched microdomains (TEMs) on retraction fibers and migrasomes. SCIMP (like TSPAN4) modulates retraction fiber and migrasome formation. Transmembrane TNF is delivered via SNARE-mediated carriers to the surfaces of retraction fibers and migrasomes, identifying retraction fibers as cytokine secretion sites.\",\n      \"method\": \"In vitro and in vivo macrophage imaging (mouse and zebrafish), TSPAN4 and SCIMP expression modulation, live cell microscopy\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, imaging-based localization with partial functional follow-up, single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"TSPAN4 is a tetraspanin membrane protein that localizes to tetraspanin-enriched microdomains (TEMs), retraction fibers, and migrasomes; its palmitoylation at juxtamembrane cysteines (written by DHHC6, erased by PPT1) drives cholesterol recruitment and TEM-to-macrodomain assembly required for migrasome formation and function, while its large extracellular loop 2 governs membrane curvature sensing; TSPAN4 forms protein complexes with integrins (indirectly via CD81/CD151), the histamine H4 receptor, EGFR (stabilizing it and activating downstream MEK/ERK, STAT3, and AKT), tropomyosin-1 (regulating cytoskeletal organization and VSMC phenotypic switching), and PD-L1 (negatively regulating its stability and surface availability).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TSPAN4 is a tetraspanin that organizes specialized plasma-membrane microdomains and drives the biogenesis of migration-associated membrane structures, including retraction fibers and migrasomes [#7, #10]. It is incorporated into tetraspanin-enriched microdomains through lateral associations with other tetraspanins (CD9, CD81) and, via these partners, indirectly couples to integrins (alpha3beta1, alpha6beta1) rather than binding them directly [#0, #1]. Palmitoylation at six juxtamembrane cysteines—installed by DHHC6 and removed by PPT1—is critical for TSPAN4 clustering and cholesterol recruitment, enabling the conversion of tetraspanin-enriched microdomains into the macrodomains required for migrasome formation; palmitoylation-deficient TSPAN4 acts as a dominant negative that suppresses migrasome biogenesis and disrupts left-right asymmetry and organ morphogenesis in zebrafish [#7]. Its large extracellular loop 2 confers membrane-curvature sensitivity and governs curvature-induced lateral interactions that enrich TSPAN4 in tubular membranes [#6]. Through these microdomains TSPAN4 modulates the surface fate of partner proteins: it associates with PD-L1 on melanoma cells and limits PD-L1 stability, lateral mobility, and surface availability for PD-1 engagement [#8]. TSPAN4 also engages signaling and cytoskeletal partners in disease contexts, stabilizing EGFR and sustaining MEK/ERK, STAT3, and AKT signaling to promote glioblastoma proliferation and invasion [#4], and binding tropomyosin-1 to drive vascular smooth muscle cell phenotypic switching and neointimal formation [#5]. TGF-beta1 induces TSPAN4 expression via Smad2/3 to promote migrasome production in retinal pigmented epithelium during proliferative vitreoretinopathy [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established that TSPAN4 is not an isolated membrane protein but a participant in the tetraspanin web, defining its membership in TM4SF-TM4SF and TM4SF-integrin complexes.\",\n      \"evidence\": \"Co-immunoprecipitation and two-color immunofluorescence in HT1080 cells showing association with CD9, CD81, and integrins alpha3beta1/alpha6beta1\",\n      \"pmids\": [\"9360996\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not distinguish direct from indirect binding\", \"No functional consequence of the complexes tested\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Resolved the topology of TSPAN4-integrin association, showing the integrin link is indirect and bridged by other tetraspanins.\",\n      \"evidence\": \"Digitonin detergent immunoprecipitation across multiple cell lines disrupting tetraspan-tetraspan but not direct interactions\",\n      \"pmids\": [\"10229664\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which tetraspanin (CD81 vs CD151) provides the primary bridge not resolved\", \"No structural basis for the lateral interaction\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linked TSPAN4 expression to migratory cell populations during development, hinting at a role in cell migration before any mechanism was known.\",\n      \"evidence\": \"qRT-PCR and in situ hybridization in Xenopus laevis embryos\",\n      \"pmids\": [\"22940433\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Expression correlation only, no functional perturbation of TSPAN4\", \"No molecular mechanism addressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified a GPCR partner, showing TSPAN4 can physically engage the histamine H4 receptor in a ligand-sensitive manner.\",\n      \"evidence\": \"Split-ubiquitin yeast two-hybrid, BRET, BiFC, and Co-IP in transfected cells\",\n      \"pmids\": [\"34439793\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No effect on H4R G protein signaling, so functional purpose unclear\", \"Interaction shown only in overexpression systems\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed TSPAN4 downstream of TGF-beta1/Smad2/3 signaling as an inducible driver of migrasome production with pathological consequence.\",\n      \"evidence\": \"RPE cell models, siRNA knockdown, electron microscopy, and an in vivo proliferative vitreoretinopathy model\",\n      \"pmids\": [\"36494806\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of migrasome internalization not defined\", \"Direct transcriptional regulation of TSPAN4 by Smad not shown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected TSPAN4 to oncogenic receptor signaling by showing it stabilizes EGFR and sustains downstream proliferative/invasive pathways.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, western blot, in vitro proliferation/invasion assays, and xenografts in glioblastoma\",\n      \"pmids\": [\"39108703\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of EGFR stabilization (degradation vs trafficking) not defined\", \"Direct vs tetraspanin-bridged EGFR contact not resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided a biophysical basis for TSPAN4 enrichment in curved membranes, assigning curvature sensing to extracellular loop 2.\",\n      \"evidence\": \"Micropipette aspiration and membrane tube pulling from giant plasma membrane vesicles with EC2 deletion mutants\",\n      \"pmids\": [\"38562622\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural mechanism by which EC2 senses curvature unknown\", \"Link between curvature sensing and full migrasome assembly not directly tested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined the post-translational switch controlling TSPAN4 microdomain assembly, identifying palmitoylation writer/eraser enzymes and cholesterol recruitment as the trigger for TEM-to-macrodomain conversion and migrasome formation.\",\n      \"evidence\": \"Palmitoylation and cysteine mutagenesis, DHHC6/PPT1 knockout/knockdown, live imaging, and zebrafish morphogenesis assays\",\n      \"pmids\": [\"41984101\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and dynamics of palmitoylation cycling in vivo not quantified\", \"How cholesterol is recruited mechanistically not fully resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Revealed an immune-regulatory role at the membrane, showing TSPAN4 restrains PD-L1 surface availability and checkpoint engagement.\",\n      \"evidence\": \"Surface proximity biotinylation-MS, Co-IP, TSPAN4 knockdown, and PD-1/PD-L1 binding and T cell assays in melanoma\",\n      \"pmids\": [\"41525200\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which TSPAN4 enhances PD-L1 degradation not defined\", \"Interplay between TSPAN4 and CMTM6 at the molecular level unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TSPAN4's diverse partner-specific roles (EGFR, PD-L1, TPM1, H4R) integrate with its core migrasome-organizing function under a single mechanistic framework remains open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking microdomain assembly to partner-protein fate\", \"Structural data on TSPAN4 complexes lacking\", \"Whether partner regulation is migrasome-dependent untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [7, 6]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 7, 8, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": []}\n    ],\n    \"complexes\": [\"tetraspanin-enriched microdomain\"],\n    \"partners\": [\"CD81\", \"CD9\", \"EGFR\", \"TPM1\", \"PD-L1\", \"HRH4\", \"ITGA3\", \"ITGA6\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}