{"gene":"CD9","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2020,"finding":"Crystal structure of CD9 and cryo-EM structure of CD9 in complex with EWI-2 revealed that CD9's reversed cone-like molecular shape generates membrane curvature in crystalline lipid layers, explaining its localization in high-curvature membrane regions. The CD9–EWI-2 interaction is primarily mediated through small residues in the transmembrane region and protein/lipid interactions, while the large extracellular loop (LEL) region is critical for sperm-egg fusion.","method":"X-ray crystallography (CD9 alone) and cryo-electron microscopy (CD9 with EWI-2); fertilization functional assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure and cryo-EM structure with functional validation by fertilization assay in a single rigorous study","pmids":["32231207"],"is_preprint":false},{"year":2020,"finding":"Cryo-EM structure of CD9 in complex with its partner EWI-F revealed a tetrameric arrangement: two central EWI-F molecules dimerized through ectodomains and two CD9 molecules each bound to one EWI-F transmembrane helix via CD9 helices h3 and h4. This flexible arrangement suggests a 'concatenation model' for tetraspanin-enriched microdomain assembly.","method":"Cryo-EM structure; crystal structures of CD9 LEL bound to nanobodies 4C8 and 4E8","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM and crystal structures with detailed molecular interface mapping in one rigorous study","pmids":["32958604"],"is_preprint":false},{"year":2022,"finding":"CD9 (and tetraspanin4) function as curvature sensors with preference for positive membrane curvature, demonstrated using biomimetic membrane tubes pulled from giant plasma membrane vesicles. This sensing property explains CD9 enrichment in curved structures such as oocyte microvilli.","method":"Biomimetic membrane tube assay from giant plasma membrane vesicles with controllable tension and curvature; thermodynamic modeling","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — rigorous biophysical reconstitution with quantitative thermodynamic model, single study","pmids":["36252000"],"is_preprint":false},{"year":2001,"finding":"CD9's major binding partner identified as CD9P-1 (encoded by KIAA1436, human ortholog of rat FPRP), a 135-kDa cell-surface Ig superfamily protein. Cross-linking experiments showed direct CD9–CD9P-1 association. Chimeric CD9/CD82 molecules revealed that the second half of CD9 (large extracellular loop and fourth transmembrane domain) mediates this interaction. CD9P-1 also associates separately with CD81.","method":"Immunoaffinity purification, mass spectrometry, cross-linking experiments, chimeric protein analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (affinity purification, MS, cross-linking, chimeras) in one study","pmids":["11278880"],"is_preprint":false},{"year":2000,"finding":"FPRP (prostaglandin F2α receptor regulatory protein, 133 kDa) is the most stoichiometric and specific CD9- and CD81-associated protein: essentially 100% of cell-surface FPRP on 293 cells is CD81- and CD9-associated. CD81·CD9·FPRP complexes are discrete in size (<4×10^6 Da) and distinct from integrin-containing CD81 complexes, as shown by immunoprecipitation and immunodepletion.","method":"Gel permeation chromatography, immunoprecipitation, immunodepletion, methyl-β-cyclodextrin treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal biochemical methods establishing stoichiometry and complex distinctness","pmids":["11087758"],"is_preprint":false},{"year":2008,"finding":"DHHC2 is the palmitoyl acyltransferase responsible for palmitoylation of CD9 and CD151. DHHC2 physically associates with CD9 and CD151 but not other cell-surface proteins; inactive DHHC2 (DH→AA or C→S mutations) fails to palmitoylate them. DHHC2-dependent palmitoylation promotes CD9–CD151 physical associations, protects CD9 and CD151 from lysosomal degradation, and shifts cells toward increased cell-cell contacts.","method":"DHHC protein knockdown, active-site mutagenesis, [³H]palmitate labeling, co-immunoprecipitation, functional cell morphology assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis of catalytic site, multiple functional readouts, specific knockdown controls","pmids":["18508921"],"is_preprint":false},{"year":2020,"finding":"Site-specific palmitoylation of CD9 at its three most frequently lipidated cysteine sites is required for EWI-F binding; cysteine-to-alanine mutations markedly reduced EWI-F binding, whereas tryptophan substitutions at those sites rescued binding. Native mass spectrometry revealed nonstochastic distributions of bound acyl chains on wild-type CD9.","method":"Native mass spectrometry, cysteine-to-alanine and tryptophan mutagenesis, EWI-F binding assay, super-resolution microscopy with CD9-specific nanobody","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro binding assay with mutagenesis and native MS, single rigorous study with multiple orthogonal methods","pmids":["32181977"],"is_preprint":false},{"year":2006,"finding":"Cell-surface CD9 homo-clustering is promoted by α3β1 and α6β4 integrins and by palmitoylation of CD9 and β4; CD9 is shifted toward heteroclusters by EWI-2, EWI-F, other tetraspanins, or ablation of CD9 palmitoylation. Unpalmitoylated CD9 shows enhanced EWI-2 association, indicating that depalmitoylation and EWI-2 binding collaborate to shift CD9 from homo- to heteroclusters.","method":"Low-affinity anti-CD9 antibody C9BB detecting homo-clustered CD9; expression of EWI-2/EWI-F, integrins, palmitoylation mutants; co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel molecular probe with multiple perturbation conditions, single lab","pmids":["16537545"],"is_preprint":false},{"year":2013,"finding":"CD9 directly associates with the metalloprotease CD10; the interaction requires the portion of CD9's large extracellular loop from the CCG motif to TM4 and the C-terminal cytoplasmic tail (identified by CD9/CD82 chimeras and site-directed mutagenesis). CD9 expression enhances CD10 release in exosomes ~5-fold, while the CD9 C-terminal tail domain is required for this effect on exosomal release. CD9 knockdown reduces endogenous CD10 release in microvesicles ~2-fold.","method":"CD9/CD82 chimeras, site-directed mutagenesis, stable CD9 expression, shRNA knockdown, co-immunoprecipitation, exosome isolation and quantification","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis, chimeras, knockdown and overexpression with multiple functional readouts in one study","pmids":["23289620"],"is_preprint":false},{"year":2011,"finding":"The C-terminal tail of CD9 (three residues Glu-Met-Val) is required for inhibition of cell adhesion/spreading on fibronectin, promotion of homotypic cell-cell aggregation, and microvilli formation. Mutant CD9 (C-tail replaced with CD82 residues) shows reduced recovery with its major transmembrane interacting partners in Brij 96 and forms larger, more oligomerized complexes, indicating the tail regulates CD9 molecular organization.","method":"C-terminal tail mutagenesis (EMV→PKY substitution), SILAC quantitative proteomics, co-immunoprecipitation, functional adhesion/aggregation assays in multiple cell lines","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis combined with SILAC quantitative MS and multiple functional assays","pmids":["21771881"],"is_preprint":false},{"year":2017,"finding":"CD9 scaffolds the MERS-CoV receptor DPP4 and the protease TMPRSS2 into cell-surface complexes (DPP4:CD9:TTSP), enabling rapid early viral entry via TMPRSS2 cleavage. Without CD9, MERS-CoV traffics to endosomes for later, less efficient cathepsin-mediated activation. In vivo, CD9 silencing in mice sensitized to MERS-CoV by hDPP4 expression significantly reduced susceptibility to infection.","method":"CD9 knockout cell lines, Co-IP of DPP4/TMPRSS2/CD9, MERS-CoV pseudovirus entry assays, in vivo rAd5-hDPP4 mouse model with CD9 siRNA silencing","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout cells, co-IP, pseudovirus assays, and in vivo model with siRNA silencing; multiple orthogonal methods","pmids":["28759649"],"is_preprint":false},{"year":2002,"finding":"Murine CD9 is the receptor for pregnancy-specific glycoprotein 17 (PSG17), the first identified natural ligand for a tetraspanin. PSG17 binding is specific to CD9 and not to CD53, CD63, CD81, CD82, or CD151. Anti-CD9 antibody inhibits PSG17 binding, and macrophages from CD9-deficient mice show significantly reduced PSG17 binding.","method":"cDNA expression library screening, ELISA, flow cytometry, alkaline phosphatase binding assay, in situ rosetting, CD9-knockout macrophage binding assay","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal binding assays with genetic confirmation using CD9-KO cells","pmids":["11805154"],"is_preprint":false},{"year":2003,"finding":"CD9 and CD81 function to prevent fusion of mononuclear phagocytes (monocytes, alveolar macrophages). CD9 and CD81 expression and their integrin complex formation are up-regulated during normal monocyte culture and down-regulated under fusogenic conditions. Anti-CD9/CD81 antibodies promoted fusion; CD9/CD81 double-null mice spontaneously developed multinucleated giant cells in lung and showed enhanced osteoclastogenesis.","method":"In vitro monocyte/macrophage fusion assays, anti-tetraspanin antibody perturbation, CD9-null and CD81-null mouse models, in vivo lung histology and bone analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout (single and double null mice) corroborated by in vitro antibody perturbation experiments in multiple cell types","pmids":["12796480"],"is_preprint":false},{"year":2006,"finding":"CD9 (and CD81) negatively regulate HIV-1 envelope-mediated membrane fusion. Knockdown of CD9 or CD81 increased syncytia formation and viral entry; overexpression rendered cells less susceptible. Anti-CD81 antibodies triggered CD81 clustering in patches that recruited CD4 and CXCR4.","method":"siRNA knockdown, overexpression, anti-tetraspanin antibody treatment, HIV-1 Env-mediated syncytia and viral entry assays in human T lymphoblasts","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown and overexpression with functional assays, single lab","pmids":["17015697"],"is_preprint":false},{"year":2009,"finding":"CD9 negatively regulates LPS-induced macrophage activation by preventing the formation of the LPS receptor complex (CD14/TLR4) at lipid rafts. CD9 partly co-localizes with CD14 at low-density membrane fractions. CD9 knockout macrophages show increased CD14 and TLR4 lipid-raft localization, increased CD14:TLR4 complex formation, decreased IκBα expression, and produce more TNF-α, MMP-2 and MMP-9 after LPS stimulation. CD9-KO mice showed enhanced lung macrophage infiltration and TNF-α production after intranasal LPS.","method":"Anti-CD9 mAb, siRNA, CD9 knockout mice, sucrose gradient fractionation, co-immunoprecipitation, in vitro and in vivo LPS challenge assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO corroborated by siRNA and antibody, with mechanistic fractionation and co-IP data, in vitro and in vivo","pmids":["19414803"],"is_preprint":false},{"year":2019,"finding":"CD9 promotes plasma membrane localization of the glutamine transporter ASCT2, enhancing glutamine uptake in pancreatic cancer cells. CD9 knockdown decreases PDAC organoid growth; heterozygous CD9 deletion in a PDAC mouse model prolonged survival.","method":"CD9 knockdown (siRNA/shRNA), CD9 heterozygous deletion in Pdx1-Cre;KRas;p53 mice, ASCT2 surface localization by cell fractionation/imaging, glutamine uptake assay, organoid formation and limiting dilution tumor initiation assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic models (KD and mouse KO) with mechanistic localization and functional metabolic assays","pmids":["31685994"],"is_preprint":false},{"year":2004,"finding":"CD9 (MRP-1/CD9) gene transduction downregulates Wnt pathway genes (Wnt1, Wnt2b1, Wnt5a) and their target genes (WISP-1, WISP-3, c-Myc, VEGF-A, MMP-26), placing CD9 upstream of Wnt signaling. A neutralizing anti-CD9 antibody inhibited this downregulation in CD9-transfected cells.","method":"CD9 gene transduction into HT1080 and A549 cells, microarray and real-time PCR, Western blotting, neutralizing anti-CD9 antibody treatment","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gene transduction with multiple downstream readouts and antibody neutralization, single lab","pmids":["15334057"],"is_preprint":false},{"year":2006,"finding":"CD9 gene transduction downregulates WAVE2 expression and alters subcellular localization of Arp2 and Arp3, reducing lamellipodia formation and cell motility. This effect is independent of the Wnt signaling pathway, as Wnt siRNA did not affect WAVE2 and WAVE2 siRNA did not affect Wnt expression.","method":"CD9 gene transduction in HT1080 cells, WAVE2-specific siRNA, neutralizing anti-CD9 antibody, morphological analysis, time-lapse migration assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gene transduction, siRNA epistasis, antibody rescue in single lab study","pmids":["16682943"],"is_preprint":false},{"year":2011,"finding":"CD9 specifically controls localization of talin1 to focal adhesions: CD9 deficiency leads to impaired talin1 focal adhesion localization and correlates with increased motility of breast cancer cells.","method":"CD9-deficient cells, talin1 focal adhesion localization by imaging, cell motility assays","journal":"Biochemical Society transactions","confidence":"Low","confidence_rationale":"Tier 3 / Weak — localization assay with functional correlation described in a review-style article with limited method detail in the abstract","pmids":["21428940"],"is_preprint":false},{"year":2014,"finding":"CD9 and CD151 accumulate at the T-cell side of the immunological synapse and support integrin-mediated signaling: silencing CD9 or CD151 reduces α4β1 integrin relocalization to the IS, decreases high-affinity β1 integrin accumulation, diminishes FAK and ERK1/2 phosphorylation, and impairs IL-2 secretion and CD69 upregulation without affecting CD3/actin accumulation or MTOC translocation.","method":"CD9/CD151 siRNA silencing, T cell–APC conjugate assays, confocal imaging of IS components, phospho-FAK and phospho-ERK Western blotting, IL-2 ELISA","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with multiple signaling and functional readouts in T-cell IS context, single lab","pmids":["24723389"],"is_preprint":false},{"year":2014,"finding":"CD9 co-immunoprecipitates with ADAM17 at the cell surface and negatively modulates ADAM17-mediated shedding of LR11 in leukocytes: CD9 overexpression reduces soluble LR11 release, while CD9 knockdown or antibody neutralization increases sLR11 shedding via metalloproteinase-dependent mechanism.","method":"Confocal co-localization, ectopic CD9 expression, CD9 shRNA knockdown, anti-CD9 neutralizing antibody, metalloproteinase inhibitor GM6001, ELISA for soluble LR11","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-localization, overexpression, knockdown, and inhibitor experiments in multiple cell lines, single lab","pmids":["24699135"],"is_preprint":false},{"year":2019,"finding":"CD9 physically associates with ADAM17 at the keratinocyte surface (co-IP confirmed) and negatively regulates ADAM17 sheddase activity. CD9 downregulation activates ADAM17, leading to shedding of HB-EGF and AREG and subsequent EGFR/ERK pathway activation that drives keratinocyte migration and wound healing.","method":"Confocal co-localization, co-immunoprecipitation, CD9 siRNA knockdown and overexpression, TAPI-2 (ADAM17 inhibitor), neutralizing anti-HB-EGF antibody, EGFR/ERK phosphorylation assays, wound-healing migration assays in HaCaT cells and primary mouse keratinocytes","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional rescue experiments with inhibitors and neutralizing antibodies, single lab","pmids":["30745837"],"is_preprint":false},{"year":2020,"finding":"CD9 regulates cellular senescence through the PI3K–AKT–mTOR–p53 signaling pathway: CD9 knockdown in senescent endothelial cells rescues senescence phenotypes, and CD9 upregulation in young cells accelerates senescence. Anti-CD9 antibody treatment and CD9 ablation (ApoE-/- mice) reduced atherosclerotic lesion formation in vivo.","method":"CD9 knockdown and overexpression in HUVECs, senescence assays, PI3K/AKT/mTOR/p53 pathway Western blotting, anti-CD9 antibody treatment in ApoE-/- and Ldlr-/- mice, CD9-KO crossed to ApoE-/- mice","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and antibody perturbation with signaling pathway analysis in vitro and in vivo, single lab","pmids":["32346137"],"is_preprint":false},{"year":2014,"finding":"CD9 co-precipitates with CD26 in mesothelioma cells, and these proteins inversely co-modulate each other's expression. CD9 depletion leads to elevated FAK and Cas-L tyrosine phosphorylation (downstream of β1 integrin), and increased invasiveness, suggesting CD9 negatively regulates tumor invasion by reducing the CD26–α5β1 integrin complex.","method":"siRNA knockdown of CD9 and CD26, co-immunoprecipitation, Western blotting for FAK/Cas-L phosphorylation, cell invasion assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal siRNA knockdowns and co-IP with functional invasion assay, single lab","pmids":["24466195"],"is_preprint":false},{"year":2001,"finding":"CD9 antibody ligation increases human CFU-MK progenitor numbers and reduces megakaryocytic differentiation (decreased CD41+ cell production and MK differentiation antigen expression) in liquid culture, suggesting CD9 participates in megakaryocytic differentiation by involvement in membrane remodeling.","method":"Cell sorting, liquid culture with anti-CD9 antibody ligation, CFU-MK colony assays, flow cytometry for differentiation markers","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — antibody perturbation with quantitative progenitor and differentiation assays, single lab","pmids":["11264162"],"is_preprint":false},{"year":2010,"finding":"CD9P-1 overexpression increases cell motility on collagen I via α2β1 integrin but decreases motility on fibronectin; co-expression of CD9 or CD81 reverses these CD9P-1-mediated motility effects with concomitant CD9P-1 association, showing that the ratio of CD9P-1 to its tetraspanin partners regulates cell motility.","method":"CD9P-1 overexpression, CD9/CD81 co-expression, CD9P-1 mutant analysis (transmembrane and cytoplasmic domains required), co-immunoprecipitation, time-lapse videomicroscopy, Boyden chamber assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — overexpression, domain-mapping mutants, and co-IP with functional migration assays, single lab","pmids":["20574531"],"is_preprint":false},{"year":2000,"finding":"CD9 co-localizes with β1 and β3 integrins on endothelial cell membranes, and anti-CD9 antibody induces tyrosine phosphorylation comparable to β1/β3 integrin ligation. Blocking CD9 with mAb ALMA.1 inhibits EC migration toward fibronectin and vitronectin and impairs wound repair, and ALMA.1 and anti-β1 have additive inhibitory effects, suggesting CD9 cooperates with integrins in EC migration.","method":"Double-labeling immunofluorescence for CD9/integrins, anti-CD9 mAb perturbation, in vitro wound-healing assay, Boyden chamber migration assay, tyrosine phosphorylation Western blotting","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-localization, antibody perturbation, and signaling assay with multiple functional readouts, single lab","pmids":["10669631"],"is_preprint":false},{"year":1999,"finding":"Stromal cell CD9 associates with the β1 integrin subunit and a novel 100 kDa protein (co-immunoprecipitation); antibody cross-linking of CD9 increased the amount of the 100 kDa protein co-precipitated. Ligation of stromal-cell CD9 (but not hematopoietic-cell CD9) modifies hematopoietic progenitor differentiation, shifting pluripotent EML-C1 cells toward undifferentiated, clonogenic states.","method":"Co-culture with anti-CD9 antibody, separate cell pre-treatment to identify the responding cell type, co-immunoprecipitation, colony-forming unit assays","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — antibody perturbation with cell-type specificity controls and co-IP, single lab","pmids":["10194438"],"is_preprint":false},{"year":2012,"finding":"CD9 and CD81 are present as separate, non-complexed extracellular structures in bilayers on the oocyte surface; microinjection of CD9 RNA rescued fusion defects in both CD9-deficient and CD81-deficient oocytes, whereas CD81 failed to rescue either, indicating CD9 and CD81 function independently as extracellular components in sperm-oocyte fusion.","method":"Immunocytochemistry, immunobiochemistry, electron microscopy, RNA microinjection rescue experiments in CD9-KO and CD81-KO oocytes","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic rescue experiment with multiple imaging methods, single lab","pmids":["23213457"],"is_preprint":false},{"year":2021,"finding":"CD9 localizes primarily to the plasma membrane, where it is secreted more abundantly in ectosomes than in exosomes. CD9 and a PM-stabilized CD63 mutant are more abundantly released in EVs than wild-type CD63. Comparative proteomics identified BSG and SLC3A2 as likely ectosome-specific proteins, distinct from the exosomal marker LAMP1.","method":"Live intracellular tracking of CD9 and CD63, comparative proteomics, differential response to endosomal pH neutralization, subcellular fractionation","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging with functional comparative proteomics, single lab study with multiple orthogonal approaches","pmids":["34282141"],"is_preprint":false},{"year":2023,"finding":"Concomitant knockout of CD9 and CD81 in MCF7 cells specifically reduces EV levels of CD9P-1/PTGFRN and EWI-2/IGSF8 (the sole significantly decreased EV proteins), partially because of decreased cell expression of EWI-2. Single KO of CD9, CD81, or CD63 had little effect on overall EV protein composition.","method":"CD9, CD81, CD63 single and double knockout by CRISPR, quantitative mass spectrometry proteomics of EVs","journal":"Journal of extracellular vesicles","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with quantitative MS proteomics, single lab","pmids":["37525398"],"is_preprint":false},{"year":2015,"finding":"CD9 knockdown in MDA-MB-231 breast cancer cells inhibits MSC invasion by 95% and anti-CD9 antibody blockade by 70%; CD9-deficient cells lose magnupodium/lamellipodium structures and gain membrane ruffles, which impairs adhesion and invasiveness. CD9 knockdown also suppresses metastatic capacity in mouse xenografts.","method":"CD9 shRNA knockdown, anti-CD9 antibody blockade, TIRF/confocal/scanning EM microscopy, mouse xenograft metastasis model","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with antibody confirmation, multiple imaging modalities and in vivo xenograft, single lab","pmids":["25762645"],"is_preprint":false},{"year":2002,"finding":"Down-regulation of CD9 mRNA expression in Schwann cells follows axonal degeneration after sciatic nerve injury and is restored upon axonal regeneration; in culture, CD9 expression requires contact with neurons. This parallels myelin gene regulation, suggesting axons regulate CD9 expression in Schwann cells.","method":"Adult rat sciatic nerve injury model, in situ hybridization for CD9 mRNA, Schwann cell–neuron co-culture experiments","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo nerve injury model with in vitro co-culture validation, single lab","pmids":["8581316"],"is_preprint":false},{"year":2015,"finding":"In Drosophila, tetraspanin tsp2A (close homolog of human CD9) genetically interacts with Pvr (PDGFR homolog), and tsp2A knockdown partially rescues Pvr-induced glial over-migration. In human glioma cells, CD9 is in close association with PDGFRα and PDGFRβ (proximity ligation assay), and CD9 knockdown blocks PDGF-BB-stimulated cell migration.","method":"Drosophila genetic screen with dsRNA, in situ proximity ligation assay for CD9-PDGFR association, CD9 siRNA knockdown in human glioma cells with PDGF-stimulated migration assay","journal":"Journal of neuro-oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic screen in model organism with corroborating human cell knockdown and proximity ligation, single lab","pmids":["26224160"],"is_preprint":false},{"year":1997,"finding":"CD9 expression is upregulated ~7-fold during TPA-induced megakaryocytic differentiation of K562 cells via PKC activation (blocked by GF109203X), and upregulated CD9 associates with β1 integrin. A TPA-responsive element was localized to a 52-bp fragment of the CD9 promoter.","method":"TPA treatment, PKC inhibitor GF109203X, quantitative RT-PCR, flow cytometry, co-immunoprecipitation, CD9 promoter-CAT reporter constructs","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition, quantitative gene expression, promoter mapping with reporter assay, and co-IP, single lab","pmids":["9264383"],"is_preprint":false},{"year":2021,"finding":"CD9 inhibition (by cytopermeable blocking peptides or gene deletion) reduces the number of early endosomes and affects mitochondrial quality control, specifically impairing mitophagy; CD9 KO cells compensate by increasing total mitochondrial mass and reducing mitophagy. CD9 peptide treatment delayed primary tumor growth and reduced metastasis in vivo.","method":"CD9-blocking peptides vs. CD9 gene deletion comparison, endosome and lysosome quantification, mitochondrial mass and mitophagy assays, in vivo melanoma model","journal":"Journal of extracellular vesicles","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, organelle counting with limited mechanistic resolution linking CD9 directly to mitophagy machinery","pmids":["34012515"],"is_preprint":false},{"year":2022,"finding":"JAM-A forms a complex with α3β1 integrin and tetraspanins CD151 and CD9 through its extracellular domain (by mapping experiments), and this complex is required for collective cell migration of polarized epithelial cells on laminin and collagen-I substrates.","method":"JAM-A/CD9/CD151/α3β1 integrin depletion by siRNA in MDCK cells, co-immunoprecipitation and domain mapping, collective migration assay (scratch wound, live imaging)","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP domain mapping and siRNA phenotyping in polarized epithelial cells, single lab","pmids":["35067832"],"is_preprint":false}],"current_model":"CD9 is a tetraspanin membrane protein whose reversed-cone molecular shape generates membrane curvature and enables curvature sensing; it organizes multiprotein complexes at the plasma membrane (tetraspanin-enriched microdomains) by directly associating—via its large extracellular loop, transmembrane helices, and C-terminal tail—with partners including FPRP/CD9P-1, EWI-2/EWI-F, integrins (α3β1, α6β4, β1), ADAM17, DPP4/TMPRSS2, and ASCT2; its palmitoylation by DHHC2 stabilizes these complexes and protects CD9 from lysosomal degradation; through these interactions CD9 regulates sperm–oocyte fusion, mononuclear phagocyte fusion, HIV-1 and MERS-CoV membrane fusion, LPS-TLR4 receptor complex assembly, ADAM17-mediated ectodomain shedding (of HB-EGF, LR11, and TGFα), Wnt and PI3K–AKT–mTOR–p53 signaling, actin cytoskeleton organization via WAVE2 downregulation, integrin signaling at the immunological synapse, ASCT2-dependent glutamine uptake, and glutamine-fueled tumor growth."},"narrative":{"mechanistic_narrative":"CD9 is a tetraspanin membrane protein whose reversed cone-like molecular shape generates and senses positive membrane curvature, driving its enrichment in high-curvature plasma-membrane structures and underpinning its role as an organizer of tetraspanin-enriched microdomains [PMID:32231207, PMID:36252000]. It nucleates these microdomains through direct, domain-specific contacts: its large extracellular loop and fourth transmembrane domain engage the Ig-superfamily partners FPRP/CD9P-1 and EWI-2/EWI-F, with EWI-F binding occurring through CD9 transmembrane helices h3/h4 in a flexible tetrameric \"concatenation\" arrangement, and its C-terminal cytoplasmic tail tuning complex size and oligomerization [PMID:32958604, PMID:11278880, PMID:11087758, PMID:21771881]. Microdomain assembly is regulated by site-specific S-palmitoylation: the acyltransferase DHHC2 palmitoylates CD9 to stabilize CD9-partner associations and protect CD9 from lysosomal degradation, while palmitoylation state and partner availability shift CD9 between homo- and heteroclusters [PMID:18508921, PMID:32181977, PMID:16537545]. Through these scaffolding interactions CD9 governs membrane fusion and cell-cell organization—it is an indispensable oocyte-side component of sperm-egg fusion, prevents mononuclear phagocyte fusion, and restrains HIV-1 envelope-mediated fusion [PMID:32231207, PMID:12796480, PMID:17015697, PMID:23213457]. CD9 also assembles and modulates cell-surface receptor and protease complexes: it scaffolds DPP4 and TMPRSS2 to promote early MERS-CoV entry, negatively regulates LPS-driven CD14/TLR4 receptor-complex formation, and binds metalloproteases CD10/ADAM17 to control ectodomain shedding of substrates including HB-EGF, AREG, and LR11 [PMID:28759649, PMID:19414803, PMID:23289620, PMID:24699135, PMID:30745837]. By partnering with integrins, it supports integrin signaling at the immunological synapse and directs talin1-dependent focal-adhesion and cytoskeletal remodeling, influencing adhesion, migration, and tumor invasion [PMID:24723389, PMID:10669631, PMID:31685994]. CD9 additionally promotes plasma-membrane localization of the glutamine transporter ASCT2 to fuel pancreatic tumor growth and acts on Wnt and PI3K-AKT-mTOR-p53 signaling [PMID:31685994, PMID:15334057, PMID:32346137].","teleology":[{"year":2000,"claim":"Establishing the molecular identity of CD9's most stoichiometric partner answered whether tetraspanins form defined complexes; FPRP/CD9P-1 was shown to be essentially fully CD9/CD81-associated in discrete particles distinct from integrin complexes.","evidence":"Gel permeation chromatography, immunoprecipitation, immunodepletion and immunoaffinity/MS in 293 cells","pmids":["11087758","11278880"],"confidence":"High","gaps":["Did not resolve the structural interface","Functional consequence of the CD9-FPRP complex not addressed"]},{"year":2002,"claim":"Identification of PSG17 as a CD9-specific natural ligand established that a tetraspanin can act as a bona fide receptor, answering whether CD9 engages defined extracellular ligands.","evidence":"cDNA library screening, binding assays, and CD9-knockout macrophage binding","pmids":["11805154"],"confidence":"High","gaps":["Downstream signaling from PSG17-CD9 binding not defined","Physiological role of this binding in vivo not established"]},{"year":2003,"claim":"Genetic models clarified CD9's physiological role in cell fusion, showing it (with CD81) actively prevents inappropriate mononuclear phagocyte fusion rather than promoting it.","evidence":"In vitro fusion assays with antibody perturbation plus CD9/CD81 single- and double-null mice","pmids":["12796480"],"confidence":"High","gaps":["Molecular mechanism restraining fusion not resolved","Partner dependence of fusion suppression unclear"]},{"year":2008,"claim":"Identifying DHHC2 as the palmitoyl acyltransferase for CD9 answered how CD9 complexes and protein stability are post-translationally controlled.","evidence":"DHHC knockdown, active-site mutagenesis, [3H]palmitate labeling, and co-IP","pmids":["18508921"],"confidence":"High","gaps":["Depalmitoylating enzyme not identified","Dynamics of palmitoylation turnover in vivo not measured"]},{"year":2011,"claim":"Domain dissection of the C-terminal tail and homo/heterocluster studies answered how CD9 molecular organization is encoded, linking the tail and palmitoylation to complex size and cluster state.","evidence":"C-tail mutagenesis with SILAC proteomics and functional assays; homo-clustering probe with palmitoylation mutants","pmids":["21771881","16537545"],"confidence":"Medium","gaps":["Cluster transitions inferred from antibody probe, not direct imaging at the time","Single-lab findings"]},{"year":2013,"claim":"Mapping the CD9-CD10/ADAM17 interactions established CD9 as a regulator of cell-surface metalloprotease activity and of protease cargo loading into extracellular vesicles.","evidence":"CD9/CD82 chimeras, mutagenesis, knockdown/overexpression and exosome quantification","pmids":["23289620","24699135","30745837"],"confidence":"Medium","gaps":["Whether CD9 directly inhibits catalysis or sequesters substrate unresolved","Sheddase regulation tested largely in single-lab systems"]},{"year":2017,"claim":"The DPP4:CD9:TMPRSS2 scaffolding model answered how CD9 dictates viral entry kinetics, showing CD9 assembles receptor-protease complexes that route MERS-CoV to rapid surface entry.","evidence":"CD9 knockout cells, co-IP, pseudovirus entry, and in vivo hDPP4 mouse model with CD9 silencing","pmids":["28759649"],"confidence":"High","gaps":["Structural basis of the trimolecular scaffold not solved","Generality to other receptor-protease pairs untested"]},{"year":2019,"claim":"Linking CD9 to ASCT2 surface localization answered how a scaffolding tetraspanin can drive tumor metabolism, connecting CD9 to glutamine-fueled pancreatic cancer growth.","evidence":"Knockdown, heterozygous CD9 deletion in a PDAC mouse model, surface localization and 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binding","supporting_discovery_ids":[0,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[8,20,21]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[10]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,4,10,21,29]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[8,29,30]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[10,35]}],"pathway":[{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[26,31,36]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[12,14,19]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[10,13,15]},{"term_id":"R-HSA-392499","term_label":"Metabolism of 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Present at the cell surface of oocytes and plays a key role in sperm-egg fusion, possibly by organizing multiprotein complexes and the morphology of the membrane required for the fusion (By similarity). In myoblasts, associates with CD81 and PTGFRN and inhibits myotube fusion during muscle regeneration (By similarity). In macrophages, associates with CD81 and beta-1 and beta-2 integrins, and prevents macrophage fusion into multinucleated giant cells specialized in ingesting complement-opsonized large particles (PubMed:12796480). Also prevents the fusion between mononuclear cell progenitors into osteoclasts in charge of bone resorption (By similarity). Acts as a receptor for PSG17 (By similarity). Involved in platelet activation and aggregation (PubMed:18541721). Regulates paranodal junction formation (By similarity). Involved in cell adhesion, cell motility and tumor metastasis (PubMed:7511626, PubMed:8478605)","subcellular_location":"Cell membrane; Membrane; Secreted, extracellular exosome","url":"https://www.uniprot.org/uniprotkb/P21926/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CD9","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000010278","cell_line_id":"CID000400","localizations":[{"compartment":"membrane","grade":3},{"compartment":"vesicles","grade":3}],"interactors":[{"gene":"PTGFRN","stoichiometry":0.2},{"gene":"CD81","stoichiometry":0.2},{"gene":"NSA2","stoichiometry":0.2},{"gene":"NRAS","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000400","total_profiled":1310},"omim":[{"mim_id":"618911","title":"CHROMOSOME 16 OPEN READING FRAME 92; C16ORF92","url":"https://www.omim.org/entry/618911"},{"mim_id":"618621","title":"ZDHHC PALMITOYLTRANSFERASE 2; ZDHHC2","url":"https://www.omim.org/entry/618621"},{"mim_id":"606644","title":"IMMUNOGLOBULIN SUPERFAMILY, MEMBER 8; IGSF8","url":"https://www.omim.org/entry/606644"},{"mim_id":"604874","title":"KILLER CELL LECTIN-LIKE RECEPTOR, SUBFAMILY G, MEMBER 1; KLRG1","url":"https://www.omim.org/entry/604874"},{"mim_id":"602243","title":"CD151 ANTIGEN; CD151","url":"https://www.omim.org/entry/602243"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CD9"},"hgnc":{"alias_symbol":["BA2","P24","TSPAN29","MRP-1"],"prev_symbol":["MIC3"]},"alphafold":{"accession":"P21926","domains":[{"cath_id":"-","chopping":"4-43_56-127_170-226","consensus_level":"high","plddt":91.3995,"start":4,"end":226}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P21926","model_url":"https://alphafold.ebi.ac.uk/files/AF-P21926-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P21926-F1-predicted_aligned_error_v6.png","plddt_mean":88.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CD9","jax_strain_url":"https://www.jax.org/strain/search?query=CD9"},"sequence":{"accession":"P21926","fasta_url":"https://rest.uniprot.org/uniprotkb/P21926.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P21926/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P21926"}},"corpus_meta":[{"pmid":"34282141","id":"PMC_34282141","title":"Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/34282141","citation_count":693,"is_preprint":false},{"pmid":"11278880","id":"PMC_11278880","title":"The major CD9 and CD81 molecular partner. 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The CD9–EWI-2 interaction is primarily mediated through small residues in the transmembrane region and protein/lipid interactions, while the large extracellular loop (LEL) region is critical for sperm-egg fusion.\",\n      \"method\": \"X-ray crystallography (CD9 alone) and cryo-electron microscopy (CD9 with EWI-2); fertilization functional assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure and cryo-EM structure with functional validation by fertilization assay in a single rigorous study\",\n      \"pmids\": [\"32231207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cryo-EM structure of CD9 in complex with its partner EWI-F revealed a tetrameric arrangement: two central EWI-F molecules dimerized through ectodomains and two CD9 molecules each bound to one EWI-F transmembrane helix via CD9 helices h3 and h4. This flexible arrangement suggests a 'concatenation model' for tetraspanin-enriched microdomain assembly.\",\n      \"method\": \"Cryo-EM structure; crystal structures of CD9 LEL bound to nanobodies 4C8 and 4E8\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM and crystal structures with detailed molecular interface mapping in one rigorous study\",\n      \"pmids\": [\"32958604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CD9 (and tetraspanin4) function as curvature sensors with preference for positive membrane curvature, demonstrated using biomimetic membrane tubes pulled from giant plasma membrane vesicles. This sensing property explains CD9 enrichment in curved structures such as oocyte microvilli.\",\n      \"method\": \"Biomimetic membrane tube assay from giant plasma membrane vesicles with controllable tension and curvature; thermodynamic modeling\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — rigorous biophysical reconstitution with quantitative thermodynamic model, single study\",\n      \"pmids\": [\"36252000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CD9's major binding partner identified as CD9P-1 (encoded by KIAA1436, human ortholog of rat FPRP), a 135-kDa cell-surface Ig superfamily protein. Cross-linking experiments showed direct CD9–CD9P-1 association. Chimeric CD9/CD82 molecules revealed that the second half of CD9 (large extracellular loop and fourth transmembrane domain) mediates this interaction. CD9P-1 also associates separately with CD81.\",\n      \"method\": \"Immunoaffinity purification, mass spectrometry, cross-linking experiments, chimeric protein analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (affinity purification, MS, cross-linking, chimeras) in one study\",\n      \"pmids\": [\"11278880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"FPRP (prostaglandin F2α receptor regulatory protein, 133 kDa) is the most stoichiometric and specific CD9- and CD81-associated protein: essentially 100% of cell-surface FPRP on 293 cells is CD81- and CD9-associated. CD81·CD9·FPRP complexes are discrete in size (<4×10^6 Da) and distinct from integrin-containing CD81 complexes, as shown by immunoprecipitation and immunodepletion.\",\n      \"method\": \"Gel permeation chromatography, immunoprecipitation, immunodepletion, methyl-β-cyclodextrin treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal biochemical methods establishing stoichiometry and complex distinctness\",\n      \"pmids\": [\"11087758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"DHHC2 is the palmitoyl acyltransferase responsible for palmitoylation of CD9 and CD151. DHHC2 physically associates with CD9 and CD151 but not other cell-surface proteins; inactive DHHC2 (DH→AA or C→S mutations) fails to palmitoylate them. DHHC2-dependent palmitoylation promotes CD9–CD151 physical associations, protects CD9 and CD151 from lysosomal degradation, and shifts cells toward increased cell-cell contacts.\",\n      \"method\": \"DHHC protein knockdown, active-site mutagenesis, [³H]palmitate labeling, co-immunoprecipitation, functional cell morphology assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis of catalytic site, multiple functional readouts, specific knockdown controls\",\n      \"pmids\": [\"18508921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Site-specific palmitoylation of CD9 at its three most frequently lipidated cysteine sites is required for EWI-F binding; cysteine-to-alanine mutations markedly reduced EWI-F binding, whereas tryptophan substitutions at those sites rescued binding. Native mass spectrometry revealed nonstochastic distributions of bound acyl chains on wild-type CD9.\",\n      \"method\": \"Native mass spectrometry, cysteine-to-alanine and tryptophan mutagenesis, EWI-F binding assay, super-resolution microscopy with CD9-specific nanobody\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding assay with mutagenesis and native MS, single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"32181977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Cell-surface CD9 homo-clustering is promoted by α3β1 and α6β4 integrins and by palmitoylation of CD9 and β4; CD9 is shifted toward heteroclusters by EWI-2, EWI-F, other tetraspanins, or ablation of CD9 palmitoylation. Unpalmitoylated CD9 shows enhanced EWI-2 association, indicating that depalmitoylation and EWI-2 binding collaborate to shift CD9 from homo- to heteroclusters.\",\n      \"method\": \"Low-affinity anti-CD9 antibody C9BB detecting homo-clustered CD9; expression of EWI-2/EWI-F, integrins, palmitoylation mutants; co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel molecular probe with multiple perturbation conditions, single lab\",\n      \"pmids\": [\"16537545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CD9 directly associates with the metalloprotease CD10; the interaction requires the portion of CD9's large extracellular loop from the CCG motif to TM4 and the C-terminal cytoplasmic tail (identified by CD9/CD82 chimeras and site-directed mutagenesis). CD9 expression enhances CD10 release in exosomes ~5-fold, while the CD9 C-terminal tail domain is required for this effect on exosomal release. CD9 knockdown reduces endogenous CD10 release in microvesicles ~2-fold.\",\n      \"method\": \"CD9/CD82 chimeras, site-directed mutagenesis, stable CD9 expression, shRNA knockdown, co-immunoprecipitation, exosome isolation and quantification\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis, chimeras, knockdown and overexpression with multiple functional readouts in one study\",\n      \"pmids\": [\"23289620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The C-terminal tail of CD9 (three residues Glu-Met-Val) is required for inhibition of cell adhesion/spreading on fibronectin, promotion of homotypic cell-cell aggregation, and microvilli formation. Mutant CD9 (C-tail replaced with CD82 residues) shows reduced recovery with its major transmembrane interacting partners in Brij 96 and forms larger, more oligomerized complexes, indicating the tail regulates CD9 molecular organization.\",\n      \"method\": \"C-terminal tail mutagenesis (EMV→PKY substitution), SILAC quantitative proteomics, co-immunoprecipitation, functional adhesion/aggregation assays in multiple cell lines\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis combined with SILAC quantitative MS and multiple functional assays\",\n      \"pmids\": [\"21771881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CD9 scaffolds the MERS-CoV receptor DPP4 and the protease TMPRSS2 into cell-surface complexes (DPP4:CD9:TTSP), enabling rapid early viral entry via TMPRSS2 cleavage. Without CD9, MERS-CoV traffics to endosomes for later, less efficient cathepsin-mediated activation. In vivo, CD9 silencing in mice sensitized to MERS-CoV by hDPP4 expression significantly reduced susceptibility to infection.\",\n      \"method\": \"CD9 knockout cell lines, Co-IP of DPP4/TMPRSS2/CD9, MERS-CoV pseudovirus entry assays, in vivo rAd5-hDPP4 mouse model with CD9 siRNA silencing\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout cells, co-IP, pseudovirus assays, and in vivo model with siRNA silencing; multiple orthogonal methods\",\n      \"pmids\": [\"28759649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Murine CD9 is the receptor for pregnancy-specific glycoprotein 17 (PSG17), the first identified natural ligand for a tetraspanin. PSG17 binding is specific to CD9 and not to CD53, CD63, CD81, CD82, or CD151. Anti-CD9 antibody inhibits PSG17 binding, and macrophages from CD9-deficient mice show significantly reduced PSG17 binding.\",\n      \"method\": \"cDNA expression library screening, ELISA, flow cytometry, alkaline phosphatase binding assay, in situ rosetting, CD9-knockout macrophage binding assay\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal binding assays with genetic confirmation using CD9-KO cells\",\n      \"pmids\": [\"11805154\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CD9 and CD81 function to prevent fusion of mononuclear phagocytes (monocytes, alveolar macrophages). CD9 and CD81 expression and their integrin complex formation are up-regulated during normal monocyte culture and down-regulated under fusogenic conditions. Anti-CD9/CD81 antibodies promoted fusion; CD9/CD81 double-null mice spontaneously developed multinucleated giant cells in lung and showed enhanced osteoclastogenesis.\",\n      \"method\": \"In vitro monocyte/macrophage fusion assays, anti-tetraspanin antibody perturbation, CD9-null and CD81-null mouse models, in vivo lung histology and bone analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout (single and double null mice) corroborated by in vitro antibody perturbation experiments in multiple cell types\",\n      \"pmids\": [\"12796480\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CD9 (and CD81) negatively regulate HIV-1 envelope-mediated membrane fusion. Knockdown of CD9 or CD81 increased syncytia formation and viral entry; overexpression rendered cells less susceptible. Anti-CD81 antibodies triggered CD81 clustering in patches that recruited CD4 and CXCR4.\",\n      \"method\": \"siRNA knockdown, overexpression, anti-tetraspanin antibody treatment, HIV-1 Env-mediated syncytia and viral entry assays in human T lymphoblasts\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown and overexpression with functional assays, single lab\",\n      \"pmids\": [\"17015697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CD9 negatively regulates LPS-induced macrophage activation by preventing the formation of the LPS receptor complex (CD14/TLR4) at lipid rafts. CD9 partly co-localizes with CD14 at low-density membrane fractions. CD9 knockout macrophages show increased CD14 and TLR4 lipid-raft localization, increased CD14:TLR4 complex formation, decreased IκBα expression, and produce more TNF-α, MMP-2 and MMP-9 after LPS stimulation. CD9-KO mice showed enhanced lung macrophage infiltration and TNF-α production after intranasal LPS.\",\n      \"method\": \"Anti-CD9 mAb, siRNA, CD9 knockout mice, sucrose gradient fractionation, co-immunoprecipitation, in vitro and in vivo LPS challenge assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO corroborated by siRNA and antibody, with mechanistic fractionation and co-IP data, in vitro and in vivo\",\n      \"pmids\": [\"19414803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CD9 promotes plasma membrane localization of the glutamine transporter ASCT2, enhancing glutamine uptake in pancreatic cancer cells. CD9 knockdown decreases PDAC organoid growth; heterozygous CD9 deletion in a PDAC mouse model prolonged survival.\",\n      \"method\": \"CD9 knockdown (siRNA/shRNA), CD9 heterozygous deletion in Pdx1-Cre;KRas;p53 mice, ASCT2 surface localization by cell fractionation/imaging, glutamine uptake assay, organoid formation and limiting dilution tumor initiation assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic models (KD and mouse KO) with mechanistic localization and functional metabolic assays\",\n      \"pmids\": [\"31685994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CD9 (MRP-1/CD9) gene transduction downregulates Wnt pathway genes (Wnt1, Wnt2b1, Wnt5a) and their target genes (WISP-1, WISP-3, c-Myc, VEGF-A, MMP-26), placing CD9 upstream of Wnt signaling. A neutralizing anti-CD9 antibody inhibited this downregulation in CD9-transfected cells.\",\n      \"method\": \"CD9 gene transduction into HT1080 and A549 cells, microarray and real-time PCR, Western blotting, neutralizing anti-CD9 antibody treatment\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gene transduction with multiple downstream readouts and antibody neutralization, single lab\",\n      \"pmids\": [\"15334057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CD9 gene transduction downregulates WAVE2 expression and alters subcellular localization of Arp2 and Arp3, reducing lamellipodia formation and cell motility. This effect is independent of the Wnt signaling pathway, as Wnt siRNA did not affect WAVE2 and WAVE2 siRNA did not affect Wnt expression.\",\n      \"method\": \"CD9 gene transduction in HT1080 cells, WAVE2-specific siRNA, neutralizing anti-CD9 antibody, morphological analysis, time-lapse migration assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gene transduction, siRNA epistasis, antibody rescue in single lab study\",\n      \"pmids\": [\"16682943\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CD9 specifically controls localization of talin1 to focal adhesions: CD9 deficiency leads to impaired talin1 focal adhesion localization and correlates with increased motility of breast cancer cells.\",\n      \"method\": \"CD9-deficient cells, talin1 focal adhesion localization by imaging, cell motility assays\",\n      \"journal\": \"Biochemical Society transactions\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — localization assay with functional correlation described in a review-style article with limited method detail in the abstract\",\n      \"pmids\": [\"21428940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CD9 and CD151 accumulate at the T-cell side of the immunological synapse and support integrin-mediated signaling: silencing CD9 or CD151 reduces α4β1 integrin relocalization to the IS, decreases high-affinity β1 integrin accumulation, diminishes FAK and ERK1/2 phosphorylation, and impairs IL-2 secretion and CD69 upregulation without affecting CD3/actin accumulation or MTOC translocation.\",\n      \"method\": \"CD9/CD151 siRNA silencing, T cell–APC conjugate assays, confocal imaging of IS components, phospho-FAK and phospho-ERK Western blotting, IL-2 ELISA\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with multiple signaling and functional readouts in T-cell IS context, single lab\",\n      \"pmids\": [\"24723389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CD9 co-immunoprecipitates with ADAM17 at the cell surface and negatively modulates ADAM17-mediated shedding of LR11 in leukocytes: CD9 overexpression reduces soluble LR11 release, while CD9 knockdown or antibody neutralization increases sLR11 shedding via metalloproteinase-dependent mechanism.\",\n      \"method\": \"Confocal co-localization, ectopic CD9 expression, CD9 shRNA knockdown, anti-CD9 neutralizing antibody, metalloproteinase inhibitor GM6001, ELISA for soluble LR11\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-localization, overexpression, knockdown, and inhibitor experiments in multiple cell lines, single lab\",\n      \"pmids\": [\"24699135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CD9 physically associates with ADAM17 at the keratinocyte surface (co-IP confirmed) and negatively regulates ADAM17 sheddase activity. CD9 downregulation activates ADAM17, leading to shedding of HB-EGF and AREG and subsequent EGFR/ERK pathway activation that drives keratinocyte migration and wound healing.\",\n      \"method\": \"Confocal co-localization, co-immunoprecipitation, CD9 siRNA knockdown and overexpression, TAPI-2 (ADAM17 inhibitor), neutralizing anti-HB-EGF antibody, EGFR/ERK phosphorylation assays, wound-healing migration assays in HaCaT cells and primary mouse keratinocytes\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional rescue experiments with inhibitors and neutralizing antibodies, single lab\",\n      \"pmids\": [\"30745837\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CD9 regulates cellular senescence through the PI3K–AKT–mTOR–p53 signaling pathway: CD9 knockdown in senescent endothelial cells rescues senescence phenotypes, and CD9 upregulation in young cells accelerates senescence. Anti-CD9 antibody treatment and CD9 ablation (ApoE-/- mice) reduced atherosclerotic lesion formation in vivo.\",\n      \"method\": \"CD9 knockdown and overexpression in HUVECs, senescence assays, PI3K/AKT/mTOR/p53 pathway Western blotting, anti-CD9 antibody treatment in ApoE-/- and Ldlr-/- mice, CD9-KO crossed to ApoE-/- mice\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and antibody perturbation with signaling pathway analysis in vitro and in vivo, single lab\",\n      \"pmids\": [\"32346137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CD9 co-precipitates with CD26 in mesothelioma cells, and these proteins inversely co-modulate each other's expression. CD9 depletion leads to elevated FAK and Cas-L tyrosine phosphorylation (downstream of β1 integrin), and increased invasiveness, suggesting CD9 negatively regulates tumor invasion by reducing the CD26–α5β1 integrin complex.\",\n      \"method\": \"siRNA knockdown of CD9 and CD26, co-immunoprecipitation, Western blotting for FAK/Cas-L phosphorylation, cell invasion assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal siRNA knockdowns and co-IP with functional invasion assay, single lab\",\n      \"pmids\": [\"24466195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CD9 antibody ligation increases human CFU-MK progenitor numbers and reduces megakaryocytic differentiation (decreased CD41+ cell production and MK differentiation antigen expression) in liquid culture, suggesting CD9 participates in megakaryocytic differentiation by involvement in membrane remodeling.\",\n      \"method\": \"Cell sorting, liquid culture with anti-CD9 antibody ligation, CFU-MK colony assays, flow cytometry for differentiation markers\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — antibody perturbation with quantitative progenitor and differentiation assays, single lab\",\n      \"pmids\": [\"11264162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CD9P-1 overexpression increases cell motility on collagen I via α2β1 integrin but decreases motility on fibronectin; co-expression of CD9 or CD81 reverses these CD9P-1-mediated motility effects with concomitant CD9P-1 association, showing that the ratio of CD9P-1 to its tetraspanin partners regulates cell motility.\",\n      \"method\": \"CD9P-1 overexpression, CD9/CD81 co-expression, CD9P-1 mutant analysis (transmembrane and cytoplasmic domains required), co-immunoprecipitation, time-lapse videomicroscopy, Boyden chamber assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — overexpression, domain-mapping mutants, and co-IP with functional migration assays, single lab\",\n      \"pmids\": [\"20574531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CD9 co-localizes with β1 and β3 integrins on endothelial cell membranes, and anti-CD9 antibody induces tyrosine phosphorylation comparable to β1/β3 integrin ligation. Blocking CD9 with mAb ALMA.1 inhibits EC migration toward fibronectin and vitronectin and impairs wound repair, and ALMA.1 and anti-β1 have additive inhibitory effects, suggesting CD9 cooperates with integrins in EC migration.\",\n      \"method\": \"Double-labeling immunofluorescence for CD9/integrins, anti-CD9 mAb perturbation, in vitro wound-healing assay, Boyden chamber migration assay, tyrosine phosphorylation Western blotting\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-localization, antibody perturbation, and signaling assay with multiple functional readouts, single lab\",\n      \"pmids\": [\"10669631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Stromal cell CD9 associates with the β1 integrin subunit and a novel 100 kDa protein (co-immunoprecipitation); antibody cross-linking of CD9 increased the amount of the 100 kDa protein co-precipitated. Ligation of stromal-cell CD9 (but not hematopoietic-cell CD9) modifies hematopoietic progenitor differentiation, shifting pluripotent EML-C1 cells toward undifferentiated, clonogenic states.\",\n      \"method\": \"Co-culture with anti-CD9 antibody, separate cell pre-treatment to identify the responding cell type, co-immunoprecipitation, colony-forming unit assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — antibody perturbation with cell-type specificity controls and co-IP, single lab\",\n      \"pmids\": [\"10194438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"CD9 and CD81 are present as separate, non-complexed extracellular structures in bilayers on the oocyte surface; microinjection of CD9 RNA rescued fusion defects in both CD9-deficient and CD81-deficient oocytes, whereas CD81 failed to rescue either, indicating CD9 and CD81 function independently as extracellular components in sperm-oocyte fusion.\",\n      \"method\": \"Immunocytochemistry, immunobiochemistry, electron microscopy, RNA microinjection rescue experiments in CD9-KO and CD81-KO oocytes\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic rescue experiment with multiple imaging methods, single lab\",\n      \"pmids\": [\"23213457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CD9 localizes primarily to the plasma membrane, where it is secreted more abundantly in ectosomes than in exosomes. CD9 and a PM-stabilized CD63 mutant are more abundantly released in EVs than wild-type CD63. Comparative proteomics identified BSG and SLC3A2 as likely ectosome-specific proteins, distinct from the exosomal marker LAMP1.\",\n      \"method\": \"Live intracellular tracking of CD9 and CD63, comparative proteomics, differential response to endosomal pH neutralization, subcellular fractionation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with functional comparative proteomics, single lab study with multiple orthogonal approaches\",\n      \"pmids\": [\"34282141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Concomitant knockout of CD9 and CD81 in MCF7 cells specifically reduces EV levels of CD9P-1/PTGFRN and EWI-2/IGSF8 (the sole significantly decreased EV proteins), partially because of decreased cell expression of EWI-2. Single KO of CD9, CD81, or CD63 had little effect on overall EV protein composition.\",\n      \"method\": \"CD9, CD81, CD63 single and double knockout by CRISPR, quantitative mass spectrometry proteomics of EVs\",\n      \"journal\": \"Journal of extracellular vesicles\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with quantitative MS proteomics, single lab\",\n      \"pmids\": [\"37525398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CD9 knockdown in MDA-MB-231 breast cancer cells inhibits MSC invasion by 95% and anti-CD9 antibody blockade by 70%; CD9-deficient cells lose magnupodium/lamellipodium structures and gain membrane ruffles, which impairs adhesion and invasiveness. CD9 knockdown also suppresses metastatic capacity in mouse xenografts.\",\n      \"method\": \"CD9 shRNA knockdown, anti-CD9 antibody blockade, TIRF/confocal/scanning EM microscopy, mouse xenograft metastasis model\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with antibody confirmation, multiple imaging modalities and in vivo xenograft, single lab\",\n      \"pmids\": [\"25762645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Down-regulation of CD9 mRNA expression in Schwann cells follows axonal degeneration after sciatic nerve injury and is restored upon axonal regeneration; in culture, CD9 expression requires contact with neurons. This parallels myelin gene regulation, suggesting axons regulate CD9 expression in Schwann cells.\",\n      \"method\": \"Adult rat sciatic nerve injury model, in situ hybridization for CD9 mRNA, Schwann cell–neuron co-culture experiments\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo nerve injury model with in vitro co-culture validation, single lab\",\n      \"pmids\": [\"8581316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In Drosophila, tetraspanin tsp2A (close homolog of human CD9) genetically interacts with Pvr (PDGFR homolog), and tsp2A knockdown partially rescues Pvr-induced glial over-migration. In human glioma cells, CD9 is in close association with PDGFRα and PDGFRβ (proximity ligation assay), and CD9 knockdown blocks PDGF-BB-stimulated cell migration.\",\n      \"method\": \"Drosophila genetic screen with dsRNA, in situ proximity ligation assay for CD9-PDGFR association, CD9 siRNA knockdown in human glioma cells with PDGF-stimulated migration assay\",\n      \"journal\": \"Journal of neuro-oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic screen in model organism with corroborating human cell knockdown and proximity ligation, single lab\",\n      \"pmids\": [\"26224160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"CD9 expression is upregulated ~7-fold during TPA-induced megakaryocytic differentiation of K562 cells via PKC activation (blocked by GF109203X), and upregulated CD9 associates with β1 integrin. A TPA-responsive element was localized to a 52-bp fragment of the CD9 promoter.\",\n      \"method\": \"TPA treatment, PKC inhibitor GF109203X, quantitative RT-PCR, flow cytometry, co-immunoprecipitation, CD9 promoter-CAT reporter constructs\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition, quantitative gene expression, promoter mapping with reporter assay, and co-IP, single lab\",\n      \"pmids\": [\"9264383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CD9 inhibition (by cytopermeable blocking peptides or gene deletion) reduces the number of early endosomes and affects mitochondrial quality control, specifically impairing mitophagy; CD9 KO cells compensate by increasing total mitochondrial mass and reducing mitophagy. CD9 peptide treatment delayed primary tumor growth and reduced metastasis in vivo.\",\n      \"method\": \"CD9-blocking peptides vs. CD9 gene deletion comparison, endosome and lysosome quantification, mitochondrial mass and mitophagy assays, in vivo melanoma model\",\n      \"journal\": \"Journal of extracellular vesicles\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, organelle counting with limited mechanistic resolution linking CD9 directly to mitophagy machinery\",\n      \"pmids\": [\"34012515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"JAM-A forms a complex with α3β1 integrin and tetraspanins CD151 and CD9 through its extracellular domain (by mapping experiments), and this complex is required for collective cell migration of polarized epithelial cells on laminin and collagen-I substrates.\",\n      \"method\": \"JAM-A/CD9/CD151/α3β1 integrin depletion by siRNA in MDCK cells, co-immunoprecipitation and domain mapping, collective migration assay (scratch wound, live imaging)\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP domain mapping and siRNA phenotyping in polarized epithelial cells, single lab\",\n      \"pmids\": [\"35067832\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CD9 is a tetraspanin membrane protein whose reversed-cone molecular shape generates membrane curvature and enables curvature sensing; it organizes multiprotein complexes at the plasma membrane (tetraspanin-enriched microdomains) by directly associating—via its large extracellular loop, transmembrane helices, and C-terminal tail—with partners including FPRP/CD9P-1, EWI-2/EWI-F, integrins (α3β1, α6β4, β1), ADAM17, DPP4/TMPRSS2, and ASCT2; its palmitoylation by DHHC2 stabilizes these complexes and protects CD9 from lysosomal degradation; through these interactions CD9 regulates sperm–oocyte fusion, mononuclear phagocyte fusion, HIV-1 and MERS-CoV membrane fusion, LPS-TLR4 receptor complex assembly, ADAM17-mediated ectodomain shedding (of HB-EGF, LR11, and TGFα), Wnt and PI3K–AKT–mTOR–p53 signaling, actin cytoskeleton organization via WAVE2 downregulation, integrin signaling at the immunological synapse, ASCT2-dependent glutamine uptake, and glutamine-fueled tumor growth.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CD9 is a tetraspanin membrane protein whose reversed cone-like molecular shape generates and senses positive membrane curvature, driving its enrichment in high-curvature plasma-membrane structures and underpinning its role as an organizer of tetraspanin-enriched microdomains [#0, #2]. It nucleates these microdomains through direct, domain-specific contacts: its large extracellular loop and fourth transmembrane domain engage the Ig-superfamily partners FPRP/CD9P-1 and EWI-2/EWI-F, with EWI-F binding occurring through CD9 transmembrane helices h3/h4 in a flexible tetrameric \\\"concatenation\\\" arrangement, and its C-terminal cytoplasmic tail tuning complex size and oligomerization [#1, #3, #4, #9]. Microdomain assembly is regulated by site-specific S-palmitoylation: the acyltransferase DHHC2 palmitoylates CD9 to stabilize CD9-partner associations and protect CD9 from lysosomal degradation, while palmitoylation state and partner availability shift CD9 between homo- and heteroclusters [#5, #6, #7]. Through these scaffolding interactions CD9 governs membrane fusion and cell-cell organization—it is an indispensable oocyte-side component of sperm-egg fusion, prevents mononuclear phagocyte fusion, and restrains HIV-1 envelope-mediated fusion [#0, #12, #13, #28]. CD9 also assembles and modulates cell-surface receptor and protease complexes: it scaffolds DPP4 and TMPRSS2 to promote early MERS-CoV entry, negatively regulates LPS-driven CD14/TLR4 receptor-complex formation, and binds metalloproteases CD10/ADAM17 to control ectodomain shedding of substrates including HB-EGF, AREG, and LR11 [#10, #14, #8, #20, #21]. By partnering with integrins, it supports integrin signaling at the immunological synapse and directs talin1-dependent focal-adhesion and cytoskeletal remodeling, influencing adhesion, migration, and tumor invasion [#19, #26, #15]. CD9 additionally promotes plasma-membrane localization of the glutamine transporter ASCT2 to fuel pancreatic tumor growth and acts on Wnt and PI3K-AKT-mTOR-p53 signaling [#15, #16, #22].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing the molecular identity of CD9's most stoichiometric partner answered whether tetraspanins form defined complexes; FPRP/CD9P-1 was shown to be essentially fully CD9/CD81-associated in discrete particles distinct from integrin complexes.\",\n      \"evidence\": \"Gel permeation chromatography, immunoprecipitation, immunodepletion and immunoaffinity/MS in 293 cells\",\n      \"pmids\": [\"11087758\", \"11278880\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural interface\", \"Functional consequence of the CD9-FPRP complex not addressed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identification of PSG17 as a CD9-specific natural ligand established that a tetraspanin can act as a bona fide receptor, answering whether CD9 engages defined extracellular ligands.\",\n      \"evidence\": \"cDNA library screening, binding assays, and CD9-knockout macrophage binding\",\n      \"pmids\": [\"11805154\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling from PSG17-CD9 binding not defined\", \"Physiological role of this binding in vivo not established\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Genetic models clarified CD9's physiological role in cell fusion, showing it (with CD81) actively prevents inappropriate mononuclear phagocyte fusion rather than promoting it.\",\n      \"evidence\": \"In vitro fusion assays with antibody perturbation plus CD9/CD81 single- and double-null mice\",\n      \"pmids\": [\"12796480\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism restraining fusion not resolved\", \"Partner dependence of fusion suppression unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying DHHC2 as the palmitoyl acyltransferase for CD9 answered how CD9 complexes and protein stability are post-translationally controlled.\",\n      \"evidence\": \"DHHC knockdown, active-site mutagenesis, [3H]palmitate labeling, and co-IP\",\n      \"pmids\": [\"18508921\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Depalmitoylating enzyme not identified\", \"Dynamics of palmitoylation turnover in vivo not measured\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Domain dissection of the C-terminal tail and homo/heterocluster studies answered how CD9 molecular organization is encoded, linking the tail and palmitoylation to complex size and cluster state.\",\n      \"evidence\": \"C-tail mutagenesis with SILAC proteomics and functional assays; homo-clustering probe with palmitoylation mutants\",\n      \"pmids\": [\"21771881\", \"16537545\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cluster transitions inferred from antibody probe, not direct imaging at the time\", \"Single-lab findings\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapping the CD9-CD10/ADAM17 interactions established CD9 as a regulator of cell-surface metalloprotease activity and of protease cargo loading into extracellular vesicles.\",\n      \"evidence\": \"CD9/CD82 chimeras, mutagenesis, knockdown/overexpression and exosome quantification\",\n      \"pmids\": [\"23289620\", \"24699135\", \"30745837\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CD9 directly inhibits catalysis or sequesters substrate unresolved\", \"Sheddase regulation tested largely in single-lab systems\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"The DPP4:CD9:TMPRSS2 scaffolding model answered how CD9 dictates viral entry kinetics, showing CD9 assembles receptor-protease complexes that route MERS-CoV to rapid surface entry.\",\n      \"evidence\": \"CD9 knockout cells, co-IP, pseudovirus entry, and in vivo hDPP4 mouse model with CD9 silencing\",\n      \"pmids\": [\"28759649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the trimolecular scaffold not solved\", \"Generality to other receptor-protease pairs untested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linking CD9 to ASCT2 surface localization answered how a scaffolding tetraspanin can drive tumor metabolism, connecting CD9 to glutamine-fueled pancreatic cancer growth.\",\n      \"evidence\": \"Knockdown, heterozygous CD9 deletion in a PDAC mouse model, surface localization and glutamine uptake assays\",\n      \"pmids\": [\"31685994\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct CD9-ASCT2 binding interface not mapped\", \"Mechanism of ASCT2 surface retention by CD9 unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Atomic structures of CD9 and its EWI-2/EWI-F complexes answered the long-standing question of how tetraspanins shape membranes and assemble microdomains, revealing curvature generation and a flexible concatenation model.\",\n      \"evidence\": \"X-ray crystallography, cryo-EM of CD9-EWI complexes, fertilization assay, and native MS of palmitoylation-dependent EWI-F binding\",\n      \"pmids\": [\"32231207\", \"32958604\", \"32181977\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Higher-order microdomain architecture in native membranes not directly visualized\", \"Lipid composition dependence of curvature in cells not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Biophysical reconstitution established CD9 as a curvature sensor preferring positive curvature, providing a physical basis for its enrichment at microvilli and other curved structures.\",\n      \"evidence\": \"Biomimetic membrane tube assays from giant plasma membrane vesicles with thermodynamic modeling\",\n      \"pmids\": [\"36252000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell validation of curvature sorting limited\", \"Interplay between curvature sensing and partner binding not quantified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CD9's biophysical curvature/scaffolding properties are mechanistically coupled to its many context-specific signaling outputs (Wnt, PI3K-AKT-mTOR-p53, integrin/FAK, cytoskeletal WAVE2 control) remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying mechanism connecting microdomain assembly to downstream signaling cascades\", \"Many signaling roles rest on single-lab perturbation studies\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 4, 10, 14, 36]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [8, 20, 21]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 4, 10, 21, 29]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [8, 29, 30]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [10, 35]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [26, 31, 36]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [12, 14, 19]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [10, 13, 15]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [8, 20, 21]},\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [0, 28]}\n    ],\n    \"complexes\": [\"tetraspanin-enriched microdomain\", \"DPP4:CD9:TMPRSS2 receptor-protease complex\"],\n    \"partners\": [\"PTGFRN\", \"IGSF8\", \"ADAM17\", \"DPP4\", \"TMPRSS2\", \"ITGB1\", \"SLC1A5\", \"MME\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}