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CD9

CD9 is a small integral membrane glycoprotein belonging to the tetraspanin family, characterized by four transmembrane-spanning domains that organize cholesterol-rich microdomains on the cell surface to facilitate protein-protein interactions essential for cellular processes such as adhesion, migration, and fusion.^[1] With a molecular mass of approximately 25-27 kDa, CD9 features a large extracellular loop (EC2) rich in conserved cysteine residues forming disulfide bonds for structural stability, a small extracellular loop (EC1), and intracellular domains subject to palmitoylation, which modulates its localization and interactions.^[2] Its crystal structure, resolved at 2.7 Å resolution, reveals a cone-shaped transmembrane bundle with a central hydrophilic cavity, enabling it to sense membrane curvature and recruit partners like the single-pass protein EWI-2 in a 2:2 stoichiometry.^[1] Expressed ubiquitously across mammalian tissues, CD9 is particularly abundant on the surface of leukocytes, platelets, endothelial cells, and oocytes, where its levels can increase upon cellular activation.^[3] In the immune system, CD9 regulates leukocyte adhesion and transmigration by associating with integrins such as LFA-1 and VLA-4, as well as immunoglobulin superfamily members like ICAM-1 and ALCAM, within tetraspanin-enriched microdomains (TEMs) that enhance ligand binding avidity and organize adhesive platforms on endothelium.^[3] It also influences signaling pathways, including ERK1/2 and FAK activation, and inhibits metalloprotease ADAM17 to prevent shedding of adhesion molecules, thereby supporting T-cell activation at the immune synapse and overall immune cell motility.^[3] Beyond immunity, CD9 plays pivotal roles in reproduction, where it is essential for sperm-egg fusion—CD9-null mice exhibit female infertility due to impaired gamete membrane fusion—and in cancer, where its expression inversely correlates with tumor metastasis by modulating integrin-dependent invasion and exosome biogenesis for cargo sorting.^[2]^[4] In viral infections, CD9 facilitates entry of pathogens like hepatitis C virus and SARS-CoV-2 by organizing receptor complexes, while in development, it contributes to myoblast fusion and neural crest migration.^[1]^[5] Dysregulation of CD9 has been linked to diseases including multiple sclerosis, through altered blood-brain barrier permeability, and various carcinomas, highlighting its therapeutic potential in modulating cell-matrix interactions.^[3]

Discovery and Nomenclature

Historical Background

The CD9 antigen was first identified in 1981 as a 24-kDa cell surface protein, termed p24, on non-T acute lymphoblastic leukemia cells and developing B-lymphocytes using the monoclonal antibody BA-2 generated against a pre-B cell line.^[6] This antibody also recognized the antigen on lymphohemopoietic progenitor cells in bone marrow, highlighting its broad expression in early hematopoietic lineages.^[6] Subsequent biochemical analyses in the mid-1980s confirmed p24 as a single polypeptide chain susceptible to glycosidase treatment, consistent with its glycoprotein nature. In parallel, CD9/p24 was detected on human platelets during the 1980s, where monoclonal antibodies against it, such as ALB6, were shown to induce calcium influx and platelet activation. These findings established CD9 as a modulator of platelet function, with anti-p24 antibodies promoting aggregation in the presence of calcium.^[7] By the mid-1980s, during the Second International Workshop on Human Leukocyte Differentiation Antigens in 1984, CD9 was formally designated within the cluster of differentiation system, unifying various monoclonal antibodies (e.g., BA-2, ALB6) that recognized the same 24-kDa structure on leukocytes and platelets.^[8] The gene encoding CD9 was cloned in 1991 from a megakaryocytic cDNA library using anti-CD9 monoclonal antibodies, revealing a protein reported as 227 amino acids (later confirmed as 228) with four transmembrane domains and sequence homology to other cell surface proteins like the melanoma-associated antigen ME491.^[9] This structural feature led to its classification within the emerging tetraspanin superfamily in the mid-1990s, a group of four-transmembrane proteins initially described by Hemler and colleagues based on shared hydrophobic domains and functional roles in cell adhesion and signaling.^[10] Independently, in 1992, the monkey homolog of CD9 was identified as DRAP-27, a 27-kDa protein associated with the diphtheria toxin receptor on Vero cells. Early studies in the 1990s linked CD9 to tumor cell behavior, particularly motility and metastasis; for instance, transfection of CD9 (also termed MRP-1 in this context) cDNA into human lung and fibrosarcoma cell lines suppressed in vitro motility and in vivo lung colonization in mouse models. These observations by Ikeyama et al. positioned CD9 as a potential metastasis suppressor, with reduced expression correlating to increased invasive potential in various carcinomas. The MRP-1 nomenclature arose from 1993 studies on cell motility before standardization as CD9.^[11]

Gene and Protein Identification

The CD9 gene is located on the short arm of human chromosome 12 at the cytogenetic band 12p13.31. It spans approximately 38 kb of genomic DNA and consists of 8 exons, with the coding sequence distributed across these exons to encode the full-length protein.^[12] The gene structure includes introns ranging in size up to 10 kb, and the promoter region lacks typical TATA or CAAT boxes, consistent with housekeeping gene characteristics.^[13] The CD9 protein is a 228-amino-acid polypeptide with a calculated molecular weight of approximately 25 kDa, though it often migrates at 23-27 kDa on SDS-PAGE due to post-translational modifications.^[14] Encoded by the CD9 gene, the protein sequence features four transmembrane domains typical of tetraspanins, with short intracellular N- and C-terminal tails and two extracellular loops.^[14] The official gene symbol is CD9, approved by the HUGO Gene Nomenclature Committee (HGNC:1709), with aliases including TSPAN29 (reflecting its tetraspanin family membership) and historical designations such as MRP-1 (motility-related protein-1).^[15] The CD9 gene was initially cloned in 1991 from a λgt11 expression cDNA library derived from human megakaryocytic mRNA, using monoclonal antibodies to isolate full-length sequences that confirmed its identity as a novel tetraspanin.^[9] Sequence analysis revealed high conservation across mammalian species, with orthologs in mouse, rat, and other mammals sharing over 80% identity in the core transmembrane and extracellular domains, underscoring its evolutionary preservation.^[16] This cross-species homology was further validated through comparative genomics, highlighting CD9's role in fundamental cellular processes.^[17]

Molecular Structure

Topology and Domains

CD9, a prototypical tetraspanin, possesses a conserved membrane topology defined by four α-helical transmembrane domains (TM1–TM4) that traverse the plasma membrane, forming the core structural scaffold of the protein.^[18] These helices are arranged such that their intracellular termini converge tightly, while the extracellular ends are more loosely packed, creating a central hydrophilic cavity that accommodates water molecules and polar residues.^[1] This arrangement embeds CD9 deeply into the lipid bilayer, with the transmembrane segments typically spanning 20–25 residues each, enabling precise orientation and stability within the membrane environment.^[19] The extracellular regions of CD9 include a short extracellular loop (SEL, also termed EC1) of approximately 13–31 residues positioned between TM1 and TM2, which is relatively unstructured and contributes minimally to inter-protein interactions.^[18] In contrast, the large extracellular loop (LEL, or EC2) connects TM3 and TM4 and comprises about 69–132 residues, forming the most prominent domain exposed to the extracellular space.^[18] Within the LEL, four to six conserved cysteine residues engage in disulfide bonds—often including a characteristic CCG motif—that rigidify the loop into a cone-like β-sheet-rich structure, approximately 30–40 Å in height, which protrudes from the membrane surface.^[18]^[1] Intracellularly, CD9 features brief N- and C-terminal tails, each roughly 10–20 residues long, which lack extensive secondary structure but may include palmitoylation sites on proximal cysteine residues for membrane anchoring.^[18] A small intracellular loop of 5–10 residues links TM2 and TM3, providing flexibility for conformational adjustments without significant exposure to the cytoplasm.^[18] This compact intracellular architecture, combined with the transmembrane bundling, supports CD9's integration into ordered membrane domains such as lipid rafts, promoting lateral clustering with partner proteins.^[1]

Post-Translational Modifications

CD9, a member of the tetraspanin family, undergoes several post-translational modifications that influence its stability, membrane anchoring, and functional localization. The primary modification is N-linked glycosylation, which occurs at a predicted site in the small extracellular loop (SEL), contributing to the protein's apparent molecular weight of approximately 24-27 kDa on SDS-PAGE, though some reports indicate shifts up to 38 kDa due to glycan heterogeneity. This glycosylation is essential for proper folding and trafficking, as evidenced by studies showing that inhibition or mutation of the glycosylation site leads to endoplasmic reticulum retention and reduced cell surface expression.^[20]^[14] Palmitoylation, a reversible lipid modification, occurs on intracellular cysteine residues in the juxtamembrane regions, specifically at positions such as Cys78, Cys79, and Cys87 in the N-terminal cytoplasmic domain and corresponding sites in the C-terminal tail. This S-palmitoylation anchors CD9 to cholesterol-rich membrane microdomains, enhancing its association with tetraspanin-enriched microdomains (TEMs) and stabilizing interactions within multiprotein complexes. Mutation of these palmitoylation sites disrupts CD9's partitioning into detergent-resistant membranes and reduces its half-life, leading to accelerated degradation via lysosomal pathways.^[21]^[22] Potential phosphorylation sites exist in the short cytoplasmic tails of CD9, including tyrosine residues that may be targeted upon cellular activation, such as in B lymphocytes where tyrosine phosphorylation follows antigen receptor stimulation. However, these sites remain less characterized compared to glycosylation and palmitoylation, with limited evidence for their direct impact on CD9 activity or trafficking. Overall, these modifications collectively regulate CD9's membrane dynamics, with glycosylation and palmitoylation playing dominant roles in ensuring surface localization and functional longevity.^[23]

Expression and Localization

Tissue and Cellular Distribution

CD9 exhibits ubiquitous expression across human tissues, with particularly high levels observed in hematopoietic cells such as platelets and leukocytes, as well as in epithelial cells and oocytes.^[14]^[3] In platelets, CD9 is abundantly present, with approximately 50,000–80,000 copies per cell, contributing to its role in membrane organization.^[24] Among leukocytes, CD9 is detected in all major subsets, including B cells, T cells, natural killer cells, granulocytes, monocytes, and dendritic cells, often at low basal levels that increase upon activation or culture.^[3] Epithelial cells in various tissues, such as keratinocytes and endothelial cells, also show elevated CD9 expression, while oocytes display prominent surface localization essential for reproductive processes.^[14]^[25] In contrast, CD9 expression is relatively low in the brain and liver compared to other tissues, though detectable isoforms are present in hepatocytes and neural cells.^[14] Reproductive tissues demonstrate elevated levels, with strong expression in the placenta—particularly in extravillous trophoblast cells—and in sperm, where CD9 localizes to the acrosomal cap and membrane.^[26]^[27] This pattern underscores CD9's broad yet tissue-specific distribution, conserved across species. At the cellular level, CD9 primarily localizes to the plasma membrane, where it is enriched within tetraspanin-enriched microdomains (TEMs), specialized cholesterol-rich platforms that facilitate protein interactions and membrane dynamics.^[28] These TEMs, containing CD9 alongside other tetraspanins like CD63 and CD81, are distinct from lipid rafts and promote compartmentalization at the cell surface.^[3] During development, CD9 expression is upregulated in neural crest cells during embryogenesis, as evidenced in mouse models where it is highly expressed in embryonic neural crest stem cells (enNCSCs).^[29] This pattern is conserved in mice, with CD9 knockout studies revealing defects in fertilization due to impaired sperm-egg fusion as well as roles in cell migration, mirroring aspects of human expression profiles.^[3]^[30]

Regulation of Expression

The CD9 gene features a TATA-less promoter region characterized by multiple transcription initiation sites and lacks typical CCAAT or TATA boxes, instead relying on GC-rich sequences for basal activity. This promoter contains numerous binding motifs for transcription factors, including nine Sp1 sites and five AP-2 sites, which facilitate transcriptional activation in various cell types such as hematopoietic cell lines. Sp1 binding, particularly in the proximal promoter region spanning nucleotides -237 to -205, has been confirmed through gel shift assays using nuclear extracts from erythroleukemia cells, underscoring its role in driving CD9 expression.^[31]^[32] Epigenetic mechanisms significantly influence CD9 expression, with hypermethylation of CpG islands in the promoter region leading to gene silencing, particularly in progressive stages of multiple myeloma. In multiple myeloma cell lines and patient samples, increased methylation at these CpG sites correlates with reduced CD9 mRNA and protein levels, contributing to disease advancement and poorer survival outcomes, as CD9-positive patients exhibit a median survival of 43 months compared to 24 months in CD9-negative cases. Treatment with DNA demethylating agents like 5-aza-2'-deoxycytidine partially restores CD9 expression by 2- to 4-fold, while histone deacetylase inhibitors achieve up to 40-fold reactivation, highlighting the interplay between DNA methylation and histone modifications in this repression. Although observed in multiple myeloma, similar promoter hypermethylation patterns occur in other malignancies, such as breast and prostate cancers, where they suppress CD9 to promote invasive phenotypes.^[33] Post-transcriptional regulation of CD9 occurs through microRNAs that target its 3' untranslated region, modulating protein levels in a context-dependent manner. For instance, miR-518f-5p directly binds to CD9 mRNA, reducing its stability and translation in breast cancer cells, where elevated miR-518f-5p correlates with decreased CD9 protein and enhanced cell migration and tumor growth in xenograft models. This interaction has been validated via luciferase reporter assays showing significant repression of CD9 expression upon miR-518f-5p overexpression, with antagomir reversal restoring CD9 levels. Similar miRNA-mediated downregulation is reported in prostate cancer, where altered miRNA profiles contribute to reduced CD9 in advanced tumors.^[34]^[35] Environmental factors exert dynamic control over CD9 expression, with hypoxia inducing upregulation through hypoxia-inducible factor 1α (HIF-1α)-mediated transcription. Under low oxygen conditions (0.2% O₂), HIF-1α directly binds the CD9 promoter in B-lymphoblast cells, elevating both mRNA and protein levels to enhance cell adhesion and dissemination, as evidenced in patient-derived acute lymphoblastic leukemia samples and mouse xenograft models. In contrast, inflammatory signals like interferon-γ downregulate CD9 in macrophages, reducing surface expression and altering immune responses during activation. A 2025 study in senescence-accelerated mouse models identified decreased CD9 expression as a hallmark of sarcopenia, with transcriptomic and proteomic analyses revealing its downregulation in aged skeletal muscle (p < 0.001 at protein level), linking it to impaired mitochondrial biogenesis and positioning CD9 as a potential diagnostic biomarker for early intervention.^[36]^[37]^[38]

Biological Functions

Cell Adhesion and Migration

CD9 plays a pivotal role in promoting cell fusion events, particularly through associations mediated by its large extracellular loop (LEL). In sperm-egg fusion, CD9 on the oocyte surface is essential for gamete interaction and membrane merger, as demonstrated by experiments where monoclonal antibodies targeting CD9 block fusion in vitro.^[39] Similarly, CD9 facilitates myotube formation during muscle differentiation by supporting myoblast-myoblast fusion, with studies showing that CD9 expression enhances the efficiency of myotube assembly in cell culture models.^[40] The influence of CD9 on cell migration is highly context-dependent, often inhibiting motility in non-transformed cells while potentially enhancing it in certain invasive scenarios. In fibrosarcoma cells, CD9 suppresses migration, reducing cell spreading and movement by stabilizing integrin-mediated attachments and limiting cytoskeletal dynamics.^[41] Conversely, in tumor cell lines, CD9 can promote invasive migration, though this varies by cell type and microenvironment. CD9 briefly associates with integrins such as α5β1 to modulate these adhesive interactions during motility.^[42] CD9 contributes to wound healing and angiogenesis by stabilizing adhesion complexes on endothelial and epithelial cells. During in vitro wound closure assays, CD9 enhances endothelial cell migration and reorganization of intercellular junctions, organizing platforms containing ICAM-1 and VCAM-1 to support leukocyte extravasation and tissue repair.^[43]^[44] In angiogenesis, CD9 promotes vessel sprouting by facilitating endothelial adhesion to the extracellular matrix, as evidenced by reduced tube formation in CD9-deficient models.^[45] Experimental evidence from CD9 knockout mice underscores these functions, revealing impaired platelet aggregation due to defective outside-in signaling via αIIbβ3 integrins, leading to unstable thrombus formation. Additionally, female CD9-null mice exhibit complete infertility from failed sperm-egg fusion, confirming CD9's non-redundant role in adhesion-dependent reproductive processes.

Signal Transduction and Trafficking

CD9 serves as a molecular scaffold within tetraspanin-enriched microdomains (TEMs), organizing transmembrane proteins such as integrins and growth factor receptors to facilitate intracellular signaling cascades. By stabilizing these associations, CD9 promotes the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in response to ligand binding, including basic fibroblast growth factor (bFGF) engagement with αvβ3 integrins. Specifically, CD9 forms a ternary complex with junctional adhesion molecule-A (JAM-A) and αvβ3 integrin, enabling JAM-A dissociation upon bFGF stimulation, which triggers ERK1/2 phosphorylation and downstream angiogenic signaling.^[46] Additionally, CD9 modulates ADAM17 sheddase activity, which influences the release of EGFR ligands like TGF-α, thereby fine-tuning ERK signaling intensity; for instance, CD9 overexpression reduces ADAM17-mediated substrate shedding, attenuating ERK phosphorylation.^[47] This scaffolding function ensures efficient signal propagation while preventing excessive activation, as evidenced by reduced phospho-ERK levels in CD9-knockdown cells during receptor engagement.^[48] In endosomal trafficking, CD9 regulates the sorting and recycling of receptors, including the epidermal growth factor receptor (EGFR), by influencing their internalization and post-endocytic routes. CD9 enhances EGF-dependent EGFR internalization into early endosomes, promoting its association with TEMs and altering downstream signaling by reducing surface receptor availability.^[49]^[50] Through these mechanisms, CD9 acts as a trafficking integrator, preventing aberrant accumulation in lysosomes and supporting receptor reuse in physiological contexts.^[48] CD9 plays a key role in modulating exosome biogenesis and release, being highly enriched in small extracellular vesicles (sEVs) derived from the endosomal pathway. It negatively regulates the trafficking of other tetraspanins, such as CD63, to TEMs, thereby controlling the packaging of cargo into multivesicular bodies (MVBs) and subsequent sEV secretion; CD9 depletion increases CD63 incorporation into sEVs, indicating its inhibitory function in biogenesis.^[51] This selective sorting ensures that CD9-positive sEVs, often exosomes, carry specific molecular payloads for intercellular communication, with CD9 facilitating MVB fusion to the plasma membrane via associations with ESCRT-independent pathways.^[52] Recent proximity labeling studies in 2025 have revealed the extensive CD9 interactome, identifying 710 enriched proteins in uninfected epithelial cells, many involved in extracellular matrix-receptor interactions and tight junctions.^[53] These interactors, including bacterial receptors like CD44 and CD147, form dynamic platforms that CD9 coordinates within TEMs, influencing infection dynamics; for example, during Neisseria meningitidis or Staphylococcus aureus adhesion, CD9-proximal proteins shift, with 12 enriched during meningococcal infection, enabling pathogen hijacking of host trafficking and signaling for efficient epithelial attachment.^[53] Disruption of these interactions, such as via CD9-derived peptides, reduces bacterial adherence without additive effects from receptor knockdowns, highlighting CD9's central role in infection-modulated trafficking networks.^[53]

Protein Interactions

Associations with Integrins

CD9, a member of the tetraspanin family, directly associates with several β1 integrins, including α3β1, α4β1, and α6β1, primarily through interactions mediated by its large extracellular loop (LEL).^[54] These associations have been demonstrated in various cell types, such as Schwann cells and keratinocytes, where co-immunoprecipitation experiments from cell lysates show specific coprecipitation of CD9 with antibodies against α3, α6, and β1 subunits.^[55] In leukocytes, CD9 similarly binds α4β1 (also known as VLA-4), facilitating its organization within membrane microdomains.^[56] This binding via the LEL forms intricate "integrin-tetraspanin webs" that link adhesion receptors into higher-order complexes on the cell surface.^[54] These interactions enhance the avidity and clustering of integrins, particularly in focal adhesions, thereby modulating cell adhesion strength. For instance, CD9 promotes the lateral association of α5β1 with the metalloproteinase ADAM17, which maintains the integrin in an inactive state and negatively regulates cell adhesion without altering intrinsic ligand affinity.^[57] In epithelial cells, such as Colo320, CD9 expression leads to the formation of larger integrin complexes that abrogate adhesions to extracellular matrix components like fibronectin.^[57] Proximity ligation assays further confirm increased integrin-integrin proximity in CD9-expressing cells, with up to a fivefold rise in colocalization signals compared to controls.^[57] The functional outcomes of CD9-integrin associations are context-dependent. In epithelial and endothelial cells, CD9 exerts a pro-adhesive effect by organizing integrins into platforms that support firm cell-matrix and cell-cell interactions.^[56] Conversely, in certain leukocytes like T cells, CD9 displays anti-migratory properties by negatively regulating α4β1 and LFA-1 clustering, thereby reducing overall adhesive capacity and motility.^[56] Evidence from knockout studies underscores these roles; CD9-deficient cells exhibit disrupted integrin signaling, with context-dependent effects such as impaired leukocyte adhesion to endothelium and enhanced migration in leukocytes due to unchecked integrin activation; in some epithelial models, adhesion to matrix is enhanced.^[56]^[57] Co-immunoprecipitation and functional assays in CD9-null models consistently show diminished integrin web integrity and downstream signaling.^[55]^[57]

Interactions with Other Tetraspanins and Partners

CD9 co-assembles with other tetraspanins, including CD81, CD63, and CD151, within tetraspanin-enriched microdomains (TEMs) on the plasma membrane and intracellular compartments.^[58] These associations form cholesterol-dependent platforms that organize membrane proteins into dynamic signaling complexes.^[59] The partitioning of CD9 into these TEMs relies on cholesterol levels and palmitoylation, facilitating interactions with partner tetraspanins.^[60] Beyond tetraspanins, CD9 associates with various receptors, such as the epidermal growth factor receptor (EGFR), heparin-binding epidermal growth factor-like growth factor (HB-EGF), and HLA-DR, contributing to ligand presentation mechanisms.^[61] Specifically, CD9 interacts with transmembrane HB-EGF to modulate its juxtacrine signaling potential.^[61] Similarly, CD9 forms complexes with EGFR, enhancing their association upon antibody ligation.^[62] In immune cells, CD9 colocalizes with HLA-DR in surface networks, supporting antigen presentation processes.^[63] CD9 exerts negative regulation on the trafficking of CD63 to exosomes by altering its incorporation into tetraspanin-enriched microdomains and small extracellular vesicles.^[51] Recent 2025 investigations have further demonstrated that CD9 influences the endocytosis of membrane proteins, limiting vesicular secretion.^[64] Proximity labeling proteomics has revealed a dynamic interactome for CD9, identifying approximately 710 proximal proteins in epithelial cells under bacterial infection conditions.^[65] Among these, CD9 interacts with proteases such as furin and TMPRSS2, particularly in viral entry contexts like SARS-CoV-2 infection, where it scaffolds these proteins at the plasma membrane alongside receptors like ACE2.^[66] These interactions highlight CD9's role in organizing protease-receptor complexes without direct involvement in downstream signaling outcomes.

Role in Physiology and Disease

Involvement in Cancer

CD9 exhibits a dual role in cancer progression, acting as a tumor suppressor in some malignancies while promoting tumorigenesis in others. In non-small cell lung cancer (NSCLC), downregulation of CD9 expression is associated with clinical progression, poor prognosis, and increased metastatic potential, as evidenced by lower disease-free survival rates in patients with reduced or absent CD9 compared to those with positive expression.^[67] Similarly, in breast cancer, decreased CD9 levels correlate with epithelial-mesenchymal transition (EMT), enhanced invasiveness, and unfavorable outcomes, including higher rates of metastasis and reduced overall survival.^[4] These observations highlight CD9's suppressive function in regulating cell adhesion and migration in these epithelial-derived tumors. Conversely, CD9 displays pro-tumorigenic effects in certain cancers. In melanoma, elevated CD9 expression facilitates tumor-endothelial cell interactions and transendothelial migration, thereby enhancing invasion and metastatic spread; blocking CD9 with monoclonal antibodies inhibits this process in vitro.^[68] In glioma, CD9 contributes to radioresistance by modulating cell migration and stemness, with its overexpression linked to poorer radiation sensitivity through pathways influencing invasive properties and tumor microenvironment interactions.^[69] CD9 also holds promise as a biomarker for cancer detection. Circulating exosomes bearing CD9 on their surface, often enriched in tumor-derived vesicles, enable non-invasive early detection of malignancies like glioma and breast cancer by reflecting tumor-specific cargo such as proteins or nucleic acids indicative of disease presence.^[70] Therapeutically, targeting CD9 offers strategies to curb cancer progression. Antibodies that block CD9-integrin complexes have demonstrated efficacy in inhibiting tumor cell migration, invasion, and metastasis in preclinical models, including reductions in exosome-mediated intercellular communication within the tumor niche.^[71]

Implications in Infectious Diseases

CD9 plays a significant role in facilitating viral entry into host cells by organizing tetraspanin-enriched microdomains (TEMs) that cluster key receptors and proteases essential for pathogen attachment and fusion. In the context of SARS-CoV-2 infection, CD9 localizes to the plasma membrane alongside the receptor ACE2, co-receptor neuropilin-1 (NRP1), and priming proteases furin and TMPRSS2, promoting their association within TEMs to enhance viral entry efficiency.^[5] This clustering is evidenced by co-fractionation in detergent-resistant membranes and proximity ligation assays showing interactions within 40 nm.^[5] Genetic knockout of CD9 in cell lines such as SW480 and A549-ACE2 reduces SARS-CoV-2 titers by 3- to 5-fold at 48-72 hours post-infection, partly due to decreased NRP1 expression levels, which further impairs infectivity by up to 10-fold when combined with NRP1 silencing.^[5] Additionally, blocking CD9 with specific antibodies significantly lowers viral replication, highlighting its potential as a therapeutic target.^[5] For HIV-1, CD9 contributes to viral assembly and release through exosomal pathways, where it incorporates into multivesicular bodies and facilitates the budding of virions into intracellular compartments enriched with tetraspanins like CD81 and CD53.^[72] In macrophages and T cells, CD9 on exosomes enhances HIV-1 entry by promoting membrane fusion and cytoskeletal interactions via ERM proteins, with exosome-mediated infectivity increasing in the presence of CD9-positive vesicles derived from infected cells.^[73] Conversely, anti-CD9 antibodies block this exosome-facilitated entry, reducing HIV-1 infection in T cell lines (A3R5.7) and macrophages (THP-1) by statistically significant margins (p=0.0006 and p<0.0001, respectively), indicating context-dependent anti-viral effects through disruption of tetraspanin networks.^[73] Overexpression of CD9 also accelerates exosome production, which can indirectly support lentiviral release, including HIV-1.^[74] CD9's involvement extends to other viruses, such as influenza A virus (IAV), where it supports proteolytic priming of the hemagglutinin glycoprotein in TEMs, enabling efficient viral entry and trafficking.^[75] Tetraspanins including CD9 organize membrane microdomains that facilitate IAV attachment and endocytosis, with their disruption impairing viral infectivity.^[76] Although direct CD9 knockout studies for IAV are limited, analogous experiments with related tetraspanins like CD81 show no effect on early entry steps but highlight TEMs' role in overall trafficking, suggesting CD9 similarly promotes pro-entry functions for tetraspanin-dependent pathogens.^[77] In bacterial infections, CD9 modulates host-pathogen interactions by altering its interactome, as revealed by proximity labeling proteomics using TurboID-CD9 fusion in epithelial cells.^[78] Upon infection with Neisseria meningitidis or Staphylococcus aureus, the CD9 interactome dynamically shifts, enriching 12 proteins for meningococci (e.g., SLC16A2, PRKCI) and 1 for staphylococci (NIPAL4) within 30-240 minutes, which facilitates bacterial adherence via receptors like CD147 and CD44.^[78] These changes impact immune responses, as CD44 enrichment links to phagocytosis in macrophages, and proteins like DGKD and CRCP regulate inflammation during meningococcal invasion, potentially altering macrophage polarization and bacterial clearance.^[78] Interfering with CD9 reduces bacterial adhesion, underscoring its role in infection dynamics.^[78]

Associations with Other Pathologies

In metabolic disorders such as atherosclerosis, CD9 contributes to disease progression through interactions that promote platelet activation and foam cell formation. CD9 associates with CD36 on macrophage surfaces, facilitating oxidized low-density lipoprotein (oxLDL) uptake and subsequent lipid accumulation that drives foam cell development, a hallmark of atherosclerotic plaques.^[79] Additionally, CD9 enhances platelet aggregation and activation, amplifying inflammatory responses and endothelial dysfunction in vascular lesions.^[80] CD9 has been implicated in multiple sclerosis (MS) through its influence on blood-brain barrier (BBB) permeability. Dysregulated CD9 expression alters leukocyte transmigration across the BBB by modulating tetraspanin-enriched microdomains that organize adhesion molecules and integrins, contributing to neuroinflammation and demyelination in MS models.^[3] Studies in experimental autoimmune encephalomyelitis (EAE), a model for MS, show that CD9 deficiency reduces immune cell infiltration into the central nervous system, suggesting a pro-inflammatory role.^[81] Neurologically, CD9 supports neural crest cell migration during embryonic development, influencing the formation of peripheral nervous system components. In neural crest-derived cells like Schwann cells, CD9 modulates migration by organizing membrane tetraspanin-enriched microdomains that regulate integrin-mediated adhesion and motility.^[82] Antibody-mediated blockade of CD9 disrupts calcium signaling and inhibits Schwann cell migration in vitro, indicating its functional importance in neural tissue organization.^[83]

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CD9 and CD81 Interactions and Their Structural Modelling in Sperm ...
A mutual position of CD9/CD81 is shown in human spermatozoa in the acrosomal cap, however in mice, CD9 and CD81 occupy a distinct area. During the acrosome ...
[24]
Mapping of tetraspanin-enriched microdomains that can function as ...
Here, we reveal the presence of tetraspanin-enriched microdomains (TEMs) containing the tetraspanins CD9, CD63, CD81, and CD82 at the plasma membrane.
[25]
A Stable Cranial Neural Crest Cell Line from Mouse - PubMed Central
The O9-1 cell line expresses Bmi1, Arpc1b, CD9, and Cyba, which are highly expressed in enNCSCs. ... expression of genes involved in neural crest development.
[26]
Organization of the Human CD9 Gene - ScienceDirect.com
The promoter region has no TATA or CCAAT box but has many putative binding motifs for various transcription factors, including nine Sp1 sites and five AP2 sites ...
[27]
Transcriptional regulation of the human CD9 gene - PubMed
The CD9 antigen is associated with surface proteins such as VLA integrins or HB-EGF precursor. Transfection of CD9 in melanoma cells reduces tumor growth ...
[28]
Epigenetic Silencing of the Tetraspanin CD9 during Disease ...
May 15, 2008 · Purpose: The purpose of this study was to investigate expression and epigenetic regulation of CD9 in multiple myeloma (MM) cells during ...Missing: EGF | Show results with:EGF
[29]
Tetraspanin CD9 is Regulated by miR-518f-5p and Functions ... - NIH
Mar 26, 2020 · Tetraspanin CD9 is regulated by miR-518f-5p and functions in breast cell migration and in vivo tumor growth.
[30]
miR-518f-5p decreases tetraspanin CD9 protein levels and ...
These results show that tetraspanin CD9 is regulated by miRNAs in prostate cell lines and that due to differential functional effects in non-tumourigenic versus ...
[31]
Hypoxia regulates CD9 expression and dissemination of B ...
We found that low oxygen levels enhance CD9 expression both at mRNA and protein levels. We further determined that Hypoxia Inducible Factor 1α (HIF1α), the ...
[32]
Down-Regulation of Macrophage CD9 Expression by Interferon-γ
The present study has focused on the role of inflammation and macrophage activation in the regulation of CD9 expression.
[33]
CD9, a novel potential biomarker of sarcopenia | Scientific Reports
Published: 30 May 2025. CD9, a novel potential biomarker of sarcopenia ... aiming to identify novel biomarkers and possible regulatory mechanisms for the early recognition of and interventions for sarcopenia.
[34]
Structural insights into tetraspanin CD9 function - Nature
Mar 30, 2020 · Consistently, the critical amino acid for the cholesterol binding (Glu219 in CD81) is not conserved in the other tetraspanin members and is ...Results · Complex Structure Of Cd9... · Methods
[35]
Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and ...
Aug 23, 1999 · TM4SF proteins such as CD9 and CD81 appear to promote muscle cell fusion and support myotube maintenance.Missing: 1990s | Show results with:1990s
[36]
Functional and biochemical studies of CD9 in fibrosarcoma cell line
We also observed CD9 reduced cell migration using FN a chemoattractant and inhibited cell colony formation in soft agar medium. To explore the biochemical ...
[37]
Tetraspanin CD9 in cell migration - PubMed
Tetraspanin CD9 is associated with integrin adhesion receptors and it was reported that CD9 regulates integrin-dependent cell migration and invasion.
[38]
CD9 participates in endothelial cell migration during in vitro wound ...
As such, CD9 is a potential target to inhibit angiogenesis in metastatic and atherosclerotic processes.
[39]
Targeting CD9 produces stimulus-independent antiangiogenic ...
Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy.
[40]
Tetraspanin CD9 links junctional adhesion molecule-A to αvβ3 integrin to mediate basic fibroblast growth factor–specific angiogenic signaling | Molecular Biology of the Cell
### Summary of CD9's Involvement in MAPK Activation via Integrin Pathways
[41]
Tetraspanin CD9 affects HPV16 infection by modulating ADAM17 ...
May 8, 2020 · Tetraspanin CD9 affects HPV16 infection by modulating ADAM17 activity and the ERK signalling pathway. Original Investigation; Open access ...
[42]
Tetraspanins Function as Regulators of Cellular Signaling - Frontiers
This study provides evidence that tetraspanins, such as CD9, can promote cellular signaling by stabilizing transmembrane proteins, thereby providing a potent ...
[43]
Tetraspanins interweave EV secretion, endosomal network ...
So, accumulating evidence points out to tetraspanins CD9 and CD63 as crucial regulators of the fate of endosomal compartments, being CD63 an inductor of the ...
[44]
The tetraspanin CD9 modulates epidermal growth factor receptor ...
CD9 can associate with EGFR in hepatocellular carcinoma cells (HepG2/CD9) and Chinese hamster ovary cancer cells (CHO-HER/CD9), which were transfected with pTJ/ ...Missing: promoter | Show results with:promoter
[45]
Tetraspanin CD9 alters cellular trafficking and endocytosis of ... - NIH
We report that CD9 negatively regulated CD63 trafficking to tetraspanin-enriched microdomains and its subsequent packaging into sEVs.Missing: EGFR | Show results with:EGFR
[46]
ALIX- and ESCRT-III–dependent sorting of tetraspanins to exosomes
Feb 12, 2020 · Our data argue for the discriminating role of the ALIX-dependent pathway in the endosomal sorting of the tetraspanins CD9, CD63, and CD81 (well- ...
[47]
FEBS Press
### Summary of 2025 CD9 Proximity Labeling Study
[48]
https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2017.00034/full
[49]
Association of the Tetraspan Protein CD9 With Integrins ... - PubMed
We demonstrate here that CD9 is specifically coprecipitated from S-16 cell extracts by antibodies against integrins alpha 3, alpha 6, and beta 1. In addition ...
[50]
Tetraspanin CD9: A Key Regulator of Cell Adhesion in the Immune ...
Apr 30, 2018 · Tetraspanins are glycoproteins which generally contain several N-glycosylation sites in the LEL domain (1), and in this regard, CD9 is ...
[51]
https://pmc.ncbi.nlm.nih.gov/articles/PMC11919600/
[52]
Tetraspanins affect membrane structures and the trafficking of ...
Mar 26, 2025 · Tetraspanins decorate and affect particular membrane structures. While many tetraspanins, such as CD9, CD81, and CD151, are predominantly ...
[53]
A physical and functional link between cholesterol and tetraspanins
Aug 4, 2003 · We show that tetraspanins interact with cholesterol as indicated by the precipitation of tetraspanin/tetraspanin complexes by digitonin, a ...
[54]
Single-molecule analysis of CD9 dynamics and partitioning reveals ...
CD9 mobility and partitioning are both dependent on its palmitoylation and plasma membrane cholesterol. Our data show the high dynamic of interactions in the ...
[55]
CD9 antigen interacts with heparin-binding EGF-like growth factor ...
The CD9 antigen potentiates the juxtacrine activity of both proHB-EGF and proamphiregulin, but has no effect on proTGF-alpha. While both HB-EGF and amphiregulin ...Falta(n): EGFR HLA- DR ligand presentation
[56]
The tetraspanin CD9 modulates epidermal growth factor receptor ...
Feb 4, 2008 · CD9 is a member of the tetraspanins, and has been shown to be involved in a variety of cellular activities such as migration, proliferation, ...
[57]
CD9, CD63, CD81, and CD82 are components of a ... - PubMed
We show that these molecules are associated with each other on cell surface and with other glycoproteins such as very late antigen (VLA) integrins and HLA-DR ...
[58]
Tetraspanin CD9 alters cellular trafficking and endocytosis of ...
Feb 3, 2025 · We report that CD9 negatively regulated CD63 trafficking to tetraspanin-enriched microdomains and its subsequent packaging into sEVs.
[59]
[PDF] Dynamics of the CD9 interactome during bacterial infection of ...
We believe this is the first study of the broader CD9 interactome by proximity labelling, identifying 710 potential proximal proteins after incubation with exog ...Missing: furin TMPRSS2
[60]
The Tetraspanin CD9 Facilitates SARS-CoV-2 Infection and Brings ...
Aug 20, 2025 · Furthermore, we show that CD9 colocalizes with ACE-2, NRP1, furin, and TMPRSS2 at the plasma membrane; that the absence of CD9 decreases the ...Missing: proximity labeling 710
[61]
Downregulation of CD9 protein expression is associated with ...
The disease-free survival rate of patients with downregulated/absent CD9 expression (48.6%) was significantly lower than that in patients with CD9-positive ...
[62]
Prognostic value and multifaceted roles of tetraspanin CD9 in cancer
Another study described higher metastatic rate in cells with decreased CD9 expression (10). CD9 downregulation was also observed in cells which underwent EMT ( ...
[63]
Regulatory role of tetraspanin CD9 in tumor–endothelial cell ...
Dec 15, 2001 · Anti-CD9 monoclonal antibodies were found to specifically inhibit the transendothelial migration of melanoma cells; the inhibitory effect was ...
[64]
A multi-gene predictive model for the radiation sensitivity of ... - eLife
Jun 18, 2025 · Moreover, CD9 may impact tumor sensitivity to radiation therapy by modulating the metastatic and invasive properties of tumor cells (Hori et al.
[65]
Exosomes in Cancer Diagnostics - MDPI
In particular, cancer derived exosomes likely serves as biomarker for early detection of cancer as they carry the cargo reflective of genetic or signaling ...Exosomes In Cancer... · 4. Exosomal Surface Proteins... · 5. Exosomal Nucleic Acids As...<|control11|><|separator|>
[66]
CD9 regulates proliferation, invasion, migration and radioresistance ...
Prognostic value and multifaceted roles of tetraspanin CD9 in cancer. ... Tetraspanin CD9 determines invasiveness and tumorigenicity of human breast cancer cells.Missing: NSCLC | Show results with:NSCLC
[67]
CD9, a tetraspanin target for cancer therapy? - PMC - NIH
CD9 has been identified as a favorable prognostic marker or predictor of metastatic potential depending on the cancer type.Missing: DRAP- | Show results with:DRAP-
[68]
The Tetraspanin CD9 Facilitates SARS-CoV-2 Infection and Brings ...
In addition, we could detect that CD9 is in close proximity to TMPRSS2 and ACE2 in the A549-ACE2/WT cells (Figure 6D), but not in the SW480/WT cells (Figure 6C) ...Missing: 710 | Show results with:710
[69]
In macrophages, HIV-1 assembles into an intracellular plasma ...
We find that HIV-1 buds into a compartment that contains the tetraspanins CD81, CD9, and CD53. On uninfected macrophages, these proteins are seen at the cell ...
[70]
Tetraspanin blockage reduces exosome-mediated HIV-1 entry - NIH
In this study, we demonstrated that exosomes can enhance HIV-1 entry into human T and monocytic cell lines via exosomal tetraspanin proteins CD81 and CD9.
[71]
The Impact of the CD9 Tetraspanin on Lentivirus Infectivity and ...
Feb 7, 2018 · These findings indicate that CD9 empowers an accelerated production and/or release of exosomes, whereas ESCRT components counteract this pathway ...
[72]
Coronavirus and Influenza Virus Proteolytic Priming Takes Place in ...
Tetraspanin antibodies inhibit coronavirus and influenza A virus infections. Tetraspanins facilitate the entry of many viruses, including hepatitis C virus ...
[73]
Tetraspanin functions during HIV-1 and influenza virus replication
Very recently, the tetraspanins CD81 and CD9 were also implicated in the influenza virus life cycle, when they were shown by Peter Palese, Megan Shaw (both ...
[74]
Distinctive function of Tetraspanins: Implication in viral infections
Notably, during the IAV entry process, CD81 had no impact on the virus binding, internalization, or trafficking to early endosomes. This finding differs ...
[75]
Dynamics of the CD9 interactome during bacterial infection of ...
Oct 17, 2025 · TEMs are involved in myriad biological processes ... CD9 to the plasma membrane in comparison to CD63 localisation within the lysosome.
[76]
CD9 Tetraspanin Interacts with CD36 on the Surface of Macrophages
Dec 21, 2011 · To investigate the role of CD9 in the biological functions of CD36, we first studied oxLDL uptake and foam cell formation using macrophages ...
[77]
CD9 induces cellular senescence and aggravates atherosclerotic ...
CD9 plays important roles in cell adhesion and migration [11], platelet activation and aggregation [12], fusion of sperm and ovum during mammalian ...
[78]
CD9 tetraspanin generates fusion competent sites on the egg ... - NIH
Jun 20, 2011 · Indeed, deletion of Cd9 gene results in a dramatic reduction of female fertility due to a lack of fusion of sperm with Cd9 null eggs (7, 8, 10).
[79]
An Antibody to the Tetraspan Membrane Protein CD9 Promotes ...
In sympathetic neurons, CD9 is expressed at E12, very soon after neural crest cells have formed ganglia. Although sympathetic neuroblasts continue to divide at ...Missing: schizophrenia | Show results with:schizophrenia
[80]
CD9 Plays a Role in Schwann Cell Migration in vitro
We provide evidence that CD9 mAb-induced changes in intra- cellular calcium influence neural crest-derived Schwann cells in an analogous manner. Using a ...