Affinage

CGN

Cingulin · UniProt Q9P2M7

Round 2 corrected
Length
1203 aa
Mass
137.1 kDa
Annotated
2026-04-28
44 papers in source corpus 7 papers cited in narrative 7 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

Cingulin (CGN) is a dimeric coiled-coil scaffolding protein localized to the cytoplasmic face of epithelial tight junctions, where it integrates junctional assembly with Rho GTPase signaling and paracellular permeability control. Its N-terminal globular head domain directly binds ZO-1, ZO-2, ZO-3, myosin, AF-6, JAM, and the RhoA-specific exchange factor GEF-H1, while the coiled-coil rod domain mediates parallel dimerization and is required together with the head for correct tight-junction targeting (PMID:10613913, PMID:10877843). Sequestration of GEF-H1 by cingulin inhibits RhoA/ROCK activation, linking tight-junction formation to suppression of cell proliferation (G1/S block) and restraint of epithelial migration; loss of CGN derepresses RhoA signaling and promotes invasion in both normal and cancer epithelial contexts (PMID:15866167, PMID:34830864). CGN also promotes junctional accumulation of the pore-forming claudin-2, thereby regulating paracellular sodium permeability independently of ZO-1 (PMID:37566083).

Mechanistic history

Synthesis pass · year-by-year structured walk · 6 steps
  1. 1999 High

    Resolving cingulin's domain architecture and direct binding partners established it as a multi-valent cytoplasmic scaffold at tight junctions, rather than a simple structural component.

    Evidence cDNA cloning of Xenopus cingulin, electron microscopy of coiled-coil dimers, in vitro pull-downs with Kd measurement for ZO-1, co-IP and transfection/localization in MDCK cells

    PMID:10613913

    Open questions at the time
    • Binding affinities for ZO-2, ZO-3, myosin, and AF-6 not quantified
    • Post-translational regulation of cingulin junctional recruitment not addressed
    • No functional assay for junctional barrier or signaling consequences
  2. 2000 High

    Identification of JAM as a transmembrane binding partner of cingulin showed that cingulin physically links integral membrane components to the tight-junction plaque, expanding its adaptor role beyond cytoplasmic scaffolds.

    Evidence Co-immunoprecipitation from Caco-2 detergent-insoluble fractions, domain-mapping in vitro binding assays, CHO cell transfection

    PMID:10877843

    Open questions at the time
    • Whether cingulin–JAM interaction is direct or bridged by ZO-1 was not fully resolved
    • Functional consequence of disrupting cingulin–JAM binding on barrier integrity unknown
  3. 2005 High

    Discovery that cingulin sequesters GEF-H1 to inhibit RhoA activation revealed a direct signaling function for a tight-junction scaffold, answering how junction formation feeds back on cell proliferation and morphology.

    Evidence Direct pull-down of GEF-H1, RhoA-GTP assays, cingulin/GEF-H1 RNAi in MDCK cells, G1/S cell-cycle analysis, dominant-negative mutant expression

    PMID:15866167

    Open questions at the time
    • Structural basis of cingulin–GEF-H1 interaction undefined
    • Whether cingulin regulates additional GEFs or GAPs not tested
    • Relative contributions of RhoA inhibition versus other cingulin interactions to barrier phenotype unresolved
  4. 2010 Medium

    Demonstration that TGFβ/Smad-driven EMT epigenetically silences CGN via promoter hypermethylation provided a mechanism for loss of cingulin's anti-migratory function during cancer progression.

    Evidence Bisulfite sequencing in breast cancer EMT models, Smad pathway disruption, DNMT1 activity assays, invasion assays

    PMID:20086175

    Open questions at the time
    • Whether CGN silencing is a cause or consequence of EMT progression not fully distinguished
    • In vivo relevance of CGN epigenetic silencing in patient tumors not assessed
    • Specific Smad complex binding to CGN promoter not mapped
  5. 2021 Medium

    Validation of miR-125b as a direct post-transcriptional repressor of CGN in colorectal cancer confirmed that CGN loss activates RhoA/ROCK to promote metastasis, and that CGN re-expression rescues the phenotype in vivo.

    Evidence Dual-luciferase 3′-UTR reporter, siRNA knockdown, overexpression rescue in vitro and in xenograft models, RhoA/ROCK activity measurement

    PMID:34830864

    Open questions at the time
    • Clinical correlation of miR-125b/CGN axis in patient cohorts limited
    • Whether additional miRNAs converge on CGN regulation unknown
    • Downstream RhoA effectors beyond ROCK not delineated
  6. 2023 Medium

    CRISPR knockout of CGN revealed a specific role in promoting claudin-2 junctional accumulation and paracellular sodium permeability, mechanistically separable from ZO-1-dependent functions.

    Evidence CRISPR/Cas9 KO of CGN and CGNL1 in MDCK cells, TER and permeability assays, rescue by CGN overexpression, immunofluorescence and western blot

    PMID:37566083

    Open questions at the time
    • Whether cingulin directly binds claudin-2 or acts via an intermediate is unknown
    • Impact on other claudin family members not systematically tested
    • In vivo kidney or intestinal barrier phenotype of CGN knockout not reported

Open questions

Synthesis pass · forward-looking unresolved questions
  • The structural basis of cingulin's multi-valent head-domain interactions, its regulation by phosphorylation, and its in vivo roles in tissue barrier function and tumor suppression remain unresolved.
  • No high-resolution structure of cingulin head domain or its complexes with GEF-H1, ZO proteins, or claudins
  • Phosphorylation sites are documented but functional consequences in signaling are not mapped
  • No conditional in vivo knockout phenotype in mammalian epithelial tissues reported in the timeline

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 3 GO:0098772 molecular function regulator activity 2 GO:0008092 cytoskeletal protein binding 1
Localization
GO:0005886 plasma membrane 3
Pathway
R-HSA-162582 Signal Transduction 3 R-HSA-1640170 Cell Cycle 1
Partners
ARHGEF2CGNL1CLDN2F11RTJP1TJP2TJP3
Complex memberships
tight junction plaque

Evidence

Reading pass · 7 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1999 Cingulin (CGN) is a ~140–160 kDa phosphoprotein at the cytoplasmic surface of epithelial tight junctions containing an N-terminal globular head domain (residues 1–439), a central α-helical rod domain (440–1,325), and a C-terminal tail (1,326–1,368). The rod domain mediates formation of coiled-coil parallel dimers via electron microscopy and pull-down assays. The N-terminal head fragment (residues 1–378) directly interacts with ZO-1 (Kd ~5 nM), ZO-2, ZO-3, myosin, and AF-6 in pull-down assays from epithelial, insect, and reticulocyte lysates, while ZO-1 and ZO-2 immunoprecipitates contain cingulin in vivo. Full-length cingulin but not individual fragments correctly localizes to tight junctions in transfected MDCK cells, indicating both head and rod domains are required for TJ targeting. Full-length Xenopus cDNA cloning, electron microscopy, in vitro pull-down assays (Kd measurement), co-immunoprecipitation from epithelial cells, transfection/localization in MDCK cells The Journal of cell biology High 10613913
2000 Junctional adhesion molecule (JAM) physically associates with cingulin (CGN) at tight junctions. Co-precipitation experiments in Caco-2 epithelial cells showed JAM in a complex with cingulin, and this interaction required the amino-terminal globular head domain of cingulin. JAM transfection into CHO cells enhanced junctional localization of endogenous ZO-1, and JAM–ZO-1 interaction required the C-terminal PDZ-binding motif of JAM. Co-immunoprecipitation from detergent-insoluble fractions of Caco-2 cells, in vitro binding assays, transfection of CHO cells The Journal of biological chemistry High 10877843
2005 Cingulin (CGN) directly binds to GEF-H1/Lfc, a RhoA-specific guanine nucleotide exchange factor, via its junctional adaptor function. Cingulin binding to GEF-H1 inhibits RhoA activation and downstream signaling. In confluent MDCK epithelial cells, increased cingulin expression sequesters GEF-H1, downregulating RhoA. RNAi depletion of cingulin causes irregular monolayers and RhoA activation, while RNAi of GEF-H1 or transfection of GEF-H1-binding cingulin mutants inhibits G1/S cell-cycle progression. Direct interaction assays (pull-down), RhoA activation assays (GTPase pull-down), regulated RNAi in MDCK cells, G1/S phase analysis, dominant-negative mutant transfection Developmental cell High 15866167
2010 CGN promoter undergoes hypermethylation during TGFβ-driven epithelial-mesenchymal transition (EMT) in breast cancer cells, leading to transcriptional silencing. Disrupting Smad signaling in mesenchymal breast cancer cells reversed CGN promoter methylation and restored CGN expression, accompanied by reacquisition of epithelial morphology and suppressed invasion. This silencing was linked to reduced DNA-binding activity of DNMT1. Breast cancer cell-based EMT model, bisulfite sequencing/methylation analysis, Smad pathway disruption, gene re-expression assay, invasion assay, DNMT1 DNA-binding activity assay Cancer research Medium 20086175
2021 miR-125b directly targets CGN (cingulin) mRNA in colorectal cancer cells, as validated by dual-luciferase reporter assays with the CGN 3′-UTR. Knockdown of CGN phenocopies miR-125b overexpression by enhancing cell migration and invasion. Mechanistically, CGN suppression by miR-125b activates the RhoA/ROCK signaling pathway, promoting CRC metastasis. CGN re-expression rescues the pro-migratory phenotype in vitro and in vivo. Dual-luciferase reporter assay, RNA-seq, siRNA knockdown, overexpression rescue, in vitro migration/invasion assays, in vivo xenograft model, RhoA/ROCK pathway activity measurement Cancers Medium 34830864
2023 Knockout of CGN (cingulin) alone, CGNL1 (paracingulin) alone, or both in MDCK kidney epithelial cells causes a modest but significant increase in transepithelial resistance (TER) and decreased sodium permeability, without affecting 3 kDa dextran permeability. This phenotype is associated with reduced protein expression and junctional accumulation of the sodium pore-forming claudin-2. Overexpression of CGN or CGNL1 (but not ZO-1) rescues claudin-2 junctional localization, indicating a CGN-specific role in claudin-2 regulation independent of ZO-1. CRISPR/Cas9 knockout in MDCK cells, transepithelial resistance measurements, paracellular permeability assays (dextran, sodium), calcium-switch assay, immunofluorescence, western blot, qRT-PCR, rescue by overexpression Cells Medium 37566083
2025 In ovarian cancer cells, polyphyllin H upregulates CGN (cingulin) expression, which in turn blocks the RhoA/Rock2 signaling pathway to inhibit cancer cell proliferation, invasion, and migration. This CGN/RhoA/Rock2 axis was identified through protein sequencing of drug-treated cells and functionally validated in cell line-derived and patient-derived xenograft mouse models. CCK-8 proliferation assay, invasion/migration assays, protein sequencing (proteomics), CDX and PDX mouse models, in vivo tumor growth measurement, pathway activity assays International journal of surgery (London, England) Low 40773225

Source papers

Stage 0 corpus · 44 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2002 Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proceedings of the National Academy of Sciences of the United States of America 1479 12477932
2017 Architecture of the human interactome defines protein communities and disease networks. Nature 1085 28514442
2015 A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 1015 26496610
2002 Tight junctions of the blood-brain barrier: development, composition and regulation. Vascular pharmacology 869 12529927
2020 A reference map of the human binary protein interactome. Nature 849 32296183
2003 Complete sequencing and characterization of 21,243 full-length human cDNAs. Nature genetics 754 14702039
2007 Large-scale mapping of human protein-protein interactions by mass spectrometry. Molecular systems biology 733 17353931
2021 Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell 705 33961781
2011 Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium. Briefings in bioinformatics 656 21873635
2018 High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies. Molecular cell 580 29395067
2003 Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nature biotechnology 485 12665801
2022 OpenCell: Endogenous tagging for the cartography of human cellular organization. Science (New York, N.Y.) 432 35271311
2004 Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization. Current biology : CB 386 15324660
2013 Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions. Science signaling 383 24255178
2000 Interaction of junctional adhesion molecule with the tight junction components ZO-1, cingulin, and occludin. The Journal of biological chemistry 361 10877843
2021 A proximity-dependent biotinylation map of a human cell. Nature 339 34079125
2004 Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nature biotechnology 266 15146197
1999 Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin. The Journal of cell biology 229 10613913
2016 An organelle-specific protein landscape identifies novel diseases and molecular mechanisms. Nature communications 211 27173435
2020 Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions. Nature cell biology 194 32203420
2004 Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins. The Journal of biological chemistry 185 15161933
2007 A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes. Proteomics 183 17979178
2005 Binding of GEF-H1 to the tight junction-associated adaptor cingulin results in inhibition of Rho signaling and G1/S phase transition. Developmental cell 177 15866167
2014 E-cadherin interactome complexity and robustness resolved by quantitative proteomics. Science signaling 162 25468996
2010 Smad signaling is required to maintain epigenetic silencing during breast cancer progression. Cancer research 154 20086175
2007 Prominent use of distal 5' transcription start sites and discovery of a large number of additional exons in ENCODE regions. Genome research 154 17567994
2005 Targeted proteomic analysis of 14-3-3 sigma, a p53 effector commonly silenced in cancer. Molecular & cellular proteomics : MCP 153 15778465
2012 Functional proteomics establishes the interaction of SIRT7 with chromatin remodeling complexes and expands its role in regulation of RNA polymerase I transcription. Molecular & cellular proteomics : MCP 145 22586326
2006 The DNA sequence and biological annotation of human chromosome 1. Nature 144 16710414
2017 RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination. BMC biology 135 29117863
2021 miR-125b Promotes Colorectal Cancer Migration and Invasion by Dual-Targeting CFTR and CGN. Cancers 30 34830864
2013 Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes. Nucleic acids research 28 23658230
2022 Single-cell RNA Sequencing Identified Novel Nr4a1+ Ear2+ Anti-Inflammatory Macrophage Phenotype under Myeloid-TLR4 Dependent Regulation in Anti-Glomerular Basement Membrane (GBM) Crescentic Glomerulonephritis (cGN). Advanced science (Weinheim, Baden-Wurttemberg, Germany) 22 35484716
2015 Transformation pathways in high-pressure solid nitrogen: from molecular N2 to polymeric cg-N. The Journal of chemical physics 15 25747092
2022 Biophysical interaction between lanthanum chloride and (CG)n or (GC)n repeats: A reversible B-to-Z DNA transition. International journal of biological macromolecules 11 35809677
2006 Isolation, mapping and identification of SNPs for four genes (ACP6, CGN, ANXA9, SLC27A3) from a bovine QTL region on BTA3. Cytogenetic and genome research 7 16717448
2023 Knock Out of CGN and CGNL1 in MDCK Cells Affects Claudin-2 but Has a Minor Impact on Tight Junction Barrier Function. Cells 6 37566083
2016 Where have all the inosines gone? Conflicting evidence for A-to-I editing of the anticodon of higher eukaryotic tRNAACGArg questions the dogma of a universal wobble-mediated decoding of CGN codons. IUBMB life 5 27029281
2021 Hepatic antioxidant activity, immunomodulation, and pro-anti-inflammatory cytokines manipulation of κ-carrageenan (κ-CGN) in cobia, Rachycentron canadum against Lactococcus garvieae. Fish & shellfish immunology 3 34562582
1996 [Evaluation of factors influencing platelet aggregation in patients with chronic glomerulonephritis (CGN)]. Przeglad lekarski 2 8754409
2024 Arginines of the CGN codon family are Achilles' heels of cancer genes. Scientific reports 1 38778164
2024 TLX3 regulates CGN progenitor proliferation during cerebellum development and its dysfunction can lead to autism. iScience 1 39628587
1996 [Evaluation of platelet membrane structure in patients with chronic glomerulonephritis (CGN)]. Przeglad lekarski 1 8754408
2025 Polyphyllin H inhibits malignant progression of ovarian cancer in patient-derived xenograft mouse models by regulating CGN/RhoA/Rock2 axis: an experimental research. International journal of surgery (London, England) 0 40773225