Affinage

EPN1

Epsin-1 · UniProt Q9Y6I3

Round 2 corrected
Length
576 aa
Mass
60.3 kDa
Annotated
2026-04-28
52 papers in source corpus 14 papers cited in narrative 14 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

EPN1 (epsin-1) is a multivalent endocytic adaptor that couples ubiquitinated cargo recognition to membrane deformation during vesicle formation. Its ENTH domain binds PtdIns(4,5)P2 and inserts an amphipathic α-helix into the lipid bilayer to initiate membrane curvature, while its intrinsically disordered regions generate additional steric pressure that drives bending independently of the helix (PMID:12353027, PMID:26204806). Ubiquitin-interacting motifs enable EPN1 to recognize K63-polyubiquitinated cargoes—including VEGFR2, ubiquitinated occludin, SNAT2, and TCR—directing their internalization via clathrin-dependent or clathrin-independent pathways (PMID:15701692, PMID:24311377, PMID:38949026, PMID:36730202). In vivo, combined loss of epsins does not impair housekeeping clathrin-mediated endocytosis but selectively disrupts Notch signaling, VEGFR2 turnover-dependent angiogenesis, podocyte adhesion through a Cdc42–SRF–β1 integrin axis, and TCR trans-endocytosis at the immunological synapse (PMID:19666558, PMID:24311377, PMID:33051360, PMID:36730202).

Mechanistic history

Synthesis pass · year-by-year structured walk · 12 steps
  1. 1998 High

    Identification of epsin-1 as an Eps15-binding, AP-2- and clathrin-coat-associated endocytic factor established it as a component of the clathrin-mediated endocytosis machinery, and dominant-negative disruption showed it is functionally required for coated-pit invagination.

    Evidence Co-IP from brain extracts, localization to clathrin coats, dominant-negative overexpression blocking endocytosis

    PMID:9723620

    Open questions at the time
    • Mechanism by which epsin promotes coated-pit invagination was unknown
    • No structural information on the ENTH domain
  2. 2001 High

    Determination of the ENTH domain structure and its PtdIns(4,5)P2-binding site answered how epsin is recruited to membranes, and mutagenesis proved this interaction is essential for receptor internalization.

    Evidence NMR structure, K76A mutagenesis, EGFR internalization assay in COS-7 cells

    PMID:11161217

    Open questions at the time
    • How PtdIns(4,5)P2 binding leads to membrane deformation was not yet resolved
    • Role of UIMs in cargo selection not yet defined
  3. 2002 High

    Reconstitution of membrane curvature on lipid monolayers revealed that PtdIns(4,5)P2 binding induces an amphipathic α-helix in the ENTH domain whose insertion into one bilayer leaflet is sufficient to curve membranes, providing the first direct mechanism for epsin-driven vesicle budding.

    Evidence In vitro lipid monolayer curvature assay, hydrophobic helix mutagenesis

    PMID:12353027

    Open questions at the time
    • Contribution of disordered regions to curvature not yet appreciated
    • In vivo relevance of helix insertion versus other curvature mechanisms unclear
  4. 2005 High

    RNAi ablation showed that epsin-1 (with Eps15) couples ubiquitinated EGFR to a clathrin-independent, lipid-raft-dependent endocytic pathway, extending epsin's role beyond canonical CME to ubiquitin-dependent alternative internalization routes.

    Evidence siRNA knockdown, EGFR ubiquitination mutants, lipid raft fractionation, internalization assays

    PMID:15701692

    Open questions at the time
    • Whether UIM-mediated cargo recognition applies to other substrates was untested
    • Relative quantitative contribution of clathrin-dependent versus independent pathways unknown
  5. 2009 High

    Epn1/Epn2 double knockout mice revealed that epsins are dispensable for housekeeping CME but essential for Notch signaling activation in vivo, redefining epsins as specialized endocytic adaptors for select signaling pathways rather than general CME factors.

    Evidence Double knockout mice with E9.5–E10 lethality, Notch target gene expression analysis, CME assays in knockout-derived cells

    PMID:19666558

    Open questions at the time
    • Which step of Notch ligand/receptor processing requires epsin remained unclear
    • Whether epsin's Notch function depends on UIM-mediated ubiquitin recognition was unresolved
  6. 2009 High

    Demonstration that VEGF-induced ubiquitination of occludin creates an EPN1-binding signal for endosomal trafficking established a UIM-dependent cargo-recognition paradigm for tight junction remodeling and vascular permeability.

    Evidence Co-IP of epsin-1 with ubiquitinated occludin, S490A phosphomutant and ubiquitin chimera approaches

    PMID:19478092

    Open questions at the time
    • In vivo relevance to vascular permeability not genetically validated
    • Whether EPN1 or EPN2 is the primary adaptor for occludin in endothelium unresolved
  7. 2013 High

    Genetic epistasis showing that VEGFR2 haploinsufficiency rescues endothelial-specific epsin loss proved that epsins mediate VEGFR2 internalization and degradation, and that their absence hyperactivates VEGF signaling to cause pathological angiogenesis.

    Evidence Endothelial-specific Epn1/Epn2 conditional double knockout with Flk1 heterozygosity, in vivo angiogenesis and tumor models

    PMID:24311377

    Open questions at the time
    • Structural basis of VEGFR2–epsin UIM interaction not defined
    • Whether epsin directly ubiquitin-sorts VEGFR2 or acts indirectly was unresolved
  8. 2015 High

    In vitro biophysical measurements revealed that the intrinsically disordered domains of epsin-1 generate steric pressure sufficient to curve membranes independently of the amphipathic helix, adding a second, complementary curvature-generating mechanism.

    Evidence In vitro membrane curvature measurements, protein diffusivity assays, transmembrane chimera expression in mammalian cells

    PMID:26204806

    Open questions at the time
    • Relative in vivo contribution of helix insertion versus steric crowding is unknown
    • Whether both mechanisms operate simultaneously at physiological concentrations not resolved
  9. 2020 High

    Podocyte-specific triple knockout of Epn1/Epn2/Epn3 linked epsins to kidney function, revealing a Cdc42→SRF→β1 integrin signaling axis that maintains podocyte adhesion and foot process architecture.

    Evidence Conditional triple knockout mice, albuminuria, primary podocyte adhesion assays, Cdc42 activation and SRF/integrin expression analysis

    PMID:33051360

    Open questions at the time
    • How epsin loss impairs Cdc42 activation mechanistically is unclear
    • Individual contributions of EPN1, EPN2, and EPN3 in podocytes not dissected
  10. 2023 High

    Live imaging at the immunological synapse showed that EPN1 recruits clathrin to ubiquitinated TCR microclusters to drive trans-endocytosis of pMHC-TCR conjugates into T cells, defining EPN1 as the endocytic arm of bidirectional membrane exchange at this interface.

    Evidence TIRF microscopy, sequential adaptor recruitment analysis, knockdown and dominant-negative experiments

    PMID:36730202

    Open questions at the time
    • In vivo immune consequences of EPN1 loss in T cells not tested
    • Whether EPN2 can compensate in this context is unknown
  11. 2024 High

    Identification of SNAT2 as an EPN1 cargo showed that K63-polyubiquitination at Lys33 serves as the EPN1-recognition signal for SNAT2 internalization, and that monoubiquitination at Lys59 suppresses this polyubiquitination to stabilize SNAT2 on the membrane, thereby increasing glutamine uptake and VEGFC-driven lymphangiogenesis in bladder cancer.

    Evidence Ubiquitination site mapping by MS, site-directed mutagenesis, EPN1 knockdown, glutamine uptake assays, xenograft models

    PMID:38949026

    Open questions at the time
    • Whether EPN1-mediated SNAT2 endocytosis is relevant beyond bladder cancer is untested
    • Structural basis of EPN1 UIM selectivity for K63-polyubiquitin versus other chain types not defined
  12. 2024 Medium

    Mitochondrial retargeting of ITSN1 showed it is sufficient to recruit EPN1 alongside AP-2, EPS15, and dynamin2 to ectopic sites, placing EPN1 downstream of ITSN1 in the hierarchical assembly of CME sites.

    Evidence Genome-edited live-cell imaging, mitochondrial targeting assay, ITSN1 knockdown

    PMID:39580802

    Open questions at the time
    • Whether ITSN1–EPN1 interaction is direct or mediated by intermediaries is unclear
    • Single study without independent replication

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the structural basis of UIM selectivity for K63-linked polyubiquitin chains, the relative in vivo contributions of amphipathic helix insertion versus disordered-domain steric pressure to membrane curvature, and the mechanistic link between epsin loss and impaired Cdc42 activation in podocytes.
  • No high-resolution structure of full-length epsin-1 or UIM–polyubiquitin complex
  • Quantitative partitioning of curvature mechanisms in living cells not measured
  • How epsin regulates Cdc42 activity remains mechanistically undefined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 6 GO:0008289 lipid binding 4
Localization
GO:0031410 cytoplasmic vesicle 4 GO:0005886 plasma membrane 3
Pathway
R-HSA-5653656 Vesicle-mediated transport 7 R-HSA-162582 Signal Transduction 3 R-HSA-168256 Immune System 1
Partners
AP2A1CLTCEPS15ITSN1SNAT2VEGFR2

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1998 Epsin 1 was identified as an EH-domain-binding partner of Eps15, concentrated in presynaptic nerve terminals. Its central region binds AP-2, its carboxy-terminal region binds Eps15, and it associates with clathrin coats in situ. Disruption of epsin function blocks clathrin-mediated endocytosis, implicating epsin in the molecular rearrangement of clathrin coats required for coated-pit invagination and vesicle fission. Co-immunoprecipitation from brain extracts, subcellular localization, dominant-negative overexpression blocking endocytosis Nature High 9723620
2001 The ENTH domain of epsin 1 binds phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] with high affinity. NMR analysis revealed that a cleft formed by positively charged residues mediates phosphoinositide binding. Overexpression of an ENTH domain mutant (Lys76→Ala76) defective in PtdIns(4,5)P2 binding blocked EGF receptor internalization in COS-7 cells, demonstrating that ENTH–PtdIns(4,5)P2 interaction is essential for clathrin-mediated endocytosis. NMR structure determination, site-directed mutagenesis, dominant-negative overexpression assay in COS-7 cells Science High 11161217
2002 Epsin 1 directly drives membrane curvature during clathrin-mediated endocytosis. Binding of PtdIns(4,5)P2 induces formation of an amphipathic alpha-helix in the ENTH domain that inserts into one leaflet of the lipid bilayer, inducing curvature. Mutation of hydrophobic residues on this helix abolishes membrane-curving ability. On lipid monolayers, epsin alone is sufficient to facilitate formation of clathrin-coated invaginations. In vitro membrane curvature assay with lipid monolayers, amphipathic helix identification, site-directed mutagenesis of hydrophobic helix residues Nature High 12353027
2005 Epsin 1 (together with Eps15 and Eps15R) couples ubiquitinated cargo to clathrin-independent, lipid raft-dependent endocytosis of the EGF receptor. Ablation of epsin (and eps15s) via RNAi blocks the non-clathrin internalization of ubiquitinated EGFR, showing that epsin's ubiquitin-interacting motif (UIM) is required for this alternative endocytic route. siRNA knockdown, EGFR ubiquitination mutants, lipid raft fractionation, internalization assays Proceedings of the National Academy of Sciences of the United States of America High 15701692
2009 Combined genetic inactivation of Epn1 and Epn2 in mice causes embryonic lethality at E9.5–E10 with developmental defects that phenocopy global impairment of Notch signaling; expression of Notch primary target genes is severely reduced in double-knockout embryos. However, housekeeping clathrin-mediated endocytosis is not impaired in cells derived from these embryos, establishing epsin as a specialized endocytic adaptor critical for Notch signaling activation rather than general CME. Epn1/Epn2 double knockout mice, gene expression analysis of Notch target genes, CME assays in knockout-derived cells Proceedings of the National Academy of Sciences of the United States of America High 19666558
2009 VEGF induces phosphorylation of occludin on Ser-490 and subsequent ubiquitination, which promotes occludin interaction with Epsin-1 (as shown by co-immunoprecipitation and immunocytochemistry). Epsin-1 binding via its ubiquitin-interacting motif facilitates trafficking of the ubiquitinated tight junction protein to early and late endosomes, contributing to vascular permeability. Co-immunoprecipitation, immunocytochemistry, phosphorylation-deficient (S490A) mutant and occludin-ubiquitin chimera overexpression The Journal of biological chemistry High 19478092
2013 Genetic reduction of VEGFR2 rescues the aberrant angiogenesis caused by endothelial-specific epsin (Epn1/Epn2) deletion. EPN1/EPN2 mediate internalization and degradation of activated VEGFR2; their loss elevates VEGFR2 and enhances VEGF signaling, causing defective embryonic angiogenesis and hyperactive tumor angiogenesis. Restoring one allele of VEGFR2 (Flk1) in epsin-deleted mice normalizes EC proliferation, migration, and network formation. Genetic epistasis with endothelial-specific conditional double knockout mice and Flk1 heterozygosity; in vitro angiogenesis assays; in vivo wound healing, inflammatory angiogenesis, and tumor angiogenesis assays Arteriosclerosis, thrombosis, and vascular biology High 24311377
2013 In C. elegans, epsin (EPN-1) and clathrin heavy chain (CHC-1) are required for apoptotic cell engulfment by phagocytes. EPN-1 loss impairs actin polymerization and pseudopod extension during engulfment; this defect is partially suppressed by inactivating the cofilin ortholog UNC-60. CHC-1 is recruited to the phagocytic cup in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same pathway as ced-1, ced-6, and dyn-1, downstream of CED-1 signaling, revealing a role for epsin in organizing clathrin as a scaffold for actin remodeling during phagocytosis. RNAi-mediated knockdown, genetic epistasis analysis, fluorescence live imaging of pseudopod formation, F-actin localization assays Development (Cambridge, England) High 23861060
2015 Intrinsically disordered domains of Epsin1 (and AP180) are potent drivers of membrane curvature independent of structured amphipathic helices or BAR domains. In vitro measurements show that the large hydrodynamic radii of these disordered domains generate steric pressure that bends membranes. When expressed as transmembrane cargo in mammalian cells, disordered adaptor domains are excluded from clathrin-coated pits, consistent with a balance of steric pressure governing membrane content. In vitro membrane curvature measurements, protein diffusivity assays, transmembrane chimera expression in mammalian cells Nature communications High 26204806
2020 Podocyte-specific triple knockout of Epn1, Epn2, and Epn3 in mice results in albuminuria and foot process effacement. Primary podocytes from these mice show defects in cell adhesion and spreading attributed to reduced activation of Cdc42 and SRF, leading to diminished β1 integrin expression. Loss of SRF alone phenocopies this defect, establishing that epsins maintain podocyte function through a Cdc42→SRF→β1 integrin signaling axis. Podocyte-specific conditional triple knockout mice, albuminuria measurement, primary cell adhesion and spreading assays, Cdc42 activation assay, SRF/integrin expression analysis Journal of the American Society of Nephrology : JASN High 33051360
2023 At the immunological synapse, EPN1 recruits clathrin to ubiquitinated TCR microclusters to enable trans-endocytosis of pMHC-TCR conjugates from the antigen-presenting cell into the T cell. This occurs sequentially after HRS/STAM2-mediated clathrin recruitment drives extracellular vesicle release of TCR, demonstrating that EPN1 governs the endocytic (internalization) arm of bidirectional membrane exchange at the immunological synapse. Live-cell imaging, TIRF microscopy at the immunological synapse, sequential recruitment analysis, knockdown/dominant-negative experiments Proceedings of the National Academy of Sciences of the United States of America High 36730202
2024 EPN1-mediated endocytosis of SNAT2 is suppressed when UBE2C promotes monoubiquitination of SNAT2 at Lys59, which in turn inhibits K63-linked polyubiquitination at Lys33. K63-polyubiquitination is the signal recognized by EPN1 for endocytic internalization; its inhibition increases SNAT2 membrane levels, elevates glutamine uptake and VEGFC secretion, and promotes lymphangiogenesis in bladder cancer. Ubiquitination site mapping by mass spectrometry, site-directed mutagenesis of ubiquitination sites, EPN1 knockdown, glutamine uptake assays, VEGFC secretion measurement, in vivo xenograft models The Journal of clinical investigation High 38949026
2024 Intersectin1 (ITSN1) organizes CME protein interaction networks and when artificially targeted to mitochondria is sufficient to assemble puncta containing EPN1 (epsin1), the AP2 adaptor complex, EPS15, FCHO2, and dynamin2. ITSN1 can recruit dynamin2 independently of EPN1, placing EPN1 downstream of ITSN1 in the sequential assembly of clathrin-mediated endocytosis sites. Live-cell imaging of genome-edited cells (endogenous tagging), mitochondrial targeting assay, ITSN1 knockdown, protein recruitment analysis Cell reports Medium 39580802
2024 The ENTH domain of epsin-1 (EPN1) has membrane vesiculation activity detectable in a free-floating liposome assay at physiologically relevant protein concentrations, consistent with its membrane-curving function. The assay also showed that ANTH domains of PICALM and Hip1R have similar membrane remodelling activity. Single-particle liposome curvature/vesiculation assay at physiological concentrations Journal of cell science Medium 39324332

Source papers

Stage 0 corpus · 52 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2006 Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2861 17081983
2012 Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 1718 22658674
2005 A human protein-protein interaction network: a resource for annotating the proteome. Cell 1704 16169070
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
2015 The BioPlex Network: A Systematic Exploration of the Human Interactome. Cell 1118 26186194
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
2014 A proteome-scale map of the human interactome network. Cell 977 25416956
2020 A reference map of the human binary protein interactome. Nature 849 32296183
2002 Curvature of clathrin-coated pits driven by epsin. Nature 806 12353027
2003 Complete sequencing and characterization of 21,243 full-length human cDNAs. Nature genetics 754 14702039
2021 Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell 705 33961781
2012 A census of human soluble protein complexes. Cell 689 22939629
2005 Clathrin-independent endocytosis of ubiquitinated cargos. Proceedings of the National Academy of Sciences of the United States of America 662 15701692
2011 Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium. Briefings in bioinformatics 656 21873635
2021 Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV. Nature 532 33845483
1998 Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis. Nature 490 9723620
2004 The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome research 438 15489334
2015 A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface. Cell 433 26638075
2022 OpenCell: Endogenous tagging for the cartography of human cellular organization. Science (New York, N.Y.) 432 35271311
2005 Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes. Genome research 409 16344560
2001 Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. Science (New York, N.Y.) 397 11161217
2021 A proximity-dependent biotinylation map of a human cell. Nature 339 34079125
2014 A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways. Cell 325 25036637
2009 Occludin phosphorylation and ubiquitination regulate tight junction trafficking and vascular endothelial growth factor-induced permeability. The Journal of biological chemistry 293 19478092
2012 A high-throughput approach for measuring temporal changes in the interactome. Nature methods 273 22863883
2006 Regulation of ubiquitin-binding proteins by monoubiquitination. Nature cell biology 271 16429130
2004 Functional proteomics mapping of a human signaling pathway. Genome research 247 15231748
2015 Intrinsically disordered proteins drive membrane curvature. Nature communications 213 26204806
2013 Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes? The EMBO journal 213 23314748
2009 Embryonic arrest at midgestation and disruption of Notch signaling produced by the absence of both epsin 1 and epsin 2 in mice. Proceedings of the National Academy of Sciences of the United States of America 96 19666558
2013 Mosquito cellular factors and functions in mediating the infectious entry of chikungunya virus. PLoS neglected tropical diseases 58 23409203
2013 Genetic reduction of vascular endothelial growth factor receptor 2 rescues aberrant angiogenesis caused by epsin deficiency. Arteriosclerosis, thrombosis, and vascular biology 42 24311377
2013 Phagocytic receptor signaling regulates clathrin and epsin-mediated cytoskeletal remodeling during apoptotic cell engulfment in C. elegans. Development (Cambridge, England) 33 23861060
2023 Clathrin mediates both internalization and vesicular release of triggered T cell receptor at the immunological synapse. Proceedings of the National Academy of Sciences of the United States of America 27 36730202
1993 Molecular analysis and overexpression of the gene encoding endothiapepsin, an aspartic protease from Cryphonectria parasitica. Gene 27 8462868
2004 Mapping of a translocation breakpoint in a Peutz-Jeghers hamartoma to the putative PJS locus at 19q13.4 and mutation analysis of candidate genes in polyp and STK11-negative PJS cases. Genes, chromosomes & cancer 20 15287029
2024 UBE2C-induced crosstalk between mono- and polyubiquitination of SNAT2 promotes lymphatic metastasis in bladder cancer. The Journal of clinical investigation 14 38949026
2023 Methylome and proteome integration in human skeletal muscle uncover group and individual responses to high-intensity interval training. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 14 37698381
2021 A C. elegans genome-wide RNAi screen for altered levamisole sensitivity identifies genes required for muscle function. G3 (Bethesda, Md.) 10 33713125
2022 Examining SNP-SNP interactions and risk of clinical outcomes in colorectal cancer using multifactor dimensionality reduction based methods. Frontiers in genetics 6 35991579
2020 Novel Nasal Epithelial Cell Markers of Parkinson's Disease Identified Using Cells Treated with α-Synuclein Preformed Fibrils. Journal of clinical medicine 6 32640699
2010 Epimorphin-derived peptide antagonists remedy epidermal parakeratosis triggered by unsaturated fatty acid. Journal of dermatological science 6 20688483
2021 m6A Regulator Expression Segregates Meningiomas Into Biologically Distinct Subtypes. Frontiers in oncology 4 35004283
2020 Murine Epsins Play an Integral Role in Podocyte Function. Journal of the American Society of Nephrology : JASN 4 33051360
2005 A refined radiation hybrid map of the telomeric region of bovine chromosome 18q25-q26 compared with human chromosome 19q13. Animal genetics 3 15771725
2025 Genome-wide association and functional annotation analyses reveal candidate genes and pathways associated with various ewe longevity indicators in U.S. Katahdin sheep. Frontiers in genetics 2 40678382
2024 Synaptic Vesicle-Related Proteins and Ubiquilin 2 in Cortical Synaptosomes Mediate Cognitive Impairment in Vascular Dementia Rats. Molecular neurobiology 2 38990251
2024 Intersectin1 promotes clathrin-mediated endocytosis by organizing and stabilizing endocytic protein interaction networks. Cell reports 2 39580802
2024 Intersectin1 promotes clathrin-mediated endocytosis by organizing and stabilizing endocytic protein interaction networks. bioRxiv : the preprint server for biology 1 38712149
2024 A single-particle analysis method for detecting membrane remodelling and curvature sensing. Journal of cell science 1 39324332
2010 Establishment of a neonate cell line from Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae) that supports replication of E. postvittana nucleopolyhedrovirus. Journal of invertebrate pathology 1 20167219