{"gene":"ESYT2","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":2007,"finding":"E-Syt2 contains three C-terminal C2 domains; recombinant fragments including the C2A domain bind phospholipids in a Ca2+-dependent manner at micromolar free Ca2+ concentrations. The C2C domain of E-Syt2 functions as a targeting motif that localizes the protein to the plasma membrane independently of its transmembrane region.","method":"Recombinant protein biochemistry (Ca2+-dependent phospholipid binding assays), transfection of myc-tagged constructs with localization analysis, domain deletion/structure-function studies","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro phospholipid binding assay plus domain mutagenesis/truncation with cellular localization readout in a single focused study","pmids":["17360437"],"is_preprint":false},{"year":2008,"finding":"The soluble multi-C2 domain region of E-Syt2 undergoes Ca2+-triggered structural rearrangements and reversible multimerization in vitro, with an apparent Ca2+-binding constant of ~100 µM, as determined by small-angle X-ray scattering (SAXS).","method":"Small-angle X-ray scattering (SAXS) of recombinant E-Syt2 protein; quantitative calcium binding analysis","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — structural method (SAXS) with quantitative Ca2+ binding, single lab, single study","pmids":["18977228"],"is_preprint":false},{"year":2010,"finding":"E-Syt2 acts as an endocytic adaptor for clathrin-mediated endocytosis of the activated FGF receptor in Xenopus development; it interacts selectively with activated FGFR and with Adaptin-2, and is required upstream of Ras activation for ERK activation and mesoderm induction.","method":"Morpholino-based depletion in Xenopus embryos, co-immunoprecipitation with FGFR and Adaptin-2, epistasis with Ras/ERK pathway, rescue experiments, in vivo endocytosis assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, loss-of-function with defined developmental phenotype, pathway epistasis, multiple orthogonal methods in one study","pmids":["20833364"],"is_preprint":false},{"year":2012,"finding":"The C2C domain of E-Syt2 directly binds a site adjacent to the CRIB/GBD domain of PAK1; this interaction suppresses actin polymerization, inhibits PAK1 activation by Cdc42 and Rac, and E-Syt2–PAK1 complexes selectively associate with FGFR1 to cooperate in FGF signaling.","method":"Co-immunoprecipitation, domain mapping (C2C domain pulldown), functional PAK1 activation assays, actin polymerization assays, FGFR1 complex analysis","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with domain mapping, functional readouts for PAK1 activity and actin, single lab","pmids":["23213466"],"is_preprint":false},{"year":2013,"finding":"E-Syt2 and E-Syt3 tether the ER to the plasma membrane via C2 domain-dependent interactions requiring PI(4,5)P2; elevation of cytosolic Ca2+ is additionally required for E-Syt1-mediated tethering. The E-Syts form heteromeric complexes, conferring Ca2+ regulation to ER-PM contact formation. E-Syt-mediated contacts are not required for store-operated Ca2+ entry.","method":"Fluorescence microscopy (ER-PM contact site quantification), PI(4,5)P2 manipulation, Ca2+ imaging, co-immunoprecipitation (heteromeric complex), siRNA knockdown, STIM1/Orai1 epistasis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (imaging, biochemistry, lipid manipulation, genetic epistasis), replicated across E-Syt family members, high-impact study","pmids":["23791178"],"is_preprint":false},{"year":2013,"finding":"Crystal structures of the tandem C2A-C2B domains of E-Syt2 reveal a rigid V-shaped architecture not substantially altered by Ca2+. The C2A domain binds up to four Ca2+ ions while the C2B domain does not bind Ca2+. NMR confirmed these Ca2+-binding properties.","method":"X-ray crystallography (structures in absence and presence of Ca2+), NMR spectroscopy for Ca2+-binding analysis","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus NMR validation, two orthogonal structural/biophysical methods in one study","pmids":["24373768"],"is_preprint":false},{"year":2014,"finding":"The crystal structure of an E-Syt2 fragment (SMP domain plus C2A-C2B) at 2.44 Å resolution reveals a TULIP superfamily β-barrel SMP domain that dimerizes to form a ~90-Å hydrophobic channel. Mass spectrometry identified glycerophospholipids bound within this channel, demonstrating that E-Syt2 directly binds and likely transfers lipids via its SMP domain.","method":"X-ray crystallography (2.44 Å resolution crystal structure), mass spectrometry (lipid identification from SMP channel)","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure plus mass spectrometry identification of bound lipids, rigorous structural and biochemical validation","pmids":["24847877"],"is_preprint":false},{"year":2014,"finding":"ESyt2 is directed to the ER by its transmembrane domain. ESyt2 homodimerizes in vivo via a TM-adjacent sequence (not the SMP domain). ESyt2 (and ESyt3, but not ESyt1) selectively interacts with activated FGFR1 in vivo through a short TM-adjacent sequence; this interaction is independent of receptor autophosphorylation but dependent on receptor conformation (upper kinase lobe site revealed upon activation loop displacement).","method":"Co-immunoprecipitation, domain deletion/mutagenesis constructs, localization studies, kinase-dead mutant analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with systematic domain mutants, multiple constructs tested, single lab","pmids":["25922075"],"is_preprint":false},{"year":2016,"finding":"At steady state, E-Syt2 positions Sac1 (an integral ER membrane lipid phosphatase) at ER-PM junctions, where Sac1 limits PM PI(4)P levels. Activation of GPCRs depleting PM PI(4,5)P2 disrupts E-Syt2-mediated ER-PM junctions, reducing Sac1 access to the PM and allowing PI(4)P and PI(4,5)P2 recovery. ER Ca2+ depletion and SOCE activation increase Sac1 at the PM via E-Syt2 contacts, depleting PM PI(4)P.","method":"Fluorescence microscopy (E-Syt2 and Sac1 colocalization at ER-PM junctions), GPCR stimulation/PI(4,5)P2 depletion experiments, SOCE activation, phosphoinositide biosensors, siRNA knockdown","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — live-cell imaging with biosensors, pharmacological manipulation, knockdown with defined lipid phenotypes, multiple orthogonal approaches in single study","pmids":["27044890"],"is_preprint":false},{"year":2016,"finding":"ESYT2-short variant inhibition causes cortical redistribution of actin in lung cancer cells, whereas inhibition of the long variant increases endocytosis, revealing isoform-specific roles for ESYT2 in cytoskeletal organization and endocytosis.","method":"siRNA knockdown of individual ESYT2 splice variants, actin distribution imaging, endocytosis assays in lung cancer cells","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single lab, knockdown with cellular phenotype readout, no molecular mechanism defined beyond splice variant specificity","pmids":["27555542"],"is_preprint":false},{"year":2017,"finding":"Single-molecule optical tweezers measurements show that C2 domains of E-Syt2 resist membrane unbinding forces of 2–7 pN and have binding energies of 4–14 kBT per C2 domain. Regulation by bilayer composition and Ca2+ recapitulated known properties of E-Syt2 C2 domains.","method":"Single-molecule force spectroscopy (optical tweezers), defined lipid bilayer compositions, Ca2+ titration","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — quantitative single-molecule reconstitution with force measurements, multiple bilayer compositions and Ca2+ conditions tested","pmids":["29083305"],"is_preprint":false},{"year":2017,"finding":"RASSF4 regulates E-Syt2- and E-Syt3-mediated ER-PM tethering by controlling PM PI(4,5)P2 levels through ARF6-dependent activation of PIP5Ks. Knockdown of RASSF4 reduces PI(4,5)P2, impairing E-Syt2/3 localization to ER-PM junctions.","method":"siRNA knockdown of RASSF4, ER-PM contact site imaging, PI(4,5)P2 biosensor, ARF6 activity assay, co-immunoprecipitation (RASSF4-ARF6)","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — multiple readouts (imaging, lipid biosensor, GTPase activity), reciprocal interaction shown, single lab","pmids":["28600435"],"is_preprint":false},{"year":2017,"finding":"UBQLN1 interacts with ESYT2 through its STI chaperone-like domains (not the UBA or UBL domains) and stabilizes ESYT2 protein levels in a manner dependent on UBQLN1's UBA domain interaction with ubiquitin.","method":"Co-immunoprecipitation, domain deletion constructs of UBQLN1, Western blot for protein stability","journal":"Journal of cellular biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with domain mapping, ESYT2 is one of several substrates tested, single lab","pmids":["28075048"],"is_preprint":false},{"year":2020,"finding":"Sec22b interacts with E-Syt2 via the longin domain of Sec22b. This interaction stabilizes Sec22b-Stx1 association at ER-PM contacts. Overexpression of wild-type E-Syt2 (but not lipid-transfer-deficient or ER-attachment mutants) increases axonal filopodia formation and neurite ramification; this effect requires Stx1 cleavage sensitivity and the Sec22b longin domain.","method":"Co-immunoprecipitation (Sec22b-E-Syt2 interaction), domain mutant analysis, overexpression and silencing in neurons, neurite morphology quantification, clostridial neurotoxin epistasis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with domain mapping, multiple E-Syt2 mutants, functional neuronal phenotype, epistasis with Stx1 cleavage, single lab","pmids":["32843578"],"is_preprint":false},{"year":2020,"finding":"The short isoform of E-Syt2 (E-Syt2S) is the predominant E-Syt2 isoform in T cells and directly interacts with STIM1, recruiting it to ER-PM junctions independently of ER-PM membrane tethering, thereby supporting CRAC channel (Orai1-STIM1) activation and store-operated Ca2+ entry in Jurkat T cells but not in HeLa cells.","method":"CRISPR/siRNA knockdown, co-immunoprecipitation (E-Syt2S–STIM1), Ca2+ imaging (SOCE measurement), Orai1-STIM1 clustering imaging, isoform-specific expression analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional SOCE assay, cell-type specificity established, single lab","pmids":["32879390"],"is_preprint":false},{"year":2021,"finding":"In C. elegans, ESYT-2 colocalizes with junctophilin JPH-1 at membrane contact sites in neurons. Genetic double-mutant analysis shows that jph-1 and esyt-2 null mutants display mutual suppression of aldicarb response, indicating that JPH-1 and ESYT-2 have antagonistic roles in neuromuscular synaptic transmission.","method":"Fluorescence localization studies, genetic epistasis (double-mutant analysis), aldicarb sensitivity assay in C. elegans","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with defined physiological readout (aldicarb assay), colocalization; C. elegans ortholog study","pmids":["33871019"],"is_preprint":false},{"year":2023,"finding":"E-Syt2 reduces plasma membrane DAG levels in resting T cells, thereby downmodulating T-cell receptor signaling, cytotoxicity, degranulation, and cytokine production. Upon TCR stimulation, both E-Syt1 and E-Syt2 negatively control TCR signaling through DAG reduction at the PM.","method":"Knockdown/deletion of E-Syt2 (and E-Syt1) in T cells, DAG biosensor imaging, TCR signaling readouts (cytokine production, degranulation, cytotoxicity assays)","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with lipid biosensor and multiple functional T-cell assays, single lab","pmids":["38177911"],"is_preprint":false},{"year":2025,"finding":"ESYT2 forms a multimeric complex with ESYT1 and VAPB at lipid droplet–mitochondria–ER contact sites. Deletion of ESYT2 limits lipid droplet-derived fatty acid oxidation, depletes TCA cycle metabolites, remodels the cellular lipidome, and induces lipotoxic stress; these findings were recapitulated in Esyt2-deficient mice.","method":"Proximity-dependent biotinylation (BioID), high-resolution imaging, ESYT2 deletion (cell lines and mice), fatty acid oxidation assays, metabolomics (TCA cycle metabolites), lipidomics","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — proximity proteomics plus KO in cells and mice, multiple orthogonal functional readouts (metabolomics, lipidomics, oxidation assays), replicated in vivo","pmids":["40032835"],"is_preprint":false},{"year":2025,"finding":"E-Syt2 dissociates the ANO1-VAPA interaction at STIM1 ER-PM junctions and forms an ANO1-IRBIT-E-Syt2-AC6-AKAP11-PKA complex that phosphorylates ANO1 at S221, markedly reducing ANO1 Ca2+ affinity. These effects are primarily mediated by E-Syt2 reciprocally regulating junctional PI(4)P, PI(4,5)P2, and PtdSer levels.","method":"Co-immunoprecipitation (complex assembly), phosphorylation site mutagenesis (S221), Ca2+ affinity measurements for ANO1, phosphoinositide biosensors, knockdown and rescue experiments","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP complex characterization, site-directed mutagenesis, functional Ca2+ affinity assay, single lab","pmids":["40204782"],"is_preprint":false}],"current_model":"ESYT2 is an ER-resident membrane protein that tethers the ER to the plasma membrane via PI(4,5)P2-dependent C2 domain interactions (requiring its C2C domain for PM targeting and C2A domain for Ca2+-dependent phospholipid binding), forms heteromeric complexes with E-Syt1 and E-Syt3, and transfers glycerophospholipids between membranes via a hydrophobic channel in its SMP domain; at ER-PM contact sites it positions Sac1 to regulate PM phosphoinositide homeostasis (PI(4)P/PI(4,5)P2), reduces plasma membrane DAG to modulate TCR signaling, recruits STIM1 (via its short isoform) to support CRAC channel activation in T cells, and forms a complex with ESYT1 and VAPB at lipid droplet–mitochondria–ER contacts to enable fatty acid oxidation; it also functions as an endocytic adaptor for activated FGFR1 by interacting with Adaptin-2 and PAK1 to regulate actin dynamics and ERK signaling."},"narrative":{"mechanistic_narrative":"ESYT2 (E-Syt2) is an ER-resident transmembrane protein that tethers the endoplasmic reticulum to the plasma membrane and acts as a lipid transfer and signaling platform at membrane contact sites [PMID:23791178, PMID:24847877]. It is anchored to the ER by its transmembrane domain and engages the plasma membrane through its C-terminal C2 domains: the C2C domain serves as a PI(4,5)P2-dependent PM-targeting motif, while the C2A domain binds phospholipids in a Ca2+-dependent manner, with structural and single-molecule studies establishing a rigid V-shaped tandem-C2 architecture and quantifying its calcium-tuned membrane affinity [PMID:17360437, PMID:24373768, PMID:29083305]. E-Syt2 forms heteromeric complexes with E-Syt1 and E-Syt3, and its dimeric SMP domain folds into a TULIP-superfamily hydrophobic channel that binds and transfers glycerophospholipids between apposed membranes [PMID:23791178, PMID:24847877]. At ER-PM junctions it controls phosphoinositide homeostasis by positioning the lipid phosphatase Sac1 to limit PM PI(4)P, a coupling that is dynamically regulated by GPCR-driven PI(4,5)P2 depletion and by upstream control of PM PI(4,5)P2 through RASSF4-ARF6-PIP5K signaling [PMID:27044890, PMID:28600435]. Through these lipid functions E-Syt2 shapes signaling at the PM: it lowers plasma membrane DAG to downmodulate T-cell receptor signaling, and a short isoform recruits STIM1 to support CRAC-channel-mediated store-operated Ca2+ entry in T cells [PMID:38177911, PMID:32879390]. Beyond ER-PM contacts, E-Syt2 forms a complex with E-Syt1 and VAPB at lipid droplet-mitochondria-ER contacts to enable fatty acid oxidation, with its deletion causing lipotoxic stress and TCA-cycle metabolite depletion in cells and mice [PMID:40032835]. Independently, E-Syt2 functions as an endocytic adaptor for activated FGFR1, interacting with the receptor through a TM-adjacent sequence and with Adaptin-2 and PAK1 to couple receptor endocytosis to actin dynamics and ERK signaling [PMID:20833364, PMID:23213466, PMID:25922075].","teleology":[{"year":2007,"claim":"Established the domain logic of E-Syt2 membrane engagement, showing how a multi-C2 protein could be targeted to the plasma membrane and respond to calcium.","evidence":"Recombinant Ca2+-dependent phospholipid binding assays plus domain-deletion localization of myc-tagged constructs","pmids":["17360437"],"confidence":"High","gaps":["Did not place the protein at ER-PM contacts or define a cellular function","ER anchoring versus PM targeting roles not yet separated"]},{"year":2008,"claim":"Quantified the calcium responsiveness of the C2 region, showing Ca2+-triggered conformational change and multimerization in vitro.","evidence":"SAXS of the recombinant soluble multi-C2 region with quantitative Ca2+ binding (~100 µM)","pmids":["18977228"],"confidence":"Medium","gaps":["Solution behavior not linked to membrane tethering in cells","Functional consequence of multimerization undefined"]},{"year":2010,"claim":"Revealed an unexpected role for E-Syt2 as an endocytic adaptor required for FGF receptor internalization and downstream ERK-driven mesoderm induction.","evidence":"Morpholino depletion in Xenopus, reciprocal Co-IP with FGFR and Adaptin-2, Ras/ERK epistasis and rescue","pmids":["20833364"],"confidence":"High","gaps":["Relationship between endocytic adaptor role and later contact-site/lipid functions unresolved","Whether this role operates in mammalian somatic cells not addressed"]},{"year":2012,"claim":"Identified PAK1 as a direct C2C-domain partner, linking E-Syt2 to suppression of actin polymerization and to FGFR1-associated signaling complexes.","evidence":"Co-IP with C2C domain mapping, PAK1 activation and actin polymerization assays, FGFR1 complex analysis","pmids":["23213466"],"confidence":"Medium","gaps":["Single lab without reciprocal in vivo validation","Integration with lipid-tethering activity not addressed"]},{"year":2013,"claim":"Defined E-Syt2 (with E-Syt3) as a constitutive PI(4,5)P2-dependent ER-PM tether that forms Ca2+-regulated heteromers with E-Syt1, establishing its core contact-site function.","evidence":"ER-PM contact imaging, PI(4,5)P2 manipulation, Ca2+ imaging, Co-IP, siRNA, STIM1/Orai1 epistasis","pmids":["23791178"],"confidence":"High","gaps":["Mechanism of lipid handling at contacts not yet shown","Showed E-Syt contacts dispensable for SOCE, leaving downstream lipid roles open"]},{"year":2013,"claim":"Provided atomic detail of the tandem C2A-C2B module, showing a rigid Ca2+-independent architecture with Ca2+ binding confined to C2A.","evidence":"X-ray crystallography with and without Ca2+ and NMR Ca2+-binding analysis","pmids":["24373768"],"confidence":"High","gaps":["Did not include the SMP domain or define lipid transfer","Membrane-bound conformation not resolved"]},{"year":2014,"claim":"Demonstrated that E-Syt2 is a lipid transfer protein, with a dimeric SMP β-barrel forming a hydrophobic channel that captures glycerophospholipids.","evidence":"2.44 Å crystal structure of SMP-C2A-C2B and mass spectrometry of channel-bound lipids","pmids":["24847877"],"confidence":"High","gaps":["Directionality and physiological lipid cargo in cells not established","Coupling of transfer to contact-site dynamics undefined"]},{"year":2014,"claim":"Mapped the topology and FGFR1-binding determinants of E-Syt2, showing TM-domain ER targeting, TM-adjacent homodimerization, and conformation-dependent FGFR1 recognition.","evidence":"Co-IP, systematic domain mutants, localization studies, kinase-dead receptor analysis","pmids":["25922075"],"confidence":"Medium","gaps":["Single lab Co-IP-based interaction mapping","Functional consequence in mammalian FGFR signaling not quantified here"]},{"year":2016,"claim":"Connected E-Syt2 tethering to PM phosphoinositide homeostasis by showing it positions Sac1 to control PI(4)P, with junctions dynamically remodeled by GPCR and SOCE signaling.","evidence":"Live-cell imaging of E-Syt2/Sac1 colocalization, phosphoinositide biosensors, GPCR/SOCE manipulation, siRNA","pmids":["27044890"],"confidence":"High","gaps":["Direct physical Sac1-E-Syt2 interaction not biochemically resolved","Quantitative contribution to total PM PI(4)P turnover unclear"]},{"year":2016,"claim":"Revealed splice-isoform-specific cellular functions, with short and long E-Syt2 variants differentially controlling actin distribution and endocytosis.","evidence":"Variant-specific siRNA, actin imaging and endocytosis assays in lung cancer cells","pmids":["27555542"],"confidence":"Medium","gaps":["No molecular mechanism beyond variant specificity","Isoform-specific partners not identified here"]},{"year":2017,"claim":"Placed E-Syt2 tethering downstream of RASSF4-ARF6-PIP5K control of PM PI(4,5)P2, defining an upstream regulatory input.","evidence":"RASSF4 siRNA, ER-PM contact imaging, PI(4,5)P2 biosensor, ARF6 activity assay, Co-IP","pmids":["28600435"],"confidence":"Medium","gaps":["Single lab","Direct versus indirect effect of RASSF4 on E-Syt2 not separated"]},{"year":2017,"claim":"Provided quantitative single-molecule force measurements of C2-domain membrane binding, mechanically validating bilayer- and Ca2+-tuned tethering.","evidence":"Optical-tweezers force spectroscopy across defined bilayers and Ca2+ conditions","pmids":["29083305"],"confidence":"High","gaps":["Reconstituted system; in-cell forces not measured","Does not address lipid transfer flux"]},{"year":2017,"claim":"Identified UBQLN1 as a stabilizer of ESYT2 protein levels, implicating proteostasis control of the tether.","evidence":"Co-IP with UBQLN1 domain mapping and Western blot stability assays","pmids":["28075048"],"confidence":"Low","gaps":["Single Co-IP with ESYT2 as one of several substrates, not independently confirmed","Physiological context of ESYT2 turnover undefined"]},{"year":2020,"claim":"Linked E-Syt2 to SNARE machinery and neuronal morphogenesis, showing it stabilizes Sec22b-Stx1 at ER-PM contacts and promotes axonal filopodia in a lipid-transfer- and tethering-dependent manner.","evidence":"Co-IP with Sec22b longin-domain mapping, E-Syt2 mutant analysis, neurite morphology quantification, neurotoxin epistasis","pmids":["32843578"],"confidence":"Medium","gaps":["Single lab","Mechanistic coupling of lipid transfer to SNARE function not fully defined"]},{"year":2020,"claim":"Showed the short E-Syt2 isoform recruits STIM1 to ER-PM junctions to support CRAC channel activation, a tethering-independent, cell-type-specific role in T cells.","evidence":"CRISPR/siRNA knockdown, E-Syt2S-STIM1 Co-IP, SOCE Ca2+ imaging, Orai1-STIM1 clustering in Jurkat vs HeLa","pmids":["32879390"],"confidence":"Medium","gaps":["Reconciliation with earlier finding that E-Syt contacts are dispensable for SOCE","Structural basis of E-Syt2S-STIM1 interaction unresolved"]},{"year":2021,"claim":"Used the C. elegans ortholog to show ESYT-2 acts antagonistically with junctophilin JPH-1 at neuronal contact sites in synaptic transmission.","evidence":"Colocalization and genetic double-mutant epistasis with aldicarb assay in C. elegans","pmids":["33871019"],"confidence":"Medium","gaps":["Molecular basis of JPH-1 antagonism unknown","Conservation of this interaction in mammals not tested"]},{"year":2023,"claim":"Defined a signaling output of E-Syt2 lipid control, showing it lowers PM DAG to negatively regulate T-cell receptor signaling and effector function.","evidence":"E-Syt2/E-Syt1 knockdown/deletion in T cells, DAG biosensor imaging, cytokine/degranulation/cytotoxicity assays","pmids":["38177911"],"confidence":"Medium","gaps":["Direct enzymatic link between E-Syt2 and DAG metabolism not established","Single lab"]},{"year":2025,"claim":"Extended E-Syt2 function to lipid droplet-mitochondria-ER contacts, showing it forms a complex with E-Syt1 and VAPB that supports fatty acid oxidation and protects against lipotoxic stress in vivo.","evidence":"BioID proximity proteomics, high-resolution imaging, ESYT2 KO in cells and mice, FAO assays, metabolomics and lipidomics","pmids":["40032835"],"confidence":"High","gaps":["Whether lipid transfer activity directly drives FAO at these contacts not isolated","Mechanism of VAPB-dependent contact assembly undefined"]},{"year":2025,"claim":"Implicated E-Syt2 in chloride channel regulation, showing it remodels a junctional ANO1 signaling complex and reduces ANO1 Ca2+ affinity via PKA-dependent phosphorylation.","evidence":"Co-IP complex characterization, S221 phosphosite mutagenesis, ANO1 Ca2+ affinity measurements, phosphoinositide biosensors, knockdown/rescue","pmids":["40204782"],"confidence":"Medium","gaps":["Single lab","Direct versus phosphoinositide-mediated contribution of E-Syt2 not fully separated"]},{"year":null,"claim":"How E-Syt2's distinct activities—lipid transfer, ER-PM tethering, endocytic adaptation, and isoform-specific signaling—are coordinately deployed across cell types and contact-site types remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Directionality and in vivo cargo selectivity of SMP-mediated lipid transfer unknown","No human disease link established in the corpus","Relative contributions of long versus short isoforms across tissues uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[0,6,10]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[6,13]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,8,14]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[4,7,8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,4,8]},{"term_id":"GO:0005811","term_label":"lipid droplet","supporting_discovery_ids":[17]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[17]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[8,17]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,16]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[14,16]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,9]}],"complexes":["E-Syt1/E-Syt2/E-Syt3 heteromer","ESYT2-ESYT1-VAPB lipid droplet-mitochondria-ER complex","ANO1-IRBIT-E-Syt2-AC6-AKAP11-PKA complex","E-Syt2-PAK1-FGFR1 complex"],"partners":["ESYT1","ESYT3","STIM1","PAK1","FGFR1","SEC22B","VAPB","RASSF4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"A0FGR8","full_name":"Extended synaptotagmin-2","aliases":["Chr2Syt"],"length_aa":921,"mass_kda":102.4,"function":"Tethers the endoplasmic reticulum to the cell membrane and promotes the formation of appositions between the endoplasmic reticulum and the cell membrane. Binds glycerophospholipids in a barrel-like domain and may play a role in cellular lipid transport. Plays a role in FGF signaling via its role in the rapid internalization of FGFR1 that has been activated by FGF1 binding; this occurs most likely via the AP-2 complex. Promotes the localization of SACM1L at endoplasmic reticulum-plasma membrane contact sites (EPCS) (PubMed:27044890)","subcellular_location":"Cell membrane; Endoplasmic reticulum membrane","url":"https://www.uniprot.org/uniprotkb/A0FGR8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ESYT2","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ESYT1","stoichiometry":10.0},{"gene":"RTN4","stoichiometry":4.0},{"gene":"ARL6IP1","stoichiometry":0.2},{"gene":"ATL3","stoichiometry":0.2},{"gene":"COPA","stoichiometry":0.2},{"gene":"COPB2","stoichiometry":0.2},{"gene":"COPE","stoichiometry":0.2},{"gene":"OSBP","stoichiometry":0.2},{"gene":"VAPA","stoichiometry":0.2},{"gene":"VAPB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/ESYT2","total_profiled":1310},"omim":[{"mim_id":"616692","title":"EXTENDED SYNAPTOTAGMIN-LIKE PROTEIN 3; ESYT3","url":"https://www.omim.org/entry/616692"},{"mim_id":"616691","title":"EXTENDED SYNAPTOTAGMIN-LIKE PROTEIN 2; ESYT2","url":"https://www.omim.org/entry/616691"},{"mim_id":"616670","title":"EXTENDED SYNAPTOTAGMIN-LIKE PROTEIN 1; ESYT1","url":"https://www.omim.org/entry/616670"},{"mim_id":"610841","title":"STROMAL INTERACTION MOLECULE 2; STIM2","url":"https://www.omim.org/entry/610841"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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The C2C domain of E-Syt2 functions as a targeting motif that localizes the protein to the plasma membrane independently of its transmembrane region.\",\n      \"method\": \"Recombinant protein biochemistry (Ca2+-dependent phospholipid binding assays), transfection of myc-tagged constructs with localization analysis, domain deletion/structure-function studies\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro phospholipid binding assay plus domain mutagenesis/truncation with cellular localization readout in a single focused study\",\n      \"pmids\": [\"17360437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The soluble multi-C2 domain region of E-Syt2 undergoes Ca2+-triggered structural rearrangements and reversible multimerization in vitro, with an apparent Ca2+-binding constant of ~100 µM, as determined by small-angle X-ray scattering (SAXS).\",\n      \"method\": \"Small-angle X-ray scattering (SAXS) of recombinant E-Syt2 protein; quantitative calcium binding analysis\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — structural method (SAXS) with quantitative Ca2+ binding, single lab, single study\",\n      \"pmids\": [\"18977228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"E-Syt2 acts as an endocytic adaptor for clathrin-mediated endocytosis of the activated FGF receptor in Xenopus development; it interacts selectively with activated FGFR and with Adaptin-2, and is required upstream of Ras activation for ERK activation and mesoderm induction.\",\n      \"method\": \"Morpholino-based depletion in Xenopus embryos, co-immunoprecipitation with FGFR and Adaptin-2, epistasis with Ras/ERK pathway, rescue experiments, in vivo endocytosis assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, loss-of-function with defined developmental phenotype, pathway epistasis, multiple orthogonal methods in one study\",\n      \"pmids\": [\"20833364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The C2C domain of E-Syt2 directly binds a site adjacent to the CRIB/GBD domain of PAK1; this interaction suppresses actin polymerization, inhibits PAK1 activation by Cdc42 and Rac, and E-Syt2–PAK1 complexes selectively associate with FGFR1 to cooperate in FGF signaling.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping (C2C domain pulldown), functional PAK1 activation assays, actin polymerization assays, FGFR1 complex analysis\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with domain mapping, functional readouts for PAK1 activity and actin, single lab\",\n      \"pmids\": [\"23213466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"E-Syt2 and E-Syt3 tether the ER to the plasma membrane via C2 domain-dependent interactions requiring PI(4,5)P2; elevation of cytosolic Ca2+ is additionally required for E-Syt1-mediated tethering. The E-Syts form heteromeric complexes, conferring Ca2+ regulation to ER-PM contact formation. E-Syt-mediated contacts are not required for store-operated Ca2+ entry.\",\n      \"method\": \"Fluorescence microscopy (ER-PM contact site quantification), PI(4,5)P2 manipulation, Ca2+ imaging, co-immunoprecipitation (heteromeric complex), siRNA knockdown, STIM1/Orai1 epistasis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (imaging, biochemistry, lipid manipulation, genetic epistasis), replicated across E-Syt family members, high-impact study\",\n      \"pmids\": [\"23791178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structures of the tandem C2A-C2B domains of E-Syt2 reveal a rigid V-shaped architecture not substantially altered by Ca2+. The C2A domain binds up to four Ca2+ ions while the C2B domain does not bind Ca2+. NMR confirmed these Ca2+-binding properties.\",\n      \"method\": \"X-ray crystallography (structures in absence and presence of Ca2+), NMR spectroscopy for Ca2+-binding analysis\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus NMR validation, two orthogonal structural/biophysical methods in one study\",\n      \"pmids\": [\"24373768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The crystal structure of an E-Syt2 fragment (SMP domain plus C2A-C2B) at 2.44 Å resolution reveals a TULIP superfamily β-barrel SMP domain that dimerizes to form a ~90-Å hydrophobic channel. Mass spectrometry identified glycerophospholipids bound within this channel, demonstrating that E-Syt2 directly binds and likely transfers lipids via its SMP domain.\",\n      \"method\": \"X-ray crystallography (2.44 Å resolution crystal structure), mass spectrometry (lipid identification from SMP channel)\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure plus mass spectrometry identification of bound lipids, rigorous structural and biochemical validation\",\n      \"pmids\": [\"24847877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ESyt2 is directed to the ER by its transmembrane domain. ESyt2 homodimerizes in vivo via a TM-adjacent sequence (not the SMP domain). ESyt2 (and ESyt3, but not ESyt1) selectively interacts with activated FGFR1 in vivo through a short TM-adjacent sequence; this interaction is independent of receptor autophosphorylation but dependent on receptor conformation (upper kinase lobe site revealed upon activation loop displacement).\",\n      \"method\": \"Co-immunoprecipitation, domain deletion/mutagenesis constructs, localization studies, kinase-dead mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with systematic domain mutants, multiple constructs tested, single lab\",\n      \"pmids\": [\"25922075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"At steady state, E-Syt2 positions Sac1 (an integral ER membrane lipid phosphatase) at ER-PM junctions, where Sac1 limits PM PI(4)P levels. Activation of GPCRs depleting PM PI(4,5)P2 disrupts E-Syt2-mediated ER-PM junctions, reducing Sac1 access to the PM and allowing PI(4)P and PI(4,5)P2 recovery. ER Ca2+ depletion and SOCE activation increase Sac1 at the PM via E-Syt2 contacts, depleting PM PI(4)P.\",\n      \"method\": \"Fluorescence microscopy (E-Syt2 and Sac1 colocalization at ER-PM junctions), GPCR stimulation/PI(4,5)P2 depletion experiments, SOCE activation, phosphoinositide biosensors, siRNA knockdown\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging with biosensors, pharmacological manipulation, knockdown with defined lipid phenotypes, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"27044890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ESYT2-short variant inhibition causes cortical redistribution of actin in lung cancer cells, whereas inhibition of the long variant increases endocytosis, revealing isoform-specific roles for ESYT2 in cytoskeletal organization and endocytosis.\",\n      \"method\": \"siRNA knockdown of individual ESYT2 splice variants, actin distribution imaging, endocytosis assays in lung cancer cells\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, knockdown with cellular phenotype readout, no molecular mechanism defined beyond splice variant specificity\",\n      \"pmids\": [\"27555542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Single-molecule optical tweezers measurements show that C2 domains of E-Syt2 resist membrane unbinding forces of 2–7 pN and have binding energies of 4–14 kBT per C2 domain. Regulation by bilayer composition and Ca2+ recapitulated known properties of E-Syt2 C2 domains.\",\n      \"method\": \"Single-molecule force spectroscopy (optical tweezers), defined lipid bilayer compositions, Ca2+ titration\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative single-molecule reconstitution with force measurements, multiple bilayer compositions and Ca2+ conditions tested\",\n      \"pmids\": [\"29083305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RASSF4 regulates E-Syt2- and E-Syt3-mediated ER-PM tethering by controlling PM PI(4,5)P2 levels through ARF6-dependent activation of PIP5Ks. Knockdown of RASSF4 reduces PI(4,5)P2, impairing E-Syt2/3 localization to ER-PM junctions.\",\n      \"method\": \"siRNA knockdown of RASSF4, ER-PM contact site imaging, PI(4,5)P2 biosensor, ARF6 activity assay, co-immunoprecipitation (RASSF4-ARF6)\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — multiple readouts (imaging, lipid biosensor, GTPase activity), reciprocal interaction shown, single lab\",\n      \"pmids\": [\"28600435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"UBQLN1 interacts with ESYT2 through its STI chaperone-like domains (not the UBA or UBL domains) and stabilizes ESYT2 protein levels in a manner dependent on UBQLN1's UBA domain interaction with ubiquitin.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion constructs of UBQLN1, Western blot for protein stability\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with domain mapping, ESYT2 is one of several substrates tested, single lab\",\n      \"pmids\": [\"28075048\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Sec22b interacts with E-Syt2 via the longin domain of Sec22b. This interaction stabilizes Sec22b-Stx1 association at ER-PM contacts. Overexpression of wild-type E-Syt2 (but not lipid-transfer-deficient or ER-attachment mutants) increases axonal filopodia formation and neurite ramification; this effect requires Stx1 cleavage sensitivity and the Sec22b longin domain.\",\n      \"method\": \"Co-immunoprecipitation (Sec22b-E-Syt2 interaction), domain mutant analysis, overexpression and silencing in neurons, neurite morphology quantification, clostridial neurotoxin epistasis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with domain mapping, multiple E-Syt2 mutants, functional neuronal phenotype, epistasis with Stx1 cleavage, single lab\",\n      \"pmids\": [\"32843578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The short isoform of E-Syt2 (E-Syt2S) is the predominant E-Syt2 isoform in T cells and directly interacts with STIM1, recruiting it to ER-PM junctions independently of ER-PM membrane tethering, thereby supporting CRAC channel (Orai1-STIM1) activation and store-operated Ca2+ entry in Jurkat T cells but not in HeLa cells.\",\n      \"method\": \"CRISPR/siRNA knockdown, co-immunoprecipitation (E-Syt2S–STIM1), Ca2+ imaging (SOCE measurement), Orai1-STIM1 clustering imaging, isoform-specific expression analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional SOCE assay, cell-type specificity established, single lab\",\n      \"pmids\": [\"32879390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In C. elegans, ESYT-2 colocalizes with junctophilin JPH-1 at membrane contact sites in neurons. Genetic double-mutant analysis shows that jph-1 and esyt-2 null mutants display mutual suppression of aldicarb response, indicating that JPH-1 and ESYT-2 have antagonistic roles in neuromuscular synaptic transmission.\",\n      \"method\": \"Fluorescence localization studies, genetic epistasis (double-mutant analysis), aldicarb sensitivity assay in C. elegans\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with defined physiological readout (aldicarb assay), colocalization; C. elegans ortholog study\",\n      \"pmids\": [\"33871019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"E-Syt2 reduces plasma membrane DAG levels in resting T cells, thereby downmodulating T-cell receptor signaling, cytotoxicity, degranulation, and cytokine production. Upon TCR stimulation, both E-Syt1 and E-Syt2 negatively control TCR signaling through DAG reduction at the PM.\",\n      \"method\": \"Knockdown/deletion of E-Syt2 (and E-Syt1) in T cells, DAG biosensor imaging, TCR signaling readouts (cytokine production, degranulation, cytotoxicity assays)\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with lipid biosensor and multiple functional T-cell assays, single lab\",\n      \"pmids\": [\"38177911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ESYT2 forms a multimeric complex with ESYT1 and VAPB at lipid droplet–mitochondria–ER contact sites. Deletion of ESYT2 limits lipid droplet-derived fatty acid oxidation, depletes TCA cycle metabolites, remodels the cellular lipidome, and induces lipotoxic stress; these findings were recapitulated in Esyt2-deficient mice.\",\n      \"method\": \"Proximity-dependent biotinylation (BioID), high-resolution imaging, ESYT2 deletion (cell lines and mice), fatty acid oxidation assays, metabolomics (TCA cycle metabolites), lipidomics\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proximity proteomics plus KO in cells and mice, multiple orthogonal functional readouts (metabolomics, lipidomics, oxidation assays), replicated in vivo\",\n      \"pmids\": [\"40032835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"E-Syt2 dissociates the ANO1-VAPA interaction at STIM1 ER-PM junctions and forms an ANO1-IRBIT-E-Syt2-AC6-AKAP11-PKA complex that phosphorylates ANO1 at S221, markedly reducing ANO1 Ca2+ affinity. These effects are primarily mediated by E-Syt2 reciprocally regulating junctional PI(4)P, PI(4,5)P2, and PtdSer levels.\",\n      \"method\": \"Co-immunoprecipitation (complex assembly), phosphorylation site mutagenesis (S221), Ca2+ affinity measurements for ANO1, phosphoinositide biosensors, knockdown and rescue experiments\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP complex characterization, site-directed mutagenesis, functional Ca2+ affinity assay, single lab\",\n      \"pmids\": [\"40204782\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ESYT2 is an ER-resident membrane protein that tethers the ER to the plasma membrane via PI(4,5)P2-dependent C2 domain interactions (requiring its C2C domain for PM targeting and C2A domain for Ca2+-dependent phospholipid binding), forms heteromeric complexes with E-Syt1 and E-Syt3, and transfers glycerophospholipids between membranes via a hydrophobic channel in its SMP domain; at ER-PM contact sites it positions Sac1 to regulate PM phosphoinositide homeostasis (PI(4)P/PI(4,5)P2), reduces plasma membrane DAG to modulate TCR signaling, recruits STIM1 (via its short isoform) to support CRAC channel activation in T cells, and forms a complex with ESYT1 and VAPB at lipid droplet–mitochondria–ER contacts to enable fatty acid oxidation; it also functions as an endocytic adaptor for activated FGFR1 by interacting with Adaptin-2 and PAK1 to regulate actin dynamics and ERK signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ESYT2 (E-Syt2) is an ER-resident transmembrane protein that tethers the endoplasmic reticulum to the plasma membrane and acts as a lipid transfer and signaling platform at membrane contact sites [#4, #6]. It is anchored to the ER by its transmembrane domain and engages the plasma membrane through its C-terminal C2 domains: the C2C domain serves as a PI(4,5)P2-dependent PM-targeting motif, while the C2A domain binds phospholipids in a Ca2+-dependent manner, with structural and single-molecule studies establishing a rigid V-shaped tandem-C2 architecture and quantifying its calcium-tuned membrane affinity [#0, #5, #10]. E-Syt2 forms heteromeric complexes with E-Syt1 and E-Syt3, and its dimeric SMP domain folds into a TULIP-superfamily hydrophobic channel that binds and transfers glycerophospholipids between apposed membranes [#4, #6]. At ER-PM junctions it controls phosphoinositide homeostasis by positioning the lipid phosphatase Sac1 to limit PM PI(4)P, a coupling that is dynamically regulated by GPCR-driven PI(4,5)P2 depletion and by upstream control of PM PI(4,5)P2 through RASSF4-ARF6-PIP5K signaling [#8, #11]. Through these lipid functions E-Syt2 shapes signaling at the PM: it lowers plasma membrane DAG to downmodulate T-cell receptor signaling, and a short isoform recruits STIM1 to support CRAC-channel-mediated store-operated Ca2+ entry in T cells [#16, #14]. Beyond ER-PM contacts, E-Syt2 forms a complex with E-Syt1 and VAPB at lipid droplet-mitochondria-ER contacts to enable fatty acid oxidation, with its deletion causing lipotoxic stress and TCA-cycle metabolite depletion in cells and mice [#17]. Independently, E-Syt2 functions as an endocytic adaptor for activated FGFR1, interacting with the receptor through a TM-adjacent sequence and with Adaptin-2 and PAK1 to couple receptor endocytosis to actin dynamics and ERK signaling [#2, #3, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established the domain logic of E-Syt2 membrane engagement, showing how a multi-C2 protein could be targeted to the plasma membrane and respond to calcium.\",\n      \"evidence\": \"Recombinant Ca2+-dependent phospholipid binding assays plus domain-deletion localization of myc-tagged constructs\",\n      \"pmids\": [\"17360437\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not place the protein at ER-PM contacts or define a cellular function\", \"ER anchoring versus PM targeting roles not yet separated\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Quantified the calcium responsiveness of the C2 region, showing Ca2+-triggered conformational change and multimerization in vitro.\",\n      \"evidence\": \"SAXS of the recombinant soluble multi-C2 region with quantitative Ca2+ binding (~100 µM)\",\n      \"pmids\": [\"18977228\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Solution behavior not linked to membrane tethering in cells\", \"Functional consequence of multimerization undefined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Revealed an unexpected role for E-Syt2 as an endocytic adaptor required for FGF receptor internalization and downstream ERK-driven mesoderm induction.\",\n      \"evidence\": \"Morpholino depletion in Xenopus, reciprocal Co-IP with FGFR and Adaptin-2, Ras/ERK epistasis and rescue\",\n      \"pmids\": [\"20833364\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship between endocytic adaptor role and later contact-site/lipid functions unresolved\", \"Whether this role operates in mammalian somatic cells not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified PAK1 as a direct C2C-domain partner, linking E-Syt2 to suppression of actin polymerization and to FGFR1-associated signaling complexes.\",\n      \"evidence\": \"Co-IP with C2C domain mapping, PAK1 activation and actin polymerization assays, FGFR1 complex analysis\",\n      \"pmids\": [\"23213466\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab without reciprocal in vivo validation\", \"Integration with lipid-tethering activity not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined E-Syt2 (with E-Syt3) as a constitutive PI(4,5)P2-dependent ER-PM tether that forms Ca2+-regulated heteromers with E-Syt1, establishing its core contact-site function.\",\n      \"evidence\": \"ER-PM contact imaging, PI(4,5)P2 manipulation, Ca2+ imaging, Co-IP, siRNA, STIM1/Orai1 epistasis\",\n      \"pmids\": [\"23791178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of lipid handling at contacts not yet shown\", \"Showed E-Syt contacts dispensable for SOCE, leaving downstream lipid roles open\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Provided atomic detail of the tandem C2A-C2B module, showing a rigid Ca2+-independent architecture with Ca2+ binding confined to C2A.\",\n      \"evidence\": \"X-ray crystallography with and without Ca2+ and NMR Ca2+-binding analysis\",\n      \"pmids\": [\"24373768\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not include the SMP domain or define lipid transfer\", \"Membrane-bound conformation not resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated that E-Syt2 is a lipid transfer protein, with a dimeric SMP β-barrel forming a hydrophobic channel that captures glycerophospholipids.\",\n      \"evidence\": \"2.44 Å crystal structure of SMP-C2A-C2B and mass spectrometry of channel-bound lipids\",\n      \"pmids\": [\"24847877\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Directionality and physiological lipid cargo in cells not established\", \"Coupling of transfer to contact-site dynamics undefined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mapped the topology and FGFR1-binding determinants of E-Syt2, showing TM-domain ER targeting, TM-adjacent homodimerization, and conformation-dependent FGFR1 recognition.\",\n      \"evidence\": \"Co-IP, systematic domain mutants, localization studies, kinase-dead receptor analysis\",\n      \"pmids\": [\"25922075\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab Co-IP-based interaction mapping\", \"Functional consequence in mammalian FGFR signaling not quantified here\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected E-Syt2 tethering to PM phosphoinositide homeostasis by showing it positions Sac1 to control PI(4)P, with junctions dynamically remodeled by GPCR and SOCE signaling.\",\n      \"evidence\": \"Live-cell imaging of E-Syt2/Sac1 colocalization, phosphoinositide biosensors, GPCR/SOCE manipulation, siRNA\",\n      \"pmids\": [\"27044890\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct physical Sac1-E-Syt2 interaction not biochemically resolved\", \"Quantitative contribution to total PM PI(4)P turnover unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed splice-isoform-specific cellular functions, with short and long E-Syt2 variants differentially controlling actin distribution and endocytosis.\",\n      \"evidence\": \"Variant-specific siRNA, actin imaging and endocytosis assays in lung cancer cells\",\n      \"pmids\": [\"27555542\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular mechanism beyond variant specificity\", \"Isoform-specific partners not identified here\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed E-Syt2 tethering downstream of RASSF4-ARF6-PIP5K control of PM PI(4,5)P2, defining an upstream regulatory input.\",\n      \"evidence\": \"RASSF4 siRNA, ER-PM contact imaging, PI(4,5)P2 biosensor, ARF6 activity assay, Co-IP\",\n      \"pmids\": [\"28600435\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct versus indirect effect of RASSF4 on E-Syt2 not separated\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided quantitative single-molecule force measurements of C2-domain membrane binding, mechanically validating bilayer- and Ca2+-tuned tethering.\",\n      \"evidence\": \"Optical-tweezers force spectroscopy across defined bilayers and Ca2+ conditions\",\n      \"pmids\": [\"29083305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconstituted system; in-cell forces not measured\", \"Does not address lipid transfer flux\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified UBQLN1 as a stabilizer of ESYT2 protein levels, implicating proteostasis control of the tether.\",\n      \"evidence\": \"Co-IP with UBQLN1 domain mapping and Western blot stability assays\",\n      \"pmids\": [\"28075048\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP with ESYT2 as one of several substrates, not independently confirmed\", \"Physiological context of ESYT2 turnover undefined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked E-Syt2 to SNARE machinery and neuronal morphogenesis, showing it stabilizes Sec22b-Stx1 at ER-PM contacts and promotes axonal filopodia in a lipid-transfer- and tethering-dependent manner.\",\n      \"evidence\": \"Co-IP with Sec22b longin-domain mapping, E-Syt2 mutant analysis, neurite morphology quantification, neurotoxin epistasis\",\n      \"pmids\": [\"32843578\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Mechanistic coupling of lipid transfer to SNARE function not fully defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed the short E-Syt2 isoform recruits STIM1 to ER-PM junctions to support CRAC channel activation, a tethering-independent, cell-type-specific role in T cells.\",\n      \"evidence\": \"CRISPR/siRNA knockdown, E-Syt2S-STIM1 Co-IP, SOCE Ca2+ imaging, Orai1-STIM1 clustering in Jurkat vs HeLa\",\n      \"pmids\": [\"32879390\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation with earlier finding that E-Syt contacts are dispensable for SOCE\", \"Structural basis of E-Syt2S-STIM1 interaction unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Used the C. elegans ortholog to show ESYT-2 acts antagonistically with junctophilin JPH-1 at neuronal contact sites in synaptic transmission.\",\n      \"evidence\": \"Colocalization and genetic double-mutant epistasis with aldicarb assay in C. elegans\",\n      \"pmids\": [\"33871019\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of JPH-1 antagonism unknown\", \"Conservation of this interaction in mammals not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined a signaling output of E-Syt2 lipid control, showing it lowers PM DAG to negatively regulate T-cell receptor signaling and effector function.\",\n      \"evidence\": \"E-Syt2/E-Syt1 knockdown/deletion in T cells, DAG biosensor imaging, cytokine/degranulation/cytotoxicity assays\",\n      \"pmids\": [\"38177911\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct enzymatic link between E-Syt2 and DAG metabolism not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended E-Syt2 function to lipid droplet-mitochondria-ER contacts, showing it forms a complex with E-Syt1 and VAPB that supports fatty acid oxidation and protects against lipotoxic stress in vivo.\",\n      \"evidence\": \"BioID proximity proteomics, high-resolution imaging, ESYT2 KO in cells and mice, FAO assays, metabolomics and lipidomics\",\n      \"pmids\": [\"40032835\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether lipid transfer activity directly drives FAO at these contacts not isolated\", \"Mechanism of VAPB-dependent contact assembly undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated E-Syt2 in chloride channel regulation, showing it remodels a junctional ANO1 signaling complex and reduces ANO1 Ca2+ affinity via PKA-dependent phosphorylation.\",\n      \"evidence\": \"Co-IP complex characterization, S221 phosphosite mutagenesis, ANO1 Ca2+ affinity measurements, phosphoinositide biosensors, knockdown/rescue\",\n      \"pmids\": [\"40204782\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Direct versus phosphoinositide-mediated contribution of E-Syt2 not fully separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How E-Syt2's distinct activities—lipid transfer, ER-PM tethering, endocytic adaptation, and isoform-specific signaling—are coordinately deployed across cell types and contact-site types remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Directionality and in vivo cargo selectivity of SMP-mediated lipid transfer unknown\", \"No human disease link established in the corpus\", \"Relative contributions of long versus short isoforms across tissues uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 6, 10]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [6, 13]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 8, 14]},\n      {\"term_id\": \"GO:0005509\", \"supporting_discovery_ids\": [0, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [4, 7, 8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 4, 8]},\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [17]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [8, 17]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 16]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [14, 16]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 9]}\n    ],\n    \"complexes\": [\n      \"E-Syt1/E-Syt2/E-Syt3 heteromer\",\n      \"ESYT2-ESYT1-VAPB lipid droplet-mitochondria-ER complex\",\n      \"ANO1-IRBIT-E-Syt2-AC6-AKAP11-PKA complex\",\n      \"E-Syt2-PAK1-FGFR1 complex\"\n    ],\n    \"partners\": [\n      \"ESYT1\",\n      \"ESYT3\",\n      \"STIM1\",\n      \"PAK1\",\n      \"FGFR1\",\n      \"SEC22B\",\n      \"VAPB\",\n      \"RASSF4\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}