{"gene":"IQSEC1","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":2007,"finding":"GEP100/BRAG2, via its pleckstrin homology (PH) domain, directly binds to Tyr1068/1086-phosphorylated EGFR and thereby activates Arf6 to induce breast cancer cell invasion.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression in MCF7 cells, in vivo metastasis assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding demonstrated, functional rescue/knockdown, replicated across multiple labs in subsequent papers","pmids":["18084281"],"is_preprint":false},{"year":2006,"finding":"BRAG2 (GEP100) activates Arf6 in vivo and controls endocytosis of β1 integrins; siRNA depletion of BRAG2 causes accumulation of β1 integrin on the cell surface and enhanced cell adhesion/spreading on fibronectin.","method":"siRNA knockdown, flow cytometry for surface integrin, cell spreading assay on fibronectin","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KD phenotype replicated and extended by multiple subsequent independent studies","pmids":["16461286"],"is_preprint":false},{"year":2006,"finding":"GEP100/BRAG2 interacts with α-catenin (identified by yeast two-hybrid and confirmed by co-immunoprecipitation of endogenous proteins), and α-catenin enhances GEP100-stimulated GTPγS binding by ARF6 in vitro. Depletion of GEP100 by siRNA increases E-cadherin levels ~3-fold and blocks HGF-induced E-cadherin redistribution. Overexpression of GEP100 (but not its GEF-inactive mutants) markedly reduces F-actin.","method":"Yeast two-hybrid, co-immunoprecipitation, siRNA knockdown, in vitro GTPγS binding assay, overexpression with GEF-inactive mutants","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro enzymatic assay combined with co-IP, mutagenesis, and cellular knockdown in a single study","pmids":["16807291"],"is_preprint":false},{"year":2010,"finding":"BRAG2 directly interacts with the GluA2 subunit of AMPA receptors; BRAG2-mediated Arf6 activation is controlled by ligand-binding and tyrosine phosphorylation of GluA2 and is required for clathrin-mediated endocytosis of synaptic AMPA receptors during LTD. Targeted deletion of BRAG2 in CA1 pyramidal neurons prevents LTD.","method":"Co-immunoprecipitation (direct GluA2-BRAG2 interaction), conditional neuron-specific knockout, electrophysiology (LTD recordings), pharmacological blockade of GluA2-BRAG2 interaction","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, conditional KO with defined electrophysiological phenotype, multiple orthogonal methods","pmids":["20547133"],"is_preprint":false},{"year":2012,"finding":"In addition to Arf6, endogenous BRAG2 also activates class II Arfs, specifically Arf5, at clathrin-coated pits; it is Arf5 (not Arf6) that mediates β1 integrin internalization via clathrin-mediated endocytosis. BRAG2 binds clathrin and the AP-2 adaptor complex.","method":"siRNA knockdown, co-immunoprecipitation of BRAG2 with clathrin/AP-2, Arf activity assays, rapid-cycling Arf5 rescue, fluorescence microscopy of clathrin-coated pits","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, Arf activation assays, epistasis rescue, localization), single lab","pmids":["22815487"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of Arf1-BRAG2 complex reveals an atypical PH domain constitutively anchored to the Sec7 domain; the PH domain potentiates nucleotide exchange ~2,000-fold by cumulative conformational and membrane-targeting contributions, and restricts BRAG2 activity to negatively charged membranes without phosphoinositide specificity via a positively charged surface peripheral to the canonical lipid-binding pocket.","method":"X-ray crystallography, quantitative exchange activity reconstituted on membranes, mutagenesis","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus in vitro reconstitution with quantitative analysis and mutagenesis","pmids":["24058294"],"is_preprint":false},{"year":2012,"finding":"Brag2 PH domain allosterically stimulates nucleotide exchange: PIP2 binding to the PH domain decreases Km and increases kcat; this effect requires the PH domain and the N-terminus of Arf and is largely independent of Arf myristoylation. The interdomain linker between Sec7 and PH domains contributes to activity.","method":"In vitro single-turnover and substrate-saturation kinetics with myristoylated Arf1·GDP, PH domain deletion mutants, NMR structural analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinetic assay with mutagenesis and NMR, single lab, multiple orthogonal approaches","pmids":["22613714"],"is_preprint":false},{"year":2011,"finding":"Overexpressed Her2, when autonomously phosphorylated at Tyr1139/Tyr1196, recruits GEP100 via the GEP100 PH domain to activate Arf6 and induce invasion independently of external ligands, analogous to the EGFR-GEP100 mechanism.","method":"Co-immunoprecipitation, PH domain deletion mutants, siRNA knockdown, invasion assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping plus functional invasion rescue, single lab","pmids":["21966491"],"is_preprint":false},{"year":2011,"finding":"Upon Sema3E activation, Plexin-D1 recruits phosphatidylinositol-4-phosphate 5-kinase; the resulting PI(4,5)P2 binds the PH domain of GEP100/BRAG2, enhancing its GEF activity toward Arf6 to disassemble integrin-mediated focal adhesions in endothelial cells.","method":"siRNA knockdown, GEF activity assays, PI(4,5)P2 lipid-binding assays, cell adhesion/collapse assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical lipid-binding, GEF activity assays, and cellular phenotypes in single lab","pmids":["21795701"],"is_preprint":false},{"year":2011,"finding":"VEGFR2, like EGFR, recruits GEP100 to activate Arf6 in HUVECs, and the GEP100-Arf6-AMAP1-cortactin pathway is essential for VEGF-induced angiogenic cell migration, tubular formation, VE-cadherin endocytosis, and increased permeability.","method":"siRNA knockdown, Co-immunoprecipitation of VEGFR2-GEP100, cell migration/tube formation assays, in vivo angiogenesis models","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional assays in vitro and in vivo, single lab","pmids":["21858086"],"is_preprint":false},{"year":2010,"finding":"GEP100 regulates phagocytosis of IgG-coated beads and serum-treated zymosan in monocyte-macrophage cells in an ARF6-dependent manner, requiring its Sec7 (ARF-activating) domain; GEP100 and ARF6 co-localize around internalized particles, and constitutively active ARF6Q67N rescues phagocytosis in GEP100-depleted cells.","method":"siRNA/shRNA depletion, rescue with ARF6 constitutively active/dominant-negative mutants, immunofluorescence microscopy, F-actin staining","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (rescue with ARF6 mutants), co-localization, functional phagocytosis assay, single lab","pmids":["20601426"],"is_preprint":false},{"year":2020,"finding":"IQSec1 forms a complex with the lipid transfer protein ORP3; Ca2+ influx via STIM1/Orai1 channels near focal adhesions triggers PKC-dependent translocation of this IQSec1-ORP3 complex to ER/plasma membrane contact sites adjacent to focal adhesions, where IQSec1 is allosterically activated by ORP3 to activate Arf5 (not Arf6), driving focal adhesion disassembly. ORP3-mediated PI4P extraction from the PM is also required for FA turnover.","method":"Co-immunoprecipitation, siRNA knockdown, live-cell calcium imaging, immunofluorescence, Arf5 activity assays, lipid transfer assays, FA disassembly quantification","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, Arf activation, lipid transfer, live imaging, KD phenotype), single lab but rigorous and comprehensive","pmids":["32234213"],"is_preprint":false},{"year":2019,"finding":"Small molecule Bragsin inhibits BRAG2-mediated Arf GTPase activation in vitro in a membrane-dependent, non-competitive manner; crystal structure reveals Bragsin binds at the interface between the BRAG2 PH domain and the lipid bilayer, preventing BRAG2 from activating lipidated Arf. In cells, Bragsin affects the trans-Golgi network in a BRAG2- and Arf-dependent manner.","method":"In vitro Arf GEF activity assays with membranes, X-ray crystallography of BRAG2-Bragsin complex, structure-activity relationship, cellular TGN disruption assays","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with in vitro reconstitution, SAR, and cellular functional validation","pmids":["30742123"],"is_preprint":false},{"year":2016,"finding":"In mature hippocampal cultures, GluN2A-containing NMDARs recruit BRAG2 to activate Arf6 upon NMDA stimulation; in young cultures, tonic Arf6 activation is mediated by GluN2B-BRAG1 instead. Knockdown of BRAG2 during postnatal weeks 4–5 reduces AMPAR miniature event frequency and quantal sizes of both AMPAR and NMDAR currents at Schaffer collateral synapses.","method":"Biochemical Arf6 activity assays in cortical cultures, shRNA knockdown, patch-clamp electrophysiology in acute hippocampal slices","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophysiology with KD and Arf6 activity assays, single lab","pmids":["26884337"],"is_preprint":false},{"year":2007,"finding":"BRAG2 cycles between the cytoplasm and nucleus in a CRM1/exportin1-dependent manner. Depletion of BRAG2 by RNAi increases the number of Cajal bodies and alters nucleolar structure (less focal fibrillarin staining). Ectopic expression of nuclear GTPase PIKE/AGAP2 causes both BRAG2 and coilin to accumulate in nucleoli, resulting in fibrillarin redistribution to the nucleolar periphery; neither PIKE GTPase activity nor BRAG2 nucleotide exchange activity is required for this nucleolar concentration.","method":"RNAi knockdown, CRM1 inhibition (leptomycin B), immunofluorescence microscopy, overexpression with catalytically inactive mutants","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — nuclear localization with CRM1 dependence demonstrated, functional consequence (nucleolar architecture) with mutagenesis, single lab","pmids":["17461797"],"is_preprint":false},{"year":2006,"finding":"Overexpression of GEP100/BRAG2a in macrophage-like cells induces apoptosis (chromatin condensation, annexin V staining, TUNEL); a Sec7-domain deletion mutant lacking ARF-activating ability still induces apoptosis to the same level, suggesting the pro-apoptotic function is independent of ARF activation. GEP100 silencing suppresses TNF-α-induced apoptosis.","method":"Overexpression with Sec7 deletion mutant, Annexin V staining, TUNEL assay, morphological analysis, siRNA knockdown","journal":"Journal of leukocyte biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method per endpoint, mechanism of Sec7-independent apoptosis not elucidated beyond domain deletion","pmids":["16877676"],"is_preprint":false},{"year":2019,"finding":"Biallelic loss-of-function variants in IQSEC1 (p.Thr343Met and p.Arg321Gln) cause intellectual disability and developmental delay; in Drosophila, these variants fail to rescue embryonic lethality caused by loss of the IQSEC1 ortholog schizo, while the reference cDNA does rescue, confirming loss-of-function. Conditional deletion of Iqsec1 in mouse cortical neurons leads to increased density of immature dendritic spines.","method":"Drosophila genetic complementation/rescue assay, mouse conditional knockout (cortical neurons), dendritic spine morphology analysis","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation in Drosophila with multiple variants, conditional KO in mouse with morphological readout, two orthogonal model organisms","pmids":["31607425"],"is_preprint":false},{"year":2024,"finding":"Iqsec1 knockout mice (CRISPR/Cas9) exhibit embryonic lethality (~99%); electron microscopy shows that Iqsec1-/- embryos at E8.5 lack large apical vacuoles in visceral endoderm cells of the yolk sac, indicating a critical role for IQSEC1 in membrane trafficking in visceral endoderm during embryogenesis.","method":"CRISPR/Cas9 knockout, electron microscopy of embryonic visceral endoderm","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined ultrastructural phenotype, single lab","pmids":["39561249"],"is_preprint":false},{"year":2013,"finding":"BRAG2 localizes to postsynaptic processes of bipolar dyads in the inner plexiform layer of the mouse retina and co-localizes preferentially with PSD-95 and AMPARs, as demonstrated by immunoelectron microscopy and double immunostaining; distinct from BRAG3 which localizes to inhibitory synapses.","method":"Immunohistochemistry, immunoelectron microscopy, double immunofluorescence","journal":"The Journal of comparative neurology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct subcellular localization by immunoelectron microscopy, replicated across retinal layers","pmids":["22886754"],"is_preprint":false}],"current_model":"IQSEC1/BRAG2/GEP100 is a guanine nucleotide exchange factor (GEF) for Arf5 and Arf6 whose PH domain is constitutively coupled to the catalytic Sec7 domain, allosterically stimulated ~2,000-fold by membrane phosphoinositides (especially PI(4,5)P2); it is recruited to activated receptor tyrosine kinases (EGFR, Her2, VEGFR2) via its PH domain binding to phosphotyrosine residues, triggering Arf6 activation and downstream invasion/metastasis signaling, and it acts at clathrin-coated pits to activate Arf5 for β1-integrin endocytosis; at synapses it directly interacts with GluA2-containing AMPA receptors, and BRAG2-mediated Arf6 activation is the convergent step required for LTD-associated AMPA receptor internalization; it also forms a Ca2+-activated complex with ORP3 that translocates to ER-PM contact sites to activate Arf5 and drive focal adhesion disassembly during cell migration, while whole-animal Iqsec1 knockout causes embryonic lethality due to defective membrane trafficking in visceral endoderm."},"narrative":{"mechanistic_narrative":"IQSEC1 (BRAG2/GEP100) is a guanine nucleotide exchange factor that activates ADP-ribosylation factor GTPases (Arf6 and the class II Arf5) on negatively charged membranes to drive receptor and adhesion-molecule trafficking across diverse cellular contexts [PMID:16461286, PMID:22815487, PMID:24058294]. Its catalytic Sec7 domain is constitutively coupled to an atypical PH domain that potentiates nucleotide exchange ~2,000-fold; PI(4,5)P2 binding to the PH domain lowers Km and raises kcat, restricting activity to phosphoinositide-rich membranes [PMID:24058294, PMID:22613714]. This PH module is the principal regulatory hub: it docks onto phosphotyrosine residues of activated receptor tyrosine kinases — EGFR, Her2, and VEGFR2 — to trigger Arf6 activation and downstream invasion, angiogenic migration, and cadherin endocytosis [PMID:18084281, PMID:21966491, PMID:21858086], and it is engaged by Plexin-D1-generated PI(4,5)P2 to disassemble focal adhesions in endothelial cells [PMID:21795701]. IQSEC1 controls integrin trafficking by binding clathrin and the AP-2 adaptor at coated pits, where it activates Arf5 to internalize β1 integrin and where it interacts with α-catenin to modulate E-cadherin and F-actin [PMID:22815487, PMID:16807291]. A Ca2+-dependent complex with the lipid transfer protein ORP3 translocates to ER–plasma membrane contact sites near focal adhesions, where ORP3 allosterically activates IQSEC1 toward Arf5 to drive focal adhesion disassembly during migration [PMID:32234213]. At synapses, IQSEC1 directly binds the GluA2 subunit of AMPA receptors and, downstream of NMDA receptor activation, provides the Arf6 activation required for clathrin-mediated AMPA receptor internalization and long-term depression [PMID:20547133, PMID:26884337]. Biallelic loss-of-function variants in IQSEC1 cause intellectual disability and developmental delay [PMID:31607425], and whole-animal knockout is embryonic lethal owing to defective membrane trafficking in visceral endoderm [PMID:39561249].","teleology":[{"year":2006,"claim":"Establishing that IQSEC1/BRAG2 is a physiological Arf6 GEF answered whether this protein actively controls membrane traffic, by linking it to β1 integrin surface levels and cell adhesion.","evidence":"siRNA depletion with surface integrin flow cytometry and fibronectin spreading assays","pmids":["16461286"],"confidence":"High","gaps":["Did not define the membrane compartment or upstream receptor triggering Arf6 activation","Did not distinguish Arf6 from other Arf isoforms"]},{"year":2006,"claim":"Identification of α-catenin as a binding partner and stimulator of GEP100 GEF activity connected Arf6 activation to cadherin-based adhesion and the actin cytoskeleton.","evidence":"Yeast two-hybrid, co-IP of endogenous proteins, in vitro GTPγS binding, and siRNA with GEF-inactive mutants","pmids":["16807291"],"confidence":"High","gaps":["Mechanism by which α-catenin enhances exchange not structurally defined","Did not establish whether E-cadherin effects are direct or downstream of Arf6"]},{"year":2007,"claim":"Demonstrating that the PH domain binds phosphotyrosine-EGFR established the direct mechanism coupling receptor tyrosine kinase signaling to Arf6 activation and cancer invasion.","evidence":"Co-IP, siRNA, overexpression in MCF7 cells, and in vivo metastasis assay","pmids":["18084281"],"confidence":"High","gaps":["Did not resolve how phosphotyrosine binding by an atypical PH domain coexists with lipid binding","Generality across other RTKs not yet tested"]},{"year":2007,"claim":"Discovery of CRM1-dependent nucleocytoplasmic shuttling and effects on Cajal body/nucleolar architecture raised the possibility of a nuclear function distinct from membrane trafficking.","evidence":"RNAi, leptomycin B inhibition, immunofluorescence, and catalytically inactive mutant overexpression","pmids":["17461797"],"confidence":"Medium","gaps":["No molecular mechanism linking BRAG2 to nucleolar/Cajal body structure","GEF activity dispensable, so the relevant biochemical activity is unknown"]},{"year":2010,"claim":"Identifying a direct GluA2-BRAG2 interaction and a conditional-KO LTD deficit defined IQSEC1 as the convergent Arf6 activator required for synaptic AMPA receptor internalization.","evidence":"Reciprocal co-IP, CA1-specific conditional knockout, LTD electrophysiology, and pharmacological interaction blockade","pmids":["20547133"],"confidence":"High","gaps":["Did not identify the upstream receptor coupling synaptic activity to BRAG2","Did not address whether class II Arfs also contribute at synapses"]},{"year":2010,"claim":"Linking GEP100 to Fcγ-mediated phagocytosis extended its Arf6-GEF role into innate immune membrane internalization.","evidence":"siRNA/shRNA depletion, rescue with ARF6 mutants, immunofluorescence, and F-actin staining in macrophages","pmids":["20601426"],"confidence":"Medium","gaps":["Upstream signal recruiting GEP100 to phagocytic cups unknown","Single-lab study without structural detail"]},{"year":2011,"claim":"Showing Her2 and VEGFR2 also recruit GEP100 via the PH domain generalized the phosphotyrosine-PH coupling mechanism beyond EGFR to other RTK-driven invasion and angiogenesis programs.","evidence":"Co-IP, PH domain deletion mutants, siRNA, invasion and tube formation assays, in vivo angiogenesis models","pmids":["21966491","21858086"],"confidence":"Medium","gaps":["Single-lab studies for each receptor","Did not quantify relative affinities across receptors"]},{"year":2011,"claim":"Demonstrating Plexin-D1-generated PI(4,5)P2 binding to the PH domain identified a lipid-driven route to GEF activation and focal adhesion disassembly distinct from phosphotyrosine docking.","evidence":"siRNA, GEF activity assays, PI(4,5)P2 lipid-binding and cell collapse assays in endothelial cells","pmids":["21795701"],"confidence":"Medium","gaps":["Single-lab biochemical study","Did not reconcile lipid versus phosphotyrosine engagement of the same PH domain"]},{"year":2012,"claim":"Defining the biochemical basis of PH-domain allostery showed that PIP2 binding lowers Km and raises kcat through contributions requiring the Arf N-terminus and the interdomain linker.","evidence":"In vitro single-turnover and substrate-saturation kinetics with myristoylated Arf1, PH deletion mutants, and NMR","pmids":["22613714"],"confidence":"High","gaps":["Did not capture the full membrane-bound enzyme-substrate complex","Phosphoinositide specificity not fully resolved"]},{"year":2012,"claim":"Discovering that BRAG2 activates Arf5 at clathrin-coated pits and binds clathrin/AP-2 revealed that β1 integrin internalization is mediated by a class II Arf rather than Arf6.","evidence":"siRNA, co-IP with clathrin/AP-2, Arf activity assays, rapid-cycling Arf5 rescue, and coated-pit microscopy","pmids":["22815487"],"confidence":"High","gaps":["Determinants selecting Arf5 versus Arf6 in different contexts unclear","Did not map the clathrin/AP-2 binding interface"]},{"year":2013,"claim":"The Arf1-BRAG2 crystal structure explained the ~2,000-fold catalytic enhancement, showing an atypical PH domain constitutively anchored to Sec7 that targets activity to negatively charged membranes.","evidence":"X-ray crystallography with reconstituted quantitative exchange kinetics and mutagenesis","pmids":["24058294"],"confidence":"High","gaps":["Structure lacked the bound phosphoinositide that confers regulation in cells","Did not include receptor or partner proteins"]},{"year":2013,"claim":"Immunoelectron localization to postsynaptic bipolar dyad processes with PSD-95 and AMPARs confirmed a defined synaptic localization for BRAG2 distinct from BRAG3.","evidence":"Immunohistochemistry, immunoelectron microscopy, and double immunofluorescence in mouse retina","pmids":["22886754"],"confidence":"Medium","gaps":["Did not establish functional consequences at retinal synapses","Localization descriptive only"]},{"year":2016,"claim":"Mapping NMDA receptor subtype-specific recruitment showed GluN2A-NMDARs engage BRAG2 to activate Arf6, defining the upstream synaptic trigger and a developmental switch from a BRAG1 pathway.","evidence":"Arf6 activity assays in cortical cultures, shRNA knockdown, and patch-clamp electrophysiology in slices","pmids":["26884337"],"confidence":"Medium","gaps":["Molecular link between NMDAR activation and BRAG2 recruitment not defined","Single-lab electrophysiology"]},{"year":2019,"claim":"The Bragsin inhibitor and its co-crystal structure provided proof that targeting the PH-domain/membrane interface blocks activation of lipidated Arf, validating the membrane-coupled mechanism pharmacologically.","evidence":"In vitro membrane GEF assays, BRAG2-Bragsin crystallography, SAR, and cellular TGN disruption assays","pmids":["30742123"],"confidence":"High","gaps":["Cellular TGN role of BRAG2 not mechanistically detailed","Selectivity over other Arf GEFs not fully characterized"]},{"year":2019,"claim":"Identifying biallelic loss-of-function IQSEC1 variants in patients, validated by Drosophila rescue failure and mouse cortical KO spine phenotypes, established IQSEC1 as a cause of intellectual disability and developmental delay.","evidence":"Drosophila genetic complementation, mouse conditional knockout, and dendritic spine morphology analysis","pmids":["31607425"],"confidence":"Medium","gaps":["Did not connect human variants directly to a specific Arf-GEF deficit","Immature spine phenotype mechanism not resolved"]},{"year":2020,"claim":"Discovery of the Ca2+-triggered IQSec1-ORP3 complex at ER-PM contact sites defined a lipid-transfer-coupled mode of Arf5 activation driving focal adhesion disassembly.","evidence":"Co-IP, siRNA, live-cell calcium imaging, Arf5 activity and lipid transfer assays, and FA disassembly quantification","pmids":["32234213"],"confidence":"High","gaps":["How ORP3 allosterically activates IQSec1 structurally undefined","Single-lab study"]},{"year":2024,"claim":"CRISPR knockout demonstrating embryonic lethality with absent apical vacuoles in visceral endoderm established an essential developmental role in membrane trafficking.","evidence":"CRISPR/Cas9 knockout and electron microscopy of embryonic visceral endoderm","pmids":["39561249"],"confidence":"Medium","gaps":["Which Arf and cargo pathway underlies the visceral endoderm defect not defined","Single-lab ultrastructural study"]},{"year":null,"claim":"It remains unresolved how the single PH domain integrates competing inputs — receptor phosphotyrosine, phosphoinositides, and partner proteins like α-catenin and ORP3 — to select between Arf5 and Arf6 in a context-specific manner.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the membrane-bound enzyme with a phosphotyrosine receptor","Determinants of Arf5 versus Arf6 substrate selection unknown","Mechanism linking nuclear shuttling and Sec7-independent apoptosis to canonical GEF function undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[6,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,6,11]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,4,11]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[14]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[14]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[12]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[4,1,11]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,7,9]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[3,13]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[1,8,11]}],"complexes":["IQSEC1-ORP3 complex","AP-2 adaptor / clathrin coat"],"partners":["ARF6","ARF5","EGFR","GRIA2","ORP3","CTNNA1","VEGFR2","ERBB2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6DN90","full_name":"IQ motif and SEC7 domain-containing protein 1","aliases":["ADP-ribosylation factors guanine nucleotide-exchange protein 100","ADP-ribosylation factors guanine nucleotide-exchange protein 2","Brefeldin-resistant Arf-GEF 2 protein","BRAG2"],"length_aa":963,"mass_kda":108.3,"function":"Guanine nucleotide exchange factor for ARF1 and ARF6 (PubMed:11226253, PubMed:24058294). Guanine nucleotide exchange factor activity is enhanced by lipid binding (PubMed:24058294). Accelerates GTP binding by ARFs of all three classes. Guanine nucleotide exchange protein for ARF6, mediating internalization of beta-1 integrin (PubMed:16461286). Involved in neuronal development (Probable). In neurons, plays a role in the control of vesicle formation by endocytoc cargo. Upon long term depression, interacts with GRIA2 and mediates the activation of ARF6 to internalize synaptic AMPAR receptors (By similarity)","subcellular_location":"Cytoplasm; Nucleus; Postsynaptic density; Cytoplasmic vesicle, secretory vesicle, synaptic vesicle","url":"https://www.uniprot.org/uniprotkb/Q6DN90/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/IQSEC1","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ANKRD28","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/IQSEC1","total_profiled":1310},"omim":[{"mim_id":"618687","title":"INTELLECTUAL DEVELOPMENTAL DISORDER WITH SHORT STATURE AND BEHAVIORAL ABNORMALITIES; IDDSSBA","url":"https://www.omim.org/entry/618687"},{"mim_id":"610166","title":"IQ MOTIF- AND SEC7 DOMAIN-CONTAINING PROTEIN 1; IQSEC1","url":"https://www.omim.org/entry/610166"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Vesicles","reliability":"Additional"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/IQSEC1"},"hgnc":{"alias_symbol":["KIAA0763","GEP100","BRAG2","ARF-GEP100"],"prev_symbol":[]},"alphafold":{"accession":"Q6DN90","domains":[{"cath_id":"1.10.1000.11","chopping":"554-710","consensus_level":"high","plddt":95.9908,"start":554,"end":710},{"cath_id":"2.30.29.30","chopping":"719-880","consensus_level":"high","plddt":90.9696,"start":719,"end":880}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6DN90","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6DN90-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6DN90-F1-predicted_aligned_error_v6.png","plddt_mean":62.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=IQSEC1","jax_strain_url":"https://www.jax.org/strain/search?query=IQSEC1"},"sequence":{"accession":"Q6DN90","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6DN90.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6DN90/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6DN90"}},"corpus_meta":[{"pmid":"18084281","id":"PMC_18084281","title":"GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion.","date":"2007","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/18084281","citation_count":189,"is_preprint":false},{"pmid":"20547133","id":"PMC_20547133","title":"AMPA receptor signaling through BRAG2 and Arf6 critical for long-term synaptic depression.","date":"2010","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/20547133","citation_count":132,"is_preprint":false},{"pmid":"16461286","id":"PMC_16461286","title":"The Arf6 GEF GEP100/BRAG2 regulates cell adhesion by controlling endocytosis of beta1 integrins.","date":"2006","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/16461286","citation_count":101,"is_preprint":false},{"pmid":"19416474","id":"PMC_19416474","title":"The EGFR-GEP100-Arf6-AMAP1 signaling pathway specific to breast cancer invasion and metastasis.","date":"2009","source":"Traffic (Copenhagen, 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Depletion of GEP100 by siRNA increases E-cadherin levels ~3-fold and blocks HGF-induced E-cadherin redistribution. Overexpression of GEP100 (but not its GEF-inactive mutants) markedly reduces F-actin.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, siRNA knockdown, in vitro GTPγS binding assay, overexpression with GEF-inactive mutants\",\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 enzymatic assay combined with co-IP, mutagenesis, and cellular knockdown in a single study\",\n      \"pmids\": [\"16807291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"BRAG2 directly interacts with the GluA2 subunit of AMPA receptors; BRAG2-mediated Arf6 activation is controlled by ligand-binding and tyrosine phosphorylation of GluA2 and is required for clathrin-mediated endocytosis of synaptic AMPA receptors during LTD. Targeted deletion of BRAG2 in CA1 pyramidal neurons prevents LTD.\",\n      \"method\": \"Co-immunoprecipitation (direct GluA2-BRAG2 interaction), conditional neuron-specific knockout, electrophysiology (LTD recordings), pharmacological blockade of GluA2-BRAG2 interaction\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, conditional KO with defined electrophysiological phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"20547133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In addition to Arf6, endogenous BRAG2 also activates class II Arfs, specifically Arf5, at clathrin-coated pits; it is Arf5 (not Arf6) that mediates β1 integrin internalization via clathrin-mediated endocytosis. BRAG2 binds clathrin and the AP-2 adaptor complex.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation of BRAG2 with clathrin/AP-2, Arf activity assays, rapid-cycling Arf5 rescue, fluorescence microscopy of clathrin-coated pits\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, Arf activation assays, epistasis rescue, localization), single lab\",\n      \"pmids\": [\"22815487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of Arf1-BRAG2 complex reveals an atypical PH domain constitutively anchored to the Sec7 domain; the PH domain potentiates nucleotide exchange ~2,000-fold by cumulative conformational and membrane-targeting contributions, and restricts BRAG2 activity to negatively charged membranes without phosphoinositide specificity via a positively charged surface peripheral to the canonical lipid-binding pocket.\",\n      \"method\": \"X-ray crystallography, quantitative exchange activity reconstituted on membranes, mutagenesis\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus in vitro reconstitution with quantitative analysis and mutagenesis\",\n      \"pmids\": [\"24058294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Brag2 PH domain allosterically stimulates nucleotide exchange: PIP2 binding to the PH domain decreases Km and increases kcat; this effect requires the PH domain and the N-terminus of Arf and is largely independent of Arf myristoylation. The interdomain linker between Sec7 and PH domains contributes to activity.\",\n      \"method\": \"In vitro single-turnover and substrate-saturation kinetics with myristoylated Arf1·GDP, PH domain deletion mutants, NMR structural analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinetic assay with mutagenesis and NMR, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"22613714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Overexpressed Her2, when autonomously phosphorylated at Tyr1139/Tyr1196, recruits GEP100 via the GEP100 PH domain to activate Arf6 and induce invasion independently of external ligands, analogous to the EGFR-GEP100 mechanism.\",\n      \"method\": \"Co-immunoprecipitation, PH domain deletion mutants, siRNA knockdown, invasion assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping plus functional invasion rescue, single lab\",\n      \"pmids\": [\"21966491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Upon Sema3E activation, Plexin-D1 recruits phosphatidylinositol-4-phosphate 5-kinase; the resulting PI(4,5)P2 binds the PH domain of GEP100/BRAG2, enhancing its GEF activity toward Arf6 to disassemble integrin-mediated focal adhesions in endothelial cells.\",\n      \"method\": \"siRNA knockdown, GEF activity assays, PI(4,5)P2 lipid-binding assays, cell adhesion/collapse assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical lipid-binding, GEF activity assays, and cellular phenotypes in single lab\",\n      \"pmids\": [\"21795701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"VEGFR2, like EGFR, recruits GEP100 to activate Arf6 in HUVECs, and the GEP100-Arf6-AMAP1-cortactin pathway is essential for VEGF-induced angiogenic cell migration, tubular formation, VE-cadherin endocytosis, and increased permeability.\",\n      \"method\": \"siRNA knockdown, Co-immunoprecipitation of VEGFR2-GEP100, cell migration/tube formation assays, in vivo angiogenesis models\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional assays in vitro and in vivo, single lab\",\n      \"pmids\": [\"21858086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"GEP100 regulates phagocytosis of IgG-coated beads and serum-treated zymosan in monocyte-macrophage cells in an ARF6-dependent manner, requiring its Sec7 (ARF-activating) domain; GEP100 and ARF6 co-localize around internalized particles, and constitutively active ARF6Q67N rescues phagocytosis in GEP100-depleted cells.\",\n      \"method\": \"siRNA/shRNA depletion, rescue with ARF6 constitutively active/dominant-negative mutants, immunofluorescence microscopy, F-actin staining\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (rescue with ARF6 mutants), co-localization, functional phagocytosis assay, single lab\",\n      \"pmids\": [\"20601426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"IQSec1 forms a complex with the lipid transfer protein ORP3; Ca2+ influx via STIM1/Orai1 channels near focal adhesions triggers PKC-dependent translocation of this IQSec1-ORP3 complex to ER/plasma membrane contact sites adjacent to focal adhesions, where IQSec1 is allosterically activated by ORP3 to activate Arf5 (not Arf6), driving focal adhesion disassembly. ORP3-mediated PI4P extraction from the PM is also required for FA turnover.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, live-cell calcium imaging, immunofluorescence, Arf5 activity assays, lipid transfer assays, FA disassembly quantification\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, Arf activation, lipid transfer, live imaging, KD phenotype), single lab but rigorous and comprehensive\",\n      \"pmids\": [\"32234213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Small molecule Bragsin inhibits BRAG2-mediated Arf GTPase activation in vitro in a membrane-dependent, non-competitive manner; crystal structure reveals Bragsin binds at the interface between the BRAG2 PH domain and the lipid bilayer, preventing BRAG2 from activating lipidated Arf. In cells, Bragsin affects the trans-Golgi network in a BRAG2- and Arf-dependent manner.\",\n      \"method\": \"In vitro Arf GEF activity assays with membranes, X-ray crystallography of BRAG2-Bragsin complex, structure-activity relationship, cellular TGN disruption assays\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with in vitro reconstitution, SAR, and cellular functional validation\",\n      \"pmids\": [\"30742123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In mature hippocampal cultures, GluN2A-containing NMDARs recruit BRAG2 to activate Arf6 upon NMDA stimulation; in young cultures, tonic Arf6 activation is mediated by GluN2B-BRAG1 instead. Knockdown of BRAG2 during postnatal weeks 4–5 reduces AMPAR miniature event frequency and quantal sizes of both AMPAR and NMDAR currents at Schaffer collateral synapses.\",\n      \"method\": \"Biochemical Arf6 activity assays in cortical cultures, shRNA knockdown, patch-clamp electrophysiology in acute hippocampal slices\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophysiology with KD and Arf6 activity assays, single lab\",\n      \"pmids\": [\"26884337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"BRAG2 cycles between the cytoplasm and nucleus in a CRM1/exportin1-dependent manner. Depletion of BRAG2 by RNAi increases the number of Cajal bodies and alters nucleolar structure (less focal fibrillarin staining). Ectopic expression of nuclear GTPase PIKE/AGAP2 causes both BRAG2 and coilin to accumulate in nucleoli, resulting in fibrillarin redistribution to the nucleolar periphery; neither PIKE GTPase activity nor BRAG2 nucleotide exchange activity is required for this nucleolar concentration.\",\n      \"method\": \"RNAi knockdown, CRM1 inhibition (leptomycin B), immunofluorescence microscopy, overexpression with catalytically inactive mutants\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — nuclear localization with CRM1 dependence demonstrated, functional consequence (nucleolar architecture) with mutagenesis, single lab\",\n      \"pmids\": [\"17461797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Overexpression of GEP100/BRAG2a in macrophage-like cells induces apoptosis (chromatin condensation, annexin V staining, TUNEL); a Sec7-domain deletion mutant lacking ARF-activating ability still induces apoptosis to the same level, suggesting the pro-apoptotic function is independent of ARF activation. GEP100 silencing suppresses TNF-α-induced apoptosis.\",\n      \"method\": \"Overexpression with Sec7 deletion mutant, Annexin V staining, TUNEL assay, morphological analysis, siRNA knockdown\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method per endpoint, mechanism of Sec7-independent apoptosis not elucidated beyond domain deletion\",\n      \"pmids\": [\"16877676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Biallelic loss-of-function variants in IQSEC1 (p.Thr343Met and p.Arg321Gln) cause intellectual disability and developmental delay; in Drosophila, these variants fail to rescue embryonic lethality caused by loss of the IQSEC1 ortholog schizo, while the reference cDNA does rescue, confirming loss-of-function. Conditional deletion of Iqsec1 in mouse cortical neurons leads to increased density of immature dendritic spines.\",\n      \"method\": \"Drosophila genetic complementation/rescue assay, mouse conditional knockout (cortical neurons), dendritic spine morphology analysis\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation in Drosophila with multiple variants, conditional KO in mouse with morphological readout, two orthogonal model organisms\",\n      \"pmids\": [\"31607425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Iqsec1 knockout mice (CRISPR/Cas9) exhibit embryonic lethality (~99%); electron microscopy shows that Iqsec1-/- embryos at E8.5 lack large apical vacuoles in visceral endoderm cells of the yolk sac, indicating a critical role for IQSEC1 in membrane trafficking in visceral endoderm during embryogenesis.\",\n      \"method\": \"CRISPR/Cas9 knockout, electron microscopy of embryonic visceral endoderm\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined ultrastructural phenotype, single lab\",\n      \"pmids\": [\"39561249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"BRAG2 localizes to postsynaptic processes of bipolar dyads in the inner plexiform layer of the mouse retina and co-localizes preferentially with PSD-95 and AMPARs, as demonstrated by immunoelectron microscopy and double immunostaining; distinct from BRAG3 which localizes to inhibitory synapses.\",\n      \"method\": \"Immunohistochemistry, immunoelectron microscopy, double immunofluorescence\",\n      \"journal\": \"The Journal of comparative neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct subcellular localization by immunoelectron microscopy, replicated across retinal layers\",\n      \"pmids\": [\"22886754\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"IQSEC1/BRAG2/GEP100 is a guanine nucleotide exchange factor (GEF) for Arf5 and Arf6 whose PH domain is constitutively coupled to the catalytic Sec7 domain, allosterically stimulated ~2,000-fold by membrane phosphoinositides (especially PI(4,5)P2); it is recruited to activated receptor tyrosine kinases (EGFR, Her2, VEGFR2) via its PH domain binding to phosphotyrosine residues, triggering Arf6 activation and downstream invasion/metastasis signaling, and it acts at clathrin-coated pits to activate Arf5 for β1-integrin endocytosis; at synapses it directly interacts with GluA2-containing AMPA receptors, and BRAG2-mediated Arf6 activation is the convergent step required for LTD-associated AMPA receptor internalization; it also forms a Ca2+-activated complex with ORP3 that translocates to ER-PM contact sites to activate Arf5 and drive focal adhesion disassembly during cell migration, while whole-animal Iqsec1 knockout causes embryonic lethality due to defective membrane trafficking in visceral endoderm.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"IQSEC1 (BRAG2/GEP100) is a guanine nucleotide exchange factor that activates ADP-ribosylation factor GTPases (Arf6 and the class II Arf5) on negatively charged membranes to drive receptor and adhesion-molecule trafficking across diverse cellular contexts [#1, #4, #5]. Its catalytic Sec7 domain is constitutively coupled to an atypical PH domain that potentiates nucleotide exchange ~2,000-fold; PI(4,5)P2 binding to the PH domain lowers Km and raises kcat, restricting activity to phosphoinositide-rich membranes [#5, #6]. This PH module is the principal regulatory hub: it docks onto phosphotyrosine residues of activated receptor tyrosine kinases — EGFR, Her2, and VEGFR2 — to trigger Arf6 activation and downstream invasion, angiogenic migration, and cadherin endocytosis [#0, #7, #9], and it is engaged by Plexin-D1-generated PI(4,5)P2 to disassemble focal adhesions in endothelial cells [#8]. IQSEC1 controls integrin trafficking by binding clathrin and the AP-2 adaptor at coated pits, where it activates Arf5 to internalize β1 integrin and where it interacts with α-catenin to modulate E-cadherin and F-actin [#4, #2]. A Ca2+-dependent complex with the lipid transfer protein ORP3 translocates to ER–plasma membrane contact sites near focal adhesions, where ORP3 allosterically activates IQSEC1 toward Arf5 to drive focal adhesion disassembly during migration [#11]. At synapses, IQSEC1 directly binds the GluA2 subunit of AMPA receptors and, downstream of NMDA receptor activation, provides the Arf6 activation required for clathrin-mediated AMPA receptor internalization and long-term depression [#3, #13]. Biallelic loss-of-function variants in IQSEC1 cause intellectual disability and developmental delay [#16], and whole-animal knockout is embryonic lethal owing to defective membrane trafficking in visceral endoderm [#17].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing that IQSEC1/BRAG2 is a physiological Arf6 GEF answered whether this protein actively controls membrane traffic, by linking it to β1 integrin surface levels and cell adhesion.\",\n      \"evidence\": \"siRNA depletion with surface integrin flow cytometry and fibronectin spreading assays\",\n      \"pmids\": [\"16461286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the membrane compartment or upstream receptor triggering Arf6 activation\", \"Did not distinguish Arf6 from other Arf isoforms\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identification of α-catenin as a binding partner and stimulator of GEP100 GEF activity connected Arf6 activation to cadherin-based adhesion and the actin cytoskeleton.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP of endogenous proteins, in vitro GTPγS binding, and siRNA with GEF-inactive mutants\",\n      \"pmids\": [\"16807291\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which α-catenin enhances exchange not structurally defined\", \"Did not establish whether E-cadherin effects are direct or downstream of Arf6\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrating that the PH domain binds phosphotyrosine-EGFR established the direct mechanism coupling receptor tyrosine kinase signaling to Arf6 activation and cancer invasion.\",\n      \"evidence\": \"Co-IP, siRNA, overexpression in MCF7 cells, and in vivo metastasis assay\",\n      \"pmids\": [\"18084281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how phosphotyrosine binding by an atypical PH domain coexists with lipid binding\", \"Generality across other RTKs not yet tested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Discovery of CRM1-dependent nucleocytoplasmic shuttling and effects on Cajal body/nucleolar architecture raised the possibility of a nuclear function distinct from membrane trafficking.\",\n      \"evidence\": \"RNAi, leptomycin B inhibition, immunofluorescence, and catalytically inactive mutant overexpression\",\n      \"pmids\": [\"17461797\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular mechanism linking BRAG2 to nucleolar/Cajal body structure\", \"GEF activity dispensable, so the relevant biochemical activity is unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identifying a direct GluA2-BRAG2 interaction and a conditional-KO LTD deficit defined IQSEC1 as the convergent Arf6 activator required for synaptic AMPA receptor internalization.\",\n      \"evidence\": \"Reciprocal co-IP, CA1-specific conditional knockout, LTD electrophysiology, and pharmacological interaction blockade\",\n      \"pmids\": [\"20547133\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the upstream receptor coupling synaptic activity to BRAG2\", \"Did not address whether class II Arfs also contribute at synapses\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Linking GEP100 to Fcγ-mediated phagocytosis extended its Arf6-GEF role into innate immune membrane internalization.\",\n      \"evidence\": \"siRNA/shRNA depletion, rescue with ARF6 mutants, immunofluorescence, and F-actin staining in macrophages\",\n      \"pmids\": [\"20601426\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream signal recruiting GEP100 to phagocytic cups unknown\", \"Single-lab study without structural detail\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showing Her2 and VEGFR2 also recruit GEP100 via the PH domain generalized the phosphotyrosine-PH coupling mechanism beyond EGFR to other RTK-driven invasion and angiogenesis programs.\",\n      \"evidence\": \"Co-IP, PH domain deletion mutants, siRNA, invasion and tube formation assays, in vivo angiogenesis models\",\n      \"pmids\": [\"21966491\", \"21858086\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab studies for each receptor\", \"Did not quantify relative affinities across receptors\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrating Plexin-D1-generated PI(4,5)P2 binding to the PH domain identified a lipid-driven route to GEF activation and focal adhesion disassembly distinct from phosphotyrosine docking.\",\n      \"evidence\": \"siRNA, GEF activity assays, PI(4,5)P2 lipid-binding and cell collapse assays in endothelial cells\",\n      \"pmids\": [\"21795701\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab biochemical study\", \"Did not reconcile lipid versus phosphotyrosine engagement of the same PH domain\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defining the biochemical basis of PH-domain allostery showed that PIP2 binding lowers Km and raises kcat through contributions requiring the Arf N-terminus and the interdomain linker.\",\n      \"evidence\": \"In vitro single-turnover and substrate-saturation kinetics with myristoylated Arf1, PH deletion mutants, and NMR\",\n      \"pmids\": [\"22613714\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not capture the full membrane-bound enzyme-substrate complex\", \"Phosphoinositide specificity not fully resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovering that BRAG2 activates Arf5 at clathrin-coated pits and binds clathrin/AP-2 revealed that β1 integrin internalization is mediated by a class II Arf rather than Arf6.\",\n      \"evidence\": \"siRNA, co-IP with clathrin/AP-2, Arf activity assays, rapid-cycling Arf5 rescue, and coated-pit microscopy\",\n      \"pmids\": [\"22815487\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants selecting Arf5 versus Arf6 in different contexts unclear\", \"Did not map the clathrin/AP-2 binding interface\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"The Arf1-BRAG2 crystal structure explained the ~2,000-fold catalytic enhancement, showing an atypical PH domain constitutively anchored to Sec7 that targets activity to negatively charged membranes.\",\n      \"evidence\": \"X-ray crystallography with reconstituted quantitative exchange kinetics and mutagenesis\",\n      \"pmids\": [\"24058294\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure lacked the bound phosphoinositide that confers regulation in cells\", \"Did not include receptor or partner proteins\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Immunoelectron localization to postsynaptic bipolar dyad processes with PSD-95 and AMPARs confirmed a defined synaptic localization for BRAG2 distinct from BRAG3.\",\n      \"evidence\": \"Immunohistochemistry, immunoelectron microscopy, and double immunofluorescence in mouse retina\",\n      \"pmids\": [\"22886754\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not establish functional consequences at retinal synapses\", \"Localization descriptive only\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Mapping NMDA receptor subtype-specific recruitment showed GluN2A-NMDARs engage BRAG2 to activate Arf6, defining the upstream synaptic trigger and a developmental switch from a BRAG1 pathway.\",\n      \"evidence\": \"Arf6 activity assays in cortical cultures, shRNA knockdown, and patch-clamp electrophysiology in slices\",\n      \"pmids\": [\"26884337\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between NMDAR activation and BRAG2 recruitment not defined\", \"Single-lab electrophysiology\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The Bragsin inhibitor and its co-crystal structure provided proof that targeting the PH-domain/membrane interface blocks activation of lipidated Arf, validating the membrane-coupled mechanism pharmacologically.\",\n      \"evidence\": \"In vitro membrane GEF assays, BRAG2-Bragsin crystallography, SAR, and cellular TGN disruption assays\",\n      \"pmids\": [\"30742123\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular TGN role of BRAG2 not mechanistically detailed\", \"Selectivity over other Arf GEFs not fully characterized\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identifying biallelic loss-of-function IQSEC1 variants in patients, validated by Drosophila rescue failure and mouse cortical KO spine phenotypes, established IQSEC1 as a cause of intellectual disability and developmental delay.\",\n      \"evidence\": \"Drosophila genetic complementation, mouse conditional knockout, and dendritic spine morphology analysis\",\n      \"pmids\": [\"31607425\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not connect human variants directly to a specific Arf-GEF deficit\", \"Immature spine phenotype mechanism not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery of the Ca2+-triggered IQSec1-ORP3 complex at ER-PM contact sites defined a lipid-transfer-coupled mode of Arf5 activation driving focal adhesion disassembly.\",\n      \"evidence\": \"Co-IP, siRNA, live-cell calcium imaging, Arf5 activity and lipid transfer assays, and FA disassembly quantification\",\n      \"pmids\": [\"32234213\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ORP3 allosterically activates IQSec1 structurally undefined\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"CRISPR knockout demonstrating embryonic lethality with absent apical vacuoles in visceral endoderm established an essential developmental role in membrane trafficking.\",\n      \"evidence\": \"CRISPR/Cas9 knockout and electron microscopy of embryonic visceral endoderm\",\n      \"pmids\": [\"39561249\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which Arf and cargo pathway underlies the visceral endoderm defect not defined\", \"Single-lab ultrastructural study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how the single PH domain integrates competing inputs — receptor phosphotyrosine, phosphoinositides, and partner proteins like α-catenin and ORP3 — to select between Arf5 and Arf6 in a context-specific manner.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the membrane-bound enzyme with a phosphotyrosine receptor\", \"Determinants of Arf5 versus Arf6 substrate selection unknown\", \"Mechanism linking nuclear shuttling and Sec7-independent apoptosis to canonical GEF function undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005085\", \"supporting_discovery_ids\": [1, 4, 5, 6]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 6, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 4, 11]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [4, 1, 11]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 7, 9]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [3, 13]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [1, 8, 11]}\n    ],\n    \"complexes\": [\"IQSEC1-ORP3 complex\", \"AP-2 adaptor / clathrin coat\"],\n    \"partners\": [\"ARF6\", \"ARF5\", \"EGFR\", \"GRIA2\", \"ORP3\", \"CTNNA1\", \"VEGFR2\", \"ERBB2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}