{"gene":"AGAP3","run_date":"2026-06-09T22:02:42","timeline":{"discoveries":[{"year":2013,"finding":"AGAP3 is a component of the NMDA receptor complex, physically associating with NMDA receptors in neurons, and regulates NMDA receptor-mediated Ras/ERK and Arf6 signaling pathways during chemically induced LTP in rat primary neuronal cultures.","method":"Co-immunoprecipitation (NMDA receptor complex isolation), signaling assays in rat primary neuronal cultures","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP establishing complex membership, signaling pathway assays, single lab with two orthogonal methods","pmids":["23904596"],"is_preprint":false},{"year":2013,"finding":"Knockdown of AGAP3 in rat primary neuronal cultures occludes AMPA receptor trafficking during chemically induced LTP, demonstrating AGAP3 is required to link NMDA receptor activation to AMPA receptor trafficking.","method":"shRNA knockdown of AGAP3 in primary neuronal cultures with AMPA receptor trafficking assay during chemically induced LTP","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotype (AMPA receptor trafficking occlusion), single lab","pmids":["23904596"],"is_preprint":false},{"year":2011,"finding":"CRAG (a short splicing variant of AGAP3) induces transcriptional activation of c-Fos-dependent AP-1 via serum response factor (SRF); the nuclear localization signal and both N- and C-terminal regions of CRAG are required for SRF-dependent c-Fos activation, whereas the full-length centaurin-γ3/AGAP3 does not have this activity.","method":"Transcriptional reporter assays, siRNA knockdown, dominant-negative mutant expression, deletion/mutation analysis in cultured neuronal cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis mapping combined with reporter assays and RNAi, single lab, multiple orthogonal methods","pmids":["21832068"],"is_preprint":false},{"year":2019,"finding":"CRAG (short splice variant of AGAP3) has intrinsic GTPase activity, interacts with the SRF co-activator ELK1, and activates SRF in an ELK1-dependent manner at promyelocytic leukaemia (PML) bodies via SUMO-interacting motifs; CRAG/centaurin-γ3 knockout mice show suppressed kainic acid-induced c-fos expression in the hippocampus.","method":"GTPase activity assay, co-immunoprecipitation (CRAG–ELK1), SUMO-interacting motif mutant analysis, knockout mouse model with in vivo c-fos expression measurement","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro enzymatic assay, co-IP, mutagenesis, and knockout validation in vivo, single lab with multiple orthogonal methods","pmids":["31882856"],"is_preprint":false},{"year":2021,"finding":"Dorsal forebrain-specific knockout of CRAG/Centaurin-γ3 (AGAP3) in mice results in maturational abnormality of hippocampal granule cells (increased immature neurons, decreased mature neurons — immature dentate gyrus phenotype) and hyperactivity, establishing CRAG as required for dentate gyrus neuron maturation.","method":"Conditional (forebrain-specific) CRAG/Centaurin-γ3 knockout mice; immunohistochemistry for doublecortin and calbindin; open-field behavioral testing","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined loss-of-function phenotype in conditional KO with cellular and behavioral readouts, single lab","pmids":["33811862"],"is_preprint":false},{"year":2017,"finding":"AGAP3 is enriched in the CLASP2 protein interactome in 3T3-L1 adipocytes and shows a preference for CLIP2 in subsequent AGAP3 interactome analysis, placing it in a microtubule plus-end tracking protein network.","method":"Affinity purification–mass spectrometry (AP-MS) with SAINT analysis; reciprocal co-immunoprecipitation","journal":"Molecular & cellular proteomics","confidence":"Low","confidence_rationale":"Tier 3 / Moderate — interactome MS with SAINT scoring and follow-up co-IP, but only interaction membership established with no direct mechanistic consequence for AGAP3 itself","pmids":["28550165"],"is_preprint":false},{"year":2019,"finding":"Insulin regulates phosphorylation of AGAP3 in 3T3-L1 adipocytes, identifying AGAP3 as a member of an insulin-responsive microtubule plus-end tracking (+TIP) protein network.","method":"Targeted quantitative phosphoproteomics in 3T3-L1 adipocytes","journal":"Molecular & cellular proteomics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single phosphoproteomic dataset, no functional follow-up for AGAP3 specifically","pmids":["31018989"],"is_preprint":false},{"year":2020,"finding":"AGAP3-BRAF fusion (with AGAP3 as 5' partner) promotes canonical oncogenic BRAF activity by replacing the auto-inhibitory N-terminal region of BRAF; the 5' AGAP3 partner influences subcellular localization of the fusion protein and intracellular signaling capacity; AGAP3-BRAF fusion confers resistance to EGFR-targeted monotherapy in colorectal cancer organoids.","method":"Patient-derived colorectal cancer organoids expressing AGAP3-BRAF and other BRAF fusion constructs; drug sensitivity assays; downstream signaling (MAPK pathway) analysis; subcellular localization studies","journal":"Molecular cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional characterization in patient-derived organoids with signaling and localization readouts, multiple fusion partners cross-compared in single study","pmids":["31911540"],"is_preprint":false},{"year":2017,"finding":"Expression of the AGAP3-BRAF fusion gene in BRAFV600E mutant melanoma cells induces resistance to vemurafenib (BRAF inhibitor) while retaining sensitivity to MEK inhibitors.","method":"Stable expression of AGAP3-BRAF fusion in BRAFV600E melanoma cell lines; vemurafenib and MEK inhibitor drug sensitivity assays","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional cell-line experiment with defined drug-resistance phenotype, single lab","pmids":["28539463"],"is_preprint":false},{"year":2026,"finding":"SRRM2 haploinsufficiency in mice causes mis-splicing and elevation of Agap3 protein; human iPSC-derived neurons deficient in SRRM2 display conserved AGAP3 splicing defects, identifying SRRM2 as a splicing regulator of AGAP3.","method":"Srrm2+/- mouse transcriptomics/splice analysis; human iPSC-derived neuron knockdown of SRRM2 with RNA splicing analysis","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function in two species (mouse KO and human iPSC neurons) with orthogonal transcriptomic and splicing readouts, single lab","pmids":["42189682"],"is_preprint":false}],"current_model":"AGAP3 is a multi-domain signaling protein (GTPase-like, pleckstrin homology, and ArfGAP domains) that resides in the NMDA receptor complex at synapses, where it couples NMDA receptor activation to Ras/ERK and Arf6 signaling and is required for AMPA receptor trafficking during LTP; its short splice variant CRAG translocates to the nucleus, displays GTPase activity, interacts with ELK1 at PML bodies to activate SRF-dependent c-Fos/AP-1 transcription, and is required for dentate gyrus neuron maturation in vivo; AGAP3 phosphorylation is regulated by insulin as part of a microtubule plus-end tracking network, and its splicing is regulated by the nuclear speckle factor SRRM2; when fused to BRAF as a 5' partner, AGAP3 displaces the auto-inhibitory N-terminal region of BRAF to produce a constitutively active kinase that drives oncogenic MAPK signaling and confers resistance to BRAF or EGFR inhibitors."},"narrative":{"mechanistic_narrative":"AGAP3 is a multi-domain signaling protein that links glutamate receptor activation to downstream small-GTPase and transcriptional outputs in neurons. At excitatory synapses it is a component of the NMDA receptor complex, where it couples receptor activation to Ras/ERK and Arf6 signaling and is required to drive AMPA receptor trafficking during chemically induced LTP [PMID:23904596]. A short splice variant, CRAG, carries intrinsic GTPase activity and acts in the nucleus: through its nuclear localization signal and SUMO-interacting motifs it localizes to PML bodies, binds the SRF co-activator ELK1, and activates SRF-dependent c-Fos/AP-1 transcription, an activity absent from the full-length protein [PMID:21832068, PMID:31882856]. Consistent with this transcriptional role, CRAG/Centaurin-γ3 knockout mice show blunted kainic acid-induced hippocampal c-fos induction, and forebrain-specific deletion produces an immature dentate gyrus phenotype with hyperactivity, establishing CRAG as required for dentate gyrus granule cell maturation in vivo [PMID:31882856, PMID:33811862]. AGAP3 expression is post-transcriptionally controlled by the nuclear speckle splicing factor SRRM2, whose loss causes AGAP3 mis-splicing and protein elevation in both mouse and human iPSC-derived neurons [PMID:42189682]. As a recurrent oncogenic event, AGAP3 serves as a 5' fusion partner to BRAF, where it displaces the auto-inhibitory N-terminal region of BRAF to generate a constitutively active kinase that drives MAPK signaling and confers resistance to BRAF and EGFR inhibitors [PMID:31911540, PMID:28539463].","teleology":[{"year":2011,"claim":"Established that the short CRAG splice variant, but not full-length AGAP3, can drive a specific transcriptional program, defining a domain-dependent nuclear function.","evidence":"Reporter assays, siRNA, dominant-negative and deletion mutagenesis in cultured neuronal cells","pmids":["21832068"],"confidence":"Medium","gaps":["Mechanism of how cytoplasmic vs nuclear isoform partitioning is controlled not defined","Direct DNA or co-factor binding by CRAG not yet mapped here","Physiological stimulus driving CRAG transcriptional activity not established"]},{"year":2013,"claim":"Placed AGAP3 within the NMDA receptor complex and showed it is the link coupling receptor activation to Ras/ERK and Arf6 signaling and AMPA receptor trafficking during LTP.","evidence":"Reciprocal co-IP for complex membership plus shRNA knockdown with AMPA trafficking and signaling assays in rat primary neurons","pmids":["23904596"],"confidence":"Medium","gaps":["ArfGAP catalytic activity toward Arf6 not directly demonstrated in this context","Direct binding partner within the NMDAR complex not resolved","Single lab, neuronal culture only"]},{"year":2017,"claim":"Identified AGAP3 as a recurrent oncogenic 5' BRAF fusion partner conferring drug resistance, extending its biology into cancer signaling.","evidence":"Stable expression of AGAP3-BRAF in BRAFV600E melanoma cells with vemurafenib and MEK inhibitor sensitivity assays","pmids":["28539463"],"confidence":"Medium","gaps":["Structural basis of N-terminal displacement not shown here","Contribution of AGAP3 domains to the fusion phenotype not dissected","Resistance demonstrated in cell lines only"]},{"year":2017,"claim":"Linked AGAP3 to a microtubule plus-end tracking protein network, suggesting a cytoskeletal-associated role outside neurons.","evidence":"AP-MS with SAINT and reciprocal co-IP in 3T3-L1 adipocytes","pmids":["28550165"],"confidence":"Low","gaps":["Interaction membership only; no mechanistic consequence for AGAP3 established","+TIP function of AGAP3 not functionally tested","Adipocyte system only"]},{"year":2019,"claim":"Defined the molecular basis of CRAG's nuclear transcriptional activity, showing it is a GTPase that activates SRF via ELK1 at PML bodies and contributes to hippocampal c-fos induction in vivo.","evidence":"In vitro GTPase assay, CRAG-ELK1 co-IP, SUMO-interacting-motif mutants, and knockout mouse c-fos measurement","pmids":["31882856"],"confidence":"Medium","gaps":["GTPase substrate/regulatory cycle of CRAG not characterized","How SUMO-interaction directs PML-body recruitment mechanistically unclear","Direct vs indirect role of ELK1 binding in SRF activation not separated"]},{"year":2019,"claim":"Showed AGAP3 phosphorylation is insulin-regulated, embedding it in an insulin-responsive +TIP network.","evidence":"Targeted quantitative phosphoproteomics in 3T3-L1 adipocytes","pmids":["31018989"],"confidence":"Low","gaps":["Single phosphoproteomic dataset with no functional follow-up for AGAP3","Responsible kinase and phosphosite consequence unknown","Physiological role of phosphorylation untested"]},{"year":2020,"claim":"Demonstrated mechanistically that the AGAP3-BRAF fusion activates BRAF by replacing its auto-inhibitory N-terminus and that the AGAP3 partner sets fusion localization and EGFR-inhibitor resistance.","evidence":"Patient-derived colorectal cancer organoids expressing fusion constructs with drug sensitivity, MAPK signaling, and localization readouts","pmids":["31911540"],"confidence":"Medium","gaps":["Precise structural mechanism of N-terminal displacement not resolved","Which AGAP3 domains drive localization not delineated","In vivo tumor relevance beyond organoids not established"]},{"year":2021,"claim":"Established a developmental requirement for CRAG/AGAP3 by linking its loss to failed dentate gyrus neuron maturation and behavioral hyperactivity.","evidence":"Forebrain-specific conditional knockout mice with doublecortin/calbindin immunohistochemistry and open-field testing","pmids":["33811862"],"confidence":"Medium","gaps":["Molecular pathway linking CRAG loss to maturation arrest not defined","Whether the synaptic vs transcriptional function underlies the phenotype unresolved","Single lab"]},{"year":2026,"claim":"Identified SRRM2 as an upstream splicing regulator of AGAP3, connecting AGAP3 dosage to a nuclear speckle splicing factor across species.","evidence":"Srrm2+/- mouse splice/transcriptomic analysis and SRRM2-deficient human iPSC-derived neuron splicing analysis","pmids":["42189682"],"confidence":"Medium","gaps":["Which AGAP3 isoform (e.g. CRAG) is affected by mis-splicing not specified","Functional consequence of AGAP3 elevation in neurons not tested","Direct vs indirect SRRM2 action on the AGAP3 transcript not distinguished"]},{"year":null,"claim":"The ArfGAP catalytic activity of AGAP3 toward its physiological Arf substrate, and how the synaptic, nuclear-transcriptional, cytoskeletal, and oncogenic-fusion functions are mechanistically integrated, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No direct demonstration of Arf6 GAP catalysis by AGAP3 in the corpus","Structural model of full-length AGAP3 domain regulation absent","Relationship between isoform usage and each functional context not unified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[3]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[2,3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,3]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,7,8]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,3]}],"complexes":["NMDA receptor complex"],"partners":["ELK1","CLASP2","CLIP2","BRAF","SRRM2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96P47","full_name":"Arf-GAP with GTPase, ANK repeat and PH domain-containing protein 3","aliases":["CRAM-associated GTPase","CRAG","Centaurin-gamma-3","Cnt-g3","MR1-interacting protein","MRIP-1"],"length_aa":875,"mass_kda":95.0,"function":"GTPase-activating protein for the ADP ribosylation factor family (Potential). GTPase which may be involved in the degradation of expanded polyglutamine proteins through the ubiquitin-proteasome pathway","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q96P47/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AGAP3","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000133612","cell_line_id":"CID000651","localizations":[{"compartment":"cell_contact","grade":3},{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"AGAP1","stoichiometry":10.0},{"gene":"C12ORF57","stoichiometry":0.2},{"gene":"UBE2O","stoichiometry":0.2},{"gene":"TANC1","stoichiometry":0.2},{"gene":"DCTN1;DKFZP686E0752","stoichiometry":0.2},{"gene":"AHCYL1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000651","total_profiled":1310},"omim":[{"mim_id":"621159","title":"ARF GTPase-ACTIVATING PROTEIN WITH GTPase DOMAIN, ANKYRIN REPEAT, AND PLECKSTRIN HOMOLOGY DOMAIN 11, NONCODING; AGAP11","url":"https://www.omim.org/entry/621159"},{"mim_id":"621158","title":"ARF GTPase-ACTIVATING PROTEIN WITH GTPase DOMAIN, ANKYRIN REPEAT, AND PLECKSTRIN HOMOLOGY DOMAIN 9; AGAP9","url":"https://www.omim.org/entry/621158"},{"mim_id":"621157","title":"ARF GTPase-ACTIVATING PROTEIN WITH GTPase DOMAIN, ANKYRIN REPEAT, AND PLECKSTRIN HOMOLOGY DOMAIN 6; AGAP6","url":"https://www.omim.org/entry/621157"},{"mim_id":"621156","title":"ARF GTPase-ACTIVATING PROTEIN WITH GTPase DOMAIN, ANKYRIN REPEAT, AND PLECKSTRIN HOMOLOGY DOMAIN 5; AGAP5","url":"https://www.omim.org/entry/621156"},{"mim_id":"621155","title":"ARF GTPase-ACTIVATING PROTEIN WITH GTPase DOMAIN, ANKYRIN REPEAT, AND PLECKSTRIN HOMOLOGY DOMAIN 4; AGAP4","url":"https://www.omim.org/entry/621155"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":264.6}],"url":"https://www.proteinatlas.org/search/AGAP3"},"hgnc":{"alias_symbol":[],"prev_symbol":["CENTG3"]},"alphafold":{"accession":"Q96P47","domains":[{"cath_id":"3.40.50.300","chopping":"90-257","consensus_level":"high","plddt":91.5896,"start":90,"end":257},{"cath_id":"2.30.29.30","chopping":"366-428_570-612","consensus_level":"medium","plddt":87.2513,"start":366,"end":612},{"cath_id":"1.10.220.150","chopping":"635-733","consensus_level":"medium","plddt":95.3881,"start":635,"end":733},{"cath_id":"1.25.40.20","chopping":"748-840","consensus_level":"medium","plddt":94.6637,"start":748,"end":840}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96P47","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96P47-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96P47-F1-predicted_aligned_error_v6.png","plddt_mean":70.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AGAP3","jax_strain_url":"https://www.jax.org/strain/search?query=AGAP3"},"sequence":{"accession":"Q96P47","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96P47.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96P47/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96P47"}},"corpus_meta":[{"pmid":"23904596","id":"PMC_23904596","title":"AGAP3 and Arf6 regulate trafficking of AMPA receptors and synaptic plasticity.","date":"2013","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/23904596","citation_count":58,"is_preprint":false},{"pmid":"28539463","id":"PMC_28539463","title":"BRAF Fusion as a Novel Mechanism of Acquired Resistance to Vemurafenib in BRAFV600E Mutant Melanoma.","date":"2017","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/28539463","citation_count":53,"is_preprint":false},{"pmid":"31018989","id":"PMC_31018989","title":"Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes.","date":"2019","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/31018989","citation_count":52,"is_preprint":false},{"pmid":"28550165","id":"PMC_28550165","title":"Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein.","date":"2017","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/28550165","citation_count":50,"is_preprint":false},{"pmid":"35871080","id":"PMC_35871080","title":"Identification of fusions with potential clinical significance in melanoma.","date":"2022","source":"Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc","url":"https://pubmed.ncbi.nlm.nih.gov/35871080","citation_count":30,"is_preprint":false},{"pmid":"34398495","id":"PMC_34398495","title":"Gastrointestinal stromal tumors with BRAF gene fusions. 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report.","date":"2024","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/39687148","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.10.10.617460","title":"<i>SRRM2</i>  haploinsufficiency drives SynGAP-γ reduction,  <i>AGAP3</i>  mis-splicing, and oligodendrocyte deficits in a mouse model of schizophrenia","date":"2024-10-12","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.10.617460","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15960,"output_tokens":2548,"usd":0.04305,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9764,"output_tokens":3548,"usd":0.06876,"stage2_stop_reason":"end_turn"},"total_usd":0.11181,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"AGAP3 is a component of the NMDA receptor complex, physically associating with NMDA receptors in neurons, and regulates NMDA receptor-mediated Ras/ERK and Arf6 signaling pathways during chemically induced LTP in rat primary neuronal cultures.\",\n      \"method\": \"Co-immunoprecipitation (NMDA receptor complex isolation), signaling assays in rat primary neuronal cultures\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP establishing complex membership, signaling pathway assays, single lab with two orthogonal methods\",\n      \"pmids\": [\"23904596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Knockdown of AGAP3 in rat primary neuronal cultures occludes AMPA receptor trafficking during chemically induced LTP, demonstrating AGAP3 is required to link NMDA receptor activation to AMPA receptor trafficking.\",\n      \"method\": \"shRNA knockdown of AGAP3 in primary neuronal cultures with AMPA receptor trafficking assay during chemically induced LTP\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotype (AMPA receptor trafficking occlusion), single lab\",\n      \"pmids\": [\"23904596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CRAG (a short splicing variant of AGAP3) induces transcriptional activation of c-Fos-dependent AP-1 via serum response factor (SRF); the nuclear localization signal and both N- and C-terminal regions of CRAG are required for SRF-dependent c-Fos activation, whereas the full-length centaurin-γ3/AGAP3 does not have this activity.\",\n      \"method\": \"Transcriptional reporter assays, siRNA knockdown, dominant-negative mutant expression, deletion/mutation analysis in cultured neuronal cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis mapping combined with reporter assays and RNAi, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21832068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CRAG (short splice variant of AGAP3) has intrinsic GTPase activity, interacts with the SRF co-activator ELK1, and activates SRF in an ELK1-dependent manner at promyelocytic leukaemia (PML) bodies via SUMO-interacting motifs; CRAG/centaurin-γ3 knockout mice show suppressed kainic acid-induced c-fos expression in the hippocampus.\",\n      \"method\": \"GTPase activity assay, co-immunoprecipitation (CRAG–ELK1), SUMO-interacting motif mutant analysis, knockout mouse model with in vivo c-fos expression measurement\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzymatic assay, co-IP, mutagenesis, and knockout validation in vivo, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31882856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Dorsal forebrain-specific knockout of CRAG/Centaurin-γ3 (AGAP3) in mice results in maturational abnormality of hippocampal granule cells (increased immature neurons, decreased mature neurons — immature dentate gyrus phenotype) and hyperactivity, establishing CRAG as required for dentate gyrus neuron maturation.\",\n      \"method\": \"Conditional (forebrain-specific) CRAG/Centaurin-γ3 knockout mice; immunohistochemistry for doublecortin and calbindin; open-field behavioral testing\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined loss-of-function phenotype in conditional KO with cellular and behavioral readouts, single lab\",\n      \"pmids\": [\"33811862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AGAP3 is enriched in the CLASP2 protein interactome in 3T3-L1 adipocytes and shows a preference for CLIP2 in subsequent AGAP3 interactome analysis, placing it in a microtubule plus-end tracking protein network.\",\n      \"method\": \"Affinity purification–mass spectrometry (AP-MS) with SAINT analysis; reciprocal co-immunoprecipitation\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — interactome MS with SAINT scoring and follow-up co-IP, but only interaction membership established with no direct mechanistic consequence for AGAP3 itself\",\n      \"pmids\": [\"28550165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Insulin regulates phosphorylation of AGAP3 in 3T3-L1 adipocytes, identifying AGAP3 as a member of an insulin-responsive microtubule plus-end tracking (+TIP) protein network.\",\n      \"method\": \"Targeted quantitative phosphoproteomics in 3T3-L1 adipocytes\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single phosphoproteomic dataset, no functional follow-up for AGAP3 specifically\",\n      \"pmids\": [\"31018989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AGAP3-BRAF fusion (with AGAP3 as 5' partner) promotes canonical oncogenic BRAF activity by replacing the auto-inhibitory N-terminal region of BRAF; the 5' AGAP3 partner influences subcellular localization of the fusion protein and intracellular signaling capacity; AGAP3-BRAF fusion confers resistance to EGFR-targeted monotherapy in colorectal cancer organoids.\",\n      \"method\": \"Patient-derived colorectal cancer organoids expressing AGAP3-BRAF and other BRAF fusion constructs; drug sensitivity assays; downstream signaling (MAPK pathway) analysis; subcellular localization studies\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional characterization in patient-derived organoids with signaling and localization readouts, multiple fusion partners cross-compared in single study\",\n      \"pmids\": [\"31911540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Expression of the AGAP3-BRAF fusion gene in BRAFV600E mutant melanoma cells induces resistance to vemurafenib (BRAF inhibitor) while retaining sensitivity to MEK inhibitors.\",\n      \"method\": \"Stable expression of AGAP3-BRAF fusion in BRAFV600E melanoma cell lines; vemurafenib and MEK inhibitor drug sensitivity assays\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional cell-line experiment with defined drug-resistance phenotype, single lab\",\n      \"pmids\": [\"28539463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"SRRM2 haploinsufficiency in mice causes mis-splicing and elevation of Agap3 protein; human iPSC-derived neurons deficient in SRRM2 display conserved AGAP3 splicing defects, identifying SRRM2 as a splicing regulator of AGAP3.\",\n      \"method\": \"Srrm2+/- mouse transcriptomics/splice analysis; human iPSC-derived neuron knockdown of SRRM2 with RNA splicing analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in two species (mouse KO and human iPSC neurons) with orthogonal transcriptomic and splicing readouts, single lab\",\n      \"pmids\": [\"42189682\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AGAP3 is a multi-domain signaling protein (GTPase-like, pleckstrin homology, and ArfGAP domains) that resides in the NMDA receptor complex at synapses, where it couples NMDA receptor activation to Ras/ERK and Arf6 signaling and is required for AMPA receptor trafficking during LTP; its short splice variant CRAG translocates to the nucleus, displays GTPase activity, interacts with ELK1 at PML bodies to activate SRF-dependent c-Fos/AP-1 transcription, and is required for dentate gyrus neuron maturation in vivo; AGAP3 phosphorylation is regulated by insulin as part of a microtubule plus-end tracking network, and its splicing is regulated by the nuclear speckle factor SRRM2; when fused to BRAF as a 5' partner, AGAP3 displaces the auto-inhibitory N-terminal region of BRAF to produce a constitutively active kinase that drives oncogenic MAPK signaling and confers resistance to BRAF or EGFR inhibitors.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AGAP3 is a multi-domain signaling protein that links glutamate receptor activation to downstream small-GTPase and transcriptional outputs in neurons. At excitatory synapses it is a component of the NMDA receptor complex, where it couples receptor activation to Ras/ERK and Arf6 signaling and is required to drive AMPA receptor trafficking during chemically induced LTP [#0, #1]. A short splice variant, CRAG, carries intrinsic GTPase activity and acts in the nucleus: through its nuclear localization signal and SUMO-interacting motifs it localizes to PML bodies, binds the SRF co-activator ELK1, and activates SRF-dependent c-Fos/AP-1 transcription, an activity absent from the full-length protein [#2, #3]. Consistent with this transcriptional role, CRAG/Centaurin-\\u03b33 knockout mice show blunted kainic acid-induced hippocampal c-fos induction, and forebrain-specific deletion produces an immature dentate gyrus phenotype with hyperactivity, establishing CRAG as required for dentate gyrus granule cell maturation in vivo [#3, #4]. AGAP3 expression is post-transcriptionally controlled by the nuclear speckle splicing factor SRRM2, whose loss causes AGAP3 mis-splicing and protein elevation in both mouse and human iPSC-derived neurons [#9]. As a recurrent oncogenic event, AGAP3 serves as a 5' fusion partner to BRAF, where it displaces the auto-inhibitory N-terminal region of BRAF to generate a constitutively active kinase that drives MAPK signaling and confers resistance to BRAF and EGFR inhibitors [#7, #8].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Established that the short CRAG splice variant, but not full-length AGAP3, can drive a specific transcriptional program, defining a domain-dependent nuclear function.\",\n      \"evidence\": \"Reporter assays, siRNA, dominant-negative and deletion mutagenesis in cultured neuronal cells\",\n      \"pmids\": [\"21832068\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism of how cytoplasmic vs nuclear isoform partitioning is controlled not defined\",\n        \"Direct DNA or co-factor binding by CRAG not yet mapped here\",\n        \"Physiological stimulus driving CRAG transcriptional activity not established\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed AGAP3 within the NMDA receptor complex and showed it is the link coupling receptor activation to Ras/ERK and Arf6 signaling and AMPA receptor trafficking during LTP.\",\n      \"evidence\": \"Reciprocal co-IP for complex membership plus shRNA knockdown with AMPA trafficking and signaling assays in rat primary neurons\",\n      \"pmids\": [\"23904596\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"ArfGAP catalytic activity toward Arf6 not directly demonstrated in this context\",\n        \"Direct binding partner within the NMDAR complex not resolved\",\n        \"Single lab, neuronal culture only\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified AGAP3 as a recurrent oncogenic 5' BRAF fusion partner conferring drug resistance, extending its biology into cancer signaling.\",\n      \"evidence\": \"Stable expression of AGAP3-BRAF in BRAFV600E melanoma cells with vemurafenib and MEK inhibitor sensitivity assays\",\n      \"pmids\": [\"28539463\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Structural basis of N-terminal displacement not shown here\",\n        \"Contribution of AGAP3 domains to the fusion phenotype not dissected\",\n        \"Resistance demonstrated in cell lines only\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked AGAP3 to a microtubule plus-end tracking protein network, suggesting a cytoskeletal-associated role outside neurons.\",\n      \"evidence\": \"AP-MS with SAINT and reciprocal co-IP in 3T3-L1 adipocytes\",\n      \"pmids\": [\"28550165\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Interaction membership only; no mechanistic consequence for AGAP3 established\",\n        \"+TIP function of AGAP3 not functionally tested\",\n        \"Adipocyte system only\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the molecular basis of CRAG's nuclear transcriptional activity, showing it is a GTPase that activates SRF via ELK1 at PML bodies and contributes to hippocampal c-fos induction in vivo.\",\n      \"evidence\": \"In vitro GTPase assay, CRAG-ELK1 co-IP, SUMO-interacting-motif mutants, and knockout mouse c-fos measurement\",\n      \"pmids\": [\"31882856\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"GTPase substrate/regulatory cycle of CRAG not characterized\",\n        \"How SUMO-interaction directs PML-body recruitment mechanistically unclear\",\n        \"Direct vs indirect role of ELK1 binding in SRF activation not separated\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed AGAP3 phosphorylation is insulin-regulated, embedding it in an insulin-responsive +TIP network.\",\n      \"evidence\": \"Targeted quantitative phosphoproteomics in 3T3-L1 adipocytes\",\n      \"pmids\": [\"31018989\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Single phosphoproteomic dataset with no functional follow-up for AGAP3\",\n        \"Responsible kinase and phosphosite consequence unknown\",\n        \"Physiological role of phosphorylation untested\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated mechanistically that the AGAP3-BRAF fusion activates BRAF by replacing its auto-inhibitory N-terminus and that the AGAP3 partner sets fusion localization and EGFR-inhibitor resistance.\",\n      \"evidence\": \"Patient-derived colorectal cancer organoids expressing fusion constructs with drug sensitivity, MAPK signaling, and localization readouts\",\n      \"pmids\": [\"31911540\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Precise structural mechanism of N-terminal displacement not resolved\",\n        \"Which AGAP3 domains drive localization not delineated\",\n        \"In vivo tumor relevance beyond organoids not established\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established a developmental requirement for CRAG/AGAP3 by linking its loss to failed dentate gyrus neuron maturation and behavioral hyperactivity.\",\n      \"evidence\": \"Forebrain-specific conditional knockout mice with doublecortin/calbindin immunohistochemistry and open-field testing\",\n      \"pmids\": [\"33811862\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular pathway linking CRAG loss to maturation arrest not defined\",\n        \"Whether the synaptic vs transcriptional function underlies the phenotype unresolved\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified SRRM2 as an upstream splicing regulator of AGAP3, connecting AGAP3 dosage to a nuclear speckle splicing factor across species.\",\n      \"evidence\": \"Srrm2+/- mouse splice/transcriptomic analysis and SRRM2-deficient human iPSC-derived neuron splicing analysis\",\n      \"pmids\": [\"42189682\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Which AGAP3 isoform (e.g. CRAG) is affected by mis-splicing not specified\",\n        \"Functional consequence of AGAP3 elevation in neurons not tested\",\n        \"Direct vs indirect SRRM2 action on the AGAP3 transcript not distinguished\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The ArfGAP catalytic activity of AGAP3 toward its physiological Arf substrate, and how the synaptic, nuclear-transcriptional, cytoskeletal, and oncogenic-fusion functions are mechanistically integrated, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No direct demonstration of Arf6 GAP catalysis by AGAP3 in the corpus\",\n        \"Structural model of full-length AGAP3 domain regulation absent\",\n        \"Relationship between isoform usage and each functional context not unified\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 7, 8]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [\n      \"NMDA receptor complex\"\n    ],\n    \"partners\": [\n      \"ELK1\",\n      \"CLASP2\",\n      \"CLIP2\",\n      \"BRAF\",\n      \"SRRM2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}