{"gene":"RGL3","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2000,"finding":"RGL3 was identified as a novel RalGEF-family protein that interacts with activated (GTP-bound) Rit and Ras in a GTP- and effector loop-dependent manner via a C-terminal 99-amino acid domain, and exhibits guanine nucleotide exchange activity toward the small GTPase Ral that is stimulated in vivo by expression of either activated Rit or Ras.","method":"Yeast two-hybrid screening for Rit-binding proteins; in vivo RalGEF activity assay; GTP-dependence and effector loop-dependence tests","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus in vivo GEF activity assay, single lab, two orthogonal methods","pmids":["10869344"],"is_preprint":false},{"year":2001,"finding":"RPM/RGL3 interacts selectively and strongly with GTP-bound M-Ras and p21 Ras; unlike other RalGEFs, transient expression of RPM/RGL3 did not activate an Elk-1 reporter gene but strongly inhibited Elk-1-dependent gene induction triggered by activated H-Ras or MEKK-1. This inhibitory effect was independent of the Ras binding domain and required a second signal from p21 Ras or MEKK-1 (but not Raf-1 or M-Ras). RPM/RGL3 also strongly inhibited growth of Src-Y527F-transformed fibroblasts.","method":"Yeast two-hybrid and co-immunoprecipitation for binding; Elk-1 reporter gene assay; domain deletion analysis; cell growth/transformation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assays plus binding studies plus domain deletions, single lab, multiple orthogonal methods","pmids":["11313946"],"is_preprint":false},{"year":2007,"finding":"Rgl3 was identified as a binding partner for Rap1, Rap2, H-Ras, N-Ras, and R-Ras (but not Ral or Rho) via yeast two-hybrid; Rgl3 localizes predominantly to the cytoplasm in fibroblasts; Rgl3 suppresses cell spreading induced by Rap1, R-Ras, or C3G-CAAX in HEK-293 cells; Rgl3 enhances focus formation by oncogenic H-Ras and N-Ras in NIH3T3 cells. Additionally, profilin II was identified as a binding partner for Rgl3 through its N-terminal proline cluster, and profilin II and Rgl3 cooperated to enhance N-Ras-induced focus formation.","method":"Yeast two-hybrid screening; subcellular localization by expression in fibroblasts; cell spreading assay; focus-formation assay; domain mapping by proline cluster mutation","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus cell-based functional assays, single lab, multiple assay types","pmids":["17382517"],"is_preprint":false},{"year":2010,"finding":"RGL3, along with RalGDS, Rgl1, and Rgl2, functions as a Ras effector RalGEF; plasma membrane targeting (mimicking persistent Ras activation) enhanced the growth-transforming activity of RGL3. Silencing of RGL3 in non-small cell lung cancer cell lines inhibited anchorage-dependent and anchorage-independent growth.","method":"Plasma membrane-targeting constructs; soft-agar growth assay; siRNA knockdown; cell growth assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional knockdown with growth phenotype plus membrane-targeting constructs, single lab, multiple assays","pmids":["20801877"],"is_preprint":false},{"year":2016,"finding":"RGL3 silencing in non-small cell lung cancer (NSCLC) cell lines inhibited cell population growth under anchorage-dependent and anchorage-independent conditions, demonstrating a functional role in supporting lung cancer cell proliferation.","method":"siRNA knockdown; anchorage-dependent and -independent growth assays","journal":"PloS one","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single knockdown approach with growth phenotype, single lab, limited mechanistic follow-up for RGL3 specifically","pmids":["27149377"],"is_preprint":false},{"year":2020,"finding":"High-resolution domain mapping (DoMY-Seq) confirmed the RIT1-RGL3 protein-protein interaction and mapped the interacting interface at high resolution, validating the interaction between RIT1 and RGL3.","method":"DoMY-Seq (yeast two-hybrid coupled with next-generation sequencing); domain mapping via fragment library","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid-based method, single lab, confirms known interaction with higher resolution but limited functional follow-up","pmids":["33410398"],"is_preprint":false},{"year":2006,"finding":"Rgl3 was identified as a phosphoprotein whose phosphorylation state significantly changed in response to short-term vasopressin treatment in rat inner medullary collecting duct cells, as detected by label-free quantitative phosphoproteomics.","method":"Phosphopeptide enrichment by IMAC; LC-MS/MS neutral loss scanning; label-free quantification","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Low","confidence_rationale":"Tier 3 / Weak — mass spectrometry-based phosphoproteomics, single study, no functional validation of the phosphorylation event for RGL3 specifically","pmids":["16641100"],"is_preprint":false}],"current_model":"RGL3 is a RalGEF-family guanine nucleotide exchange factor that activates the small GTPase Ral downstream of activated Ras and Rit GTPases: it binds GTP-loaded Ras (H-Ras, N-Ras, M-Ras), Rit, Rap1, Rap2, and R-Ras via a C-terminal Ras-binding domain, localizes predominantly to the cytoplasm, and promotes Ral activation in vivo; it also harbors an N-terminal proline cluster that recruits profilin II, influences actin-dependent cell morphology and oncogenic focus formation, and paradoxically inhibits Elk-1-dependent transcription downstream of H-Ras/MEKK-1 through a Ras-binding-domain-independent mechanism."},"narrative":{"mechanistic_narrative":"RGL3 is a RalGEF-family guanine nucleotide exchange factor that couples activated Ras-superfamily GTPases to Ral activation, functioning as a Ras effector in growth control [PMID:10869344, PMID:20801877]. Through a C-terminal Ras-binding domain it engages GTP-loaded Rit and Ras in a GTP- and effector-loop-dependent manner and catalyzes guanine nucleotide exchange on Ral in vivo when Rit or Ras is activated [PMID:10869344]; its binding repertoire extends to M-Ras, H-Ras, N-Ras, Rap1, Rap2, and R-Ras, but not Ral or Rho [PMID:11313946, PMID:17382517]. RGL3 localizes predominantly to the cytoplasm, and plasma-membrane targeting that mimics persistent Ras activation enhances its growth-transforming activity, while silencing it impairs anchorage-dependent and anchorage-independent growth of non-small cell lung cancer cells [PMID:17382517, PMID:20801877]. An N-terminal proline cluster recruits profilin II, and the two cooperate to enhance N-Ras-induced focus formation, linking RGL3 to actin-dependent control of cell spreading and morphology [PMID:17382517]. Distinct from its transforming role, RGL3 inhibits Elk-1-dependent transcription downstream of activated H-Ras or MEKK-1 through a Ras-binding-domain-independent mechanism [PMID:11313946].","teleology":[{"year":2000,"claim":"Established RGL3 as a bona fide RalGEF by showing it binds activated Rit and Ras through a defined C-terminal domain and stimulates Ral nucleotide exchange in vivo, placing it downstream of these GTPases.","evidence":"Yeast two-hybrid for Rit-binding partners with GTP- and effector-loop-dependence tests and an in vivo RalGEF activity assay","pmids":["10869344"],"confidence":"Medium","gaps":["No direct biochemical reconstitution of exchange activity with purified components","Endogenous RGL3-Ral coupling not demonstrated"]},{"year":2001,"claim":"Revealed a paradoxical signaling role: rather than activating Elk-1 like other RalGEFs, RGL3 binds GTP-bound M-Ras and Ras yet inhibits Ras/MEKK-1-driven Elk-1 transcription via a Ras-binding-domain-independent mechanism, and suppresses Src-transformed fibroblast growth.","evidence":"Yeast two-hybrid and co-immunoprecipitation binding, Elk-1 reporter assays, domain deletion analysis, and transformed cell growth assays","pmids":["11313946"],"confidence":"Medium","gaps":["The RBD-independent inhibitory mechanism and the required second signal are unidentified","Relationship between transcriptional inhibition and Ral activation unresolved"]},{"year":2007,"claim":"Broadened the GTPase interaction map and identified profilin II as an N-terminal proline-cluster partner, connecting RGL3 to actin-dependent morphology while showing opposing effects on cell spreading versus oncogenic focus formation.","evidence":"Yeast two-hybrid, cytoplasmic localization in fibroblasts, cell spreading and focus-formation assays, and proline-cluster mutation mapping","pmids":["17382517"],"confidence":"Medium","gaps":["Mechanism linking profilin II recruitment to focus formation undefined","How RGL3 suppresses spreading yet enhances transformation not reconciled"]},{"year":2010,"claim":"Demonstrated RGL3 functions in the Ras effector RalGEF family to support transformation, with membrane targeting enhancing its transforming activity and silencing impairing lung cancer cell growth.","evidence":"Plasma membrane-targeting constructs, soft-agar growth assays, and siRNA knockdown in NSCLC lines","pmids":["20801877"],"confidence":"Medium","gaps":["Whether the growth phenotype depends on Ral activation per se untested","No in vivo tumor model"]},{"year":2016,"claim":"Reinforced a functional requirement for RGL3 in lung cancer cell proliferation under both anchorage conditions.","evidence":"siRNA knockdown with anchorage-dependent and -independent growth assays in NSCLC lines","pmids":["27149377"],"confidence":"Low","gaps":["Single knockdown approach without rescue","Downstream effectors mediating the phenotype not identified"]},{"year":2020,"claim":"Resolved the RIT1-RGL3 interaction interface at high resolution, refining the structural basis of the GTPase-effector contact.","evidence":"DoMY-Seq (yeast two-hybrid coupled to next-generation sequencing) fragment-library domain mapping","pmids":["33410398"],"confidence":"Low","gaps":["Yeast two-hybrid-based mapping without structural or biochemical confirmation","Functional consequence of the mapped interface untested"]},{"year":2006,"claim":"Placed RGL3 within signal-responsive phosphoregulation by detecting vasopressin-dependent changes in its phosphorylation state.","evidence":"IMAC phosphopeptide enrichment with LC-MS/MS and label-free quantification in rat inner medullary collecting duct cells","pmids":["16641100"],"confidence":"Low","gaps":["Phosphosites and responsible kinase not defined","Functional effect of phosphorylation on RGL3 activity unknown"]},{"year":null,"claim":"How RGL3's RalGEF activity, its RBD-independent inhibition of Elk-1 transcription, and its profilin II-actin axis integrate into a single coherent function in normal and transformed cells remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of full-length RGL3","Endogenous substrate and effector hierarchy not established","Physiological context outside cancer cell lines undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,2]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,3]}],"complexes":[],"partners":["RIT1","HRAS","NRAS","MRAS","RAP1","RRAS","PFN2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q3MIN7","full_name":"Ral guanine nucleotide dissociation stimulator-like 3","aliases":[],"length_aa":710,"mass_kda":78.1,"function":"Guanine nucleotide exchange factor (GEF) for Ral-A. Potential effector of GTPase HRas and Ras-related protein M-Ras. Negatively regulates Elk-1-dependent gene induction downstream of HRas and MEKK1 (By similarity)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q3MIN7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RGL3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RGL3","total_profiled":1310},"omim":[{"mim_id":"616743","title":"RAL GUANINE NUCLEOTIDE DISSOCIATION STIMULATOR-LIKE 3; RGL3","url":"https://www.omim.org/entry/616743"},{"mim_id":"601991","title":"NOVA ALTERNATIVE SPLICING REGULATOR 2; NOVA2","url":"https://www.omim.org/entry/601991"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"kidney","ntpm":42.1},{"tissue":"parathyroid gland","ntpm":43.1},{"tissue":"thyroid gland","ntpm":61.9}],"url":"https://www.proteinatlas.org/search/RGL3"},"hgnc":{"alias_symbol":["FLJ32585"],"prev_symbol":[]},"alphafold":{"accession":"Q3MIN7","domains":[{"cath_id":"-","chopping":"13-38_60-66","consensus_level":"medium","plddt":79.5236,"start":13,"end":66},{"cath_id":"1.20.870.10","chopping":"74-118_141-200","consensus_level":"medium","plddt":91.789,"start":74,"end":200},{"cath_id":"1.10.840.10","chopping":"249-395_418-502","consensus_level":"high","plddt":91.637,"start":249,"end":502},{"cath_id":"3.10.20.90","chopping":"615-698","consensus_level":"high","plddt":81.0962,"start":615,"end":698}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q3MIN7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q3MIN7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q3MIN7-F1-predicted_aligned_error_v6.png","plddt_mean":72.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RGL3","jax_strain_url":"https://www.jax.org/strain/search?query=RGL3"},"sequence":{"accession":"Q3MIN7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q3MIN7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q3MIN7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q3MIN7"}},"corpus_meta":[{"pmid":"16641100","id":"PMC_16641100","title":"Quantitative phosphoproteomics of vasopressin-sensitive renal cells: regulation of aquaporin-2 phosphorylation at two sites.","date":"2006","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/16641100","citation_count":292,"is_preprint":false},{"pmid":"37248299","id":"PMC_37248299","title":"Polygenic prediction of preeclampsia and gestational hypertension.","date":"2023","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37248299","citation_count":105,"is_preprint":false},{"pmid":"18359847","id":"PMC_18359847","title":"Elucidating the germination transcriptional program using small molecules.","date":"2008","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18359847","citation_count":90,"is_preprint":false},{"pmid":"20478380","id":"PMC_20478380","title":"RalGDS family members couple Ras to Ral signalling and that's not all.","date":"2010","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/20478380","citation_count":66,"is_preprint":false},{"pmid":"28275852","id":"PMC_28275852","title":"GA-DELLA pathway is involved in regulation of nitrogen deficiency-induced anthocyanin accumulation.","date":"2017","source":"Plant cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/28275852","citation_count":65,"is_preprint":false},{"pmid":"10869344","id":"PMC_10869344","title":"A novel RalGEF-like protein, RGL3, as a candidate effector for rit and Ras.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10869344","citation_count":52,"is_preprint":false},{"pmid":"20801877","id":"PMC_20801877","title":"Aberrant overexpression of the Rgl2 Ral small GTPase-specific guanine nucleotide exchange factor promotes pancreatic cancer growth through Ral-dependent and Ral-independent mechanisms.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20801877","citation_count":51,"is_preprint":false},{"pmid":"21855169","id":"PMC_21855169","title":"GsGASA1 mediated root growth inhibition in response to chronic cold stress is marked by the accumulation of DELLAs.","date":"2011","source":"Journal of plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21855169","citation_count":48,"is_preprint":false},{"pmid":"23770287","id":"PMC_23770287","title":"Rit subfamily small GTPases: regulators in neuronal differentiation and survival.","date":"2013","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/23770287","citation_count":44,"is_preprint":false},{"pmid":"38548989","id":"PMC_38548989","title":"Protein-altering variants at copy number-variable regions influence diverse human phenotypes.","date":"2024","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/38548989","citation_count":36,"is_preprint":false},{"pmid":"11313946","id":"PMC_11313946","title":"A novel potential effector of M-Ras and p21 Ras negatively regulates p21 Ras-mediated gene induction and cell growth.","date":"2001","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/11313946","citation_count":32,"is_preprint":false},{"pmid":"33793917","id":"PMC_33793917","title":"The gibberellin signaling negative regulator RGA-LIKE3 promotes seed storage protein accumulation.","date":"2021","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/33793917","citation_count":29,"is_preprint":false},{"pmid":"23425858","id":"PMC_23425858","title":"The DELLA protein RGL3 positively contributes to jasmonate/ethylene defense responses.","date":"2013","source":"Plant signaling & behavior","url":"https://pubmed.ncbi.nlm.nih.gov/23425858","citation_count":22,"is_preprint":false},{"pmid":"33859359","id":"PMC_33859359","title":"Multi-ancestry genome-wide gene-sleep interactions identify novel loci for blood pressure.","date":"2021","source":"Molecular psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/33859359","citation_count":21,"is_preprint":false},{"pmid":"27149377","id":"PMC_27149377","title":"RalGPS2 Is Essential for Survival and Cell Cycle Progression of Lung Cancer Cells Independently of Its Established Substrates Ral GTPases.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27149377","citation_count":17,"is_preprint":false},{"pmid":"36219912","id":"PMC_36219912","title":"Polymorphisms of hypertension susceptibility genes as a risk factors of preeclampsia in the Caucasian population of central Russia.","date":"2022","source":"Placenta","url":"https://pubmed.ncbi.nlm.nih.gov/36219912","citation_count":16,"is_preprint":false},{"pmid":"17382517","id":"PMC_17382517","title":"Identification of Rgl3 as a potential binding partner for Rap-family small G-proteins and profilin II.","date":"2007","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/17382517","citation_count":15,"is_preprint":false},{"pmid":"36553520","id":"PMC_36553520","title":"Aggregation of Genome-Wide Association Data from FinnGen and UK Biobank Replicates Multiple Risk Loci for Pregnancy Complications.","date":"2022","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/36553520","citation_count":15,"is_preprint":false},{"pmid":"37176017","id":"PMC_37176017","title":"Risk Effects of rs1799945 Polymorphism of the HFE Gene and Intergenic Interactions of GWAS-Significant Loci for Arterial Hypertension in the Caucasian Population of Central Russia.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37176017","citation_count":13,"is_preprint":false},{"pmid":"33410398","id":"PMC_33410398","title":"DoMY-Seq: A yeast two-hybrid-based technique for precision mapping of protein-protein interaction motifs.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33410398","citation_count":8,"is_preprint":false},{"pmid":"38525669","id":"PMC_38525669","title":"Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.","date":"2024","source":"The Plant journal : for cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/38525669","citation_count":6,"is_preprint":false},{"pmid":"37333244","id":"PMC_37333244","title":"Hidden protein-altering variants influence diverse human phenotypes.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37333244","citation_count":3,"is_preprint":false},{"pmid":"38574408","id":"PMC_38574408","title":"Enhanced resolution profiling in twins reveals differential methylation signatures of type 2 diabetes with links to its complications.","date":"2024","source":"EBioMedicine","url":"https://pubmed.ncbi.nlm.nih.gov/38574408","citation_count":3,"is_preprint":false},{"pmid":"41400294","id":"PMC_41400294","title":"GA2ox1-1 modulates bioactive gibberellin levels to regulate the RGL3-MYB169 pathway in pear stone cell formation.","date":"2026","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/41400294","citation_count":2,"is_preprint":false},{"pmid":"36833422","id":"PMC_36833422","title":"Whole Exome Sequencing Reveals Novel Candidate Genes in Familial Forms of Glaucomatous Neurodegeneration.","date":"2023","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/36833422","citation_count":2,"is_preprint":false},{"pmid":"39126378","id":"PMC_39126378","title":"Morphological classification of radial glia-like cells in the postnatal mouse subventricular zone.","date":"2024","source":"The European journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/39126378","citation_count":1,"is_preprint":false},{"pmid":"41501475","id":"PMC_41501475","title":"Precision nutrition for hypertension: tea, coffee, antioxidant vitamins interactions with polygenic risk in multi-ethnic populations.","date":"2026","source":"European journal of clinical nutrition","url":"https://pubmed.ncbi.nlm.nih.gov/41501475","citation_count":1,"is_preprint":false},{"pmid":"41699318","id":"PMC_41699318","title":"Distinct radial glia subtypes regulate midbrain dopaminergic neuron development.","date":"2026","source":"Nature neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/41699318","citation_count":0,"is_preprint":false},{"pmid":"42037589","id":"PMC_42037589","title":"ARF6 integrates auxin and gibberellin signaling to promote stone cell lignification in pear via the HB49-MYB169 module.","date":"2026","source":"The New phytologist","url":"https://pubmed.ncbi.nlm.nih.gov/42037589","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16240,"output_tokens":2298,"usd":0.041595,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8816,"output_tokens":2574,"usd":0.054215,"stage2_stop_reason":"end_turn"},"total_usd":0.09581,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"Looking at the corpus, I need to distinguish between:\n1. **Human/mammalian RGL3** (a RalGEF family member, effector for Ras/Rit GTPases) - canonical protein\n2. **Arabidopsis RGL3** (RGA-LIKE3, a DELLA protein in gibberellin signaling) - plant ortholog with fundamentally different function/domain architecture\n3. **Radial glia subtypes (Rgl1, Rgl2, Rgl3)** - cellular classification, not the protein\n\nThe plant DELLA protein RGL3 is NOT an ortholog of human RGL3 - it has completely different domain architecture (DELLA domain, GRAS domain) and function (GA signaling repressor) incompatible with the mammalian RalGEF. These are symbol collisions in Arabidopsis. Papers [3], [5], [8], [12], [13], [21], [24], [29] are plant DELLA protein papers - EXCLUDE.\n\nPapers [26], [28] describe radial glia cell types (morphological/cellular classification), not the RGL3 protein - EXCLUDE.\n\nPapers [1], [2], [10], [14], [16], [17], [18], [19], [22], [23], [25], [27] - mostly GWAS/association/expression studies - largely exclude mechanistic content.\n\nPapers [4], [6], [7], [9], [11], [15], [17], [20] describe the mammalian RGL3 RalGEF protein mechanistically - INCLUDE.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"RGL3 was identified as a novel RalGEF-family protein that interacts with activated (GTP-bound) Rit and Ras in a GTP- and effector loop-dependent manner via a C-terminal 99-amino acid domain, and exhibits guanine nucleotide exchange activity toward the small GTPase Ral that is stimulated in vivo by expression of either activated Rit or Ras.\",\n      \"method\": \"Yeast two-hybrid screening for Rit-binding proteins; in vivo RalGEF activity assay; GTP-dependence and effector loop-dependence tests\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus in vivo GEF activity assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"10869344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"RPM/RGL3 interacts selectively and strongly with GTP-bound M-Ras and p21 Ras; unlike other RalGEFs, transient expression of RPM/RGL3 did not activate an Elk-1 reporter gene but strongly inhibited Elk-1-dependent gene induction triggered by activated H-Ras or MEKK-1. This inhibitory effect was independent of the Ras binding domain and required a second signal from p21 Ras or MEKK-1 (but not Raf-1 or M-Ras). RPM/RGL3 also strongly inhibited growth of Src-Y527F-transformed fibroblasts.\",\n      \"method\": \"Yeast two-hybrid and co-immunoprecipitation for binding; Elk-1 reporter gene assay; domain deletion analysis; cell growth/transformation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assays plus binding studies plus domain deletions, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"11313946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Rgl3 was identified as a binding partner for Rap1, Rap2, H-Ras, N-Ras, and R-Ras (but not Ral or Rho) via yeast two-hybrid; Rgl3 localizes predominantly to the cytoplasm in fibroblasts; Rgl3 suppresses cell spreading induced by Rap1, R-Ras, or C3G-CAAX in HEK-293 cells; Rgl3 enhances focus formation by oncogenic H-Ras and N-Ras in NIH3T3 cells. Additionally, profilin II was identified as a binding partner for Rgl3 through its N-terminal proline cluster, and profilin II and Rgl3 cooperated to enhance N-Ras-induced focus formation.\",\n      \"method\": \"Yeast two-hybrid screening; subcellular localization by expression in fibroblasts; cell spreading assay; focus-formation assay; domain mapping by proline cluster mutation\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus cell-based functional assays, single lab, multiple assay types\",\n      \"pmids\": [\"17382517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RGL3, along with RalGDS, Rgl1, and Rgl2, functions as a Ras effector RalGEF; plasma membrane targeting (mimicking persistent Ras activation) enhanced the growth-transforming activity of RGL3. Silencing of RGL3 in non-small cell lung cancer cell lines inhibited anchorage-dependent and anchorage-independent growth.\",\n      \"method\": \"Plasma membrane-targeting constructs; soft-agar growth assay; siRNA knockdown; cell growth assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional knockdown with growth phenotype plus membrane-targeting constructs, single lab, multiple assays\",\n      \"pmids\": [\"20801877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RGL3 silencing in non-small cell lung cancer (NSCLC) cell lines inhibited cell population growth under anchorage-dependent and anchorage-independent conditions, demonstrating a functional role in supporting lung cancer cell proliferation.\",\n      \"method\": \"siRNA knockdown; anchorage-dependent and -independent growth assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single knockdown approach with growth phenotype, single lab, limited mechanistic follow-up for RGL3 specifically\",\n      \"pmids\": [\"27149377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"High-resolution domain mapping (DoMY-Seq) confirmed the RIT1-RGL3 protein-protein interaction and mapped the interacting interface at high resolution, validating the interaction between RIT1 and RGL3.\",\n      \"method\": \"DoMY-Seq (yeast two-hybrid coupled with next-generation sequencing); domain mapping via fragment library\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid-based method, single lab, confirms known interaction with higher resolution but limited functional follow-up\",\n      \"pmids\": [\"33410398\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Rgl3 was identified as a phosphoprotein whose phosphorylation state significantly changed in response to short-term vasopressin treatment in rat inner medullary collecting duct cells, as detected by label-free quantitative phosphoproteomics.\",\n      \"method\": \"Phosphopeptide enrichment by IMAC; LC-MS/MS neutral loss scanning; label-free quantification\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — mass spectrometry-based phosphoproteomics, single study, no functional validation of the phosphorylation event for RGL3 specifically\",\n      \"pmids\": [\"16641100\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RGL3 is a RalGEF-family guanine nucleotide exchange factor that activates the small GTPase Ral downstream of activated Ras and Rit GTPases: it binds GTP-loaded Ras (H-Ras, N-Ras, M-Ras), Rit, Rap1, Rap2, and R-Ras via a C-terminal Ras-binding domain, localizes predominantly to the cytoplasm, and promotes Ral activation in vivo; it also harbors an N-terminal proline cluster that recruits profilin II, influences actin-dependent cell morphology and oncogenic focus formation, and paradoxically inhibits Elk-1-dependent transcription downstream of H-Ras/MEKK-1 through a Ras-binding-domain-independent mechanism.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RGL3 is a RalGEF-family guanine nucleotide exchange factor that couples activated Ras-superfamily GTPases to Ral activation, functioning as a Ras effector in growth control [#0, #3]. Through a C-terminal Ras-binding domain it engages GTP-loaded Rit and Ras in a GTP- and effector-loop-dependent manner and catalyzes guanine nucleotide exchange on Ral in vivo when Rit or Ras is activated [#0]; its binding repertoire extends to M-Ras, H-Ras, N-Ras, Rap1, Rap2, and R-Ras, but not Ral or Rho [#1, #2]. RGL3 localizes predominantly to the cytoplasm, and plasma-membrane targeting that mimics persistent Ras activation enhances its growth-transforming activity, while silencing it impairs anchorage-dependent and anchorage-independent growth of non-small cell lung cancer cells [#2, #3]. An N-terminal proline cluster recruits profilin II, and the two cooperate to enhance N-Ras-induced focus formation, linking RGL3 to actin-dependent control of cell spreading and morphology [#2]. Distinct from its transforming role, RGL3 inhibits Elk-1-dependent transcription downstream of activated H-Ras or MEKK-1 through a Ras-binding-domain-independent mechanism [#1].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established RGL3 as a bona fide RalGEF by showing it binds activated Rit and Ras through a defined C-terminal domain and stimulates Ral nucleotide exchange in vivo, placing it downstream of these GTPases.\",\n      \"evidence\": \"Yeast two-hybrid for Rit-binding partners with GTP- and effector-loop-dependence tests and an in vivo RalGEF activity assay\",\n      \"pmids\": [\"10869344\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct biochemical reconstitution of exchange activity with purified components\", \"Endogenous RGL3-Ral coupling not demonstrated\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Revealed a paradoxical signaling role: rather than activating Elk-1 like other RalGEFs, RGL3 binds GTP-bound M-Ras and Ras yet inhibits Ras/MEKK-1-driven Elk-1 transcription via a Ras-binding-domain-independent mechanism, and suppresses Src-transformed fibroblast growth.\",\n      \"evidence\": \"Yeast two-hybrid and co-immunoprecipitation binding, Elk-1 reporter assays, domain deletion analysis, and transformed cell growth assays\",\n      \"pmids\": [\"11313946\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The RBD-independent inhibitory mechanism and the required second signal are unidentified\", \"Relationship between transcriptional inhibition and Ral activation unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Broadened the GTPase interaction map and identified profilin II as an N-terminal proline-cluster partner, connecting RGL3 to actin-dependent morphology while showing opposing effects on cell spreading versus oncogenic focus formation.\",\n      \"evidence\": \"Yeast two-hybrid, cytoplasmic localization in fibroblasts, cell spreading and focus-formation assays, and proline-cluster mutation mapping\",\n      \"pmids\": [\"17382517\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking profilin II recruitment to focus formation undefined\", \"How RGL3 suppresses spreading yet enhances transformation not reconciled\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated RGL3 functions in the Ras effector RalGEF family to support transformation, with membrane targeting enhancing its transforming activity and silencing impairing lung cancer cell growth.\",\n      \"evidence\": \"Plasma membrane-targeting constructs, soft-agar growth assays, and siRNA knockdown in NSCLC lines\",\n      \"pmids\": [\"20801877\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the growth phenotype depends on Ral activation per se untested\", \"No in vivo tumor model\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Reinforced a functional requirement for RGL3 in lung cancer cell proliferation under both anchorage conditions.\",\n      \"evidence\": \"siRNA knockdown with anchorage-dependent and -independent growth assays in NSCLC lines\",\n      \"pmids\": [\"27149377\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single knockdown approach without rescue\", \"Downstream effectors mediating the phenotype not identified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved the RIT1-RGL3 interaction interface at high resolution, refining the structural basis of the GTPase-effector contact.\",\n      \"evidence\": \"DoMY-Seq (yeast two-hybrid coupled to next-generation sequencing) fragment-library domain mapping\",\n      \"pmids\": [\"33410398\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Yeast two-hybrid-based mapping without structural or biochemical confirmation\", \"Functional consequence of the mapped interface untested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed RGL3 within signal-responsive phosphoregulation by detecting vasopressin-dependent changes in its phosphorylation state.\",\n      \"evidence\": \"IMAC phosphopeptide enrichment with LC-MS/MS and label-free quantification in rat inner medullary collecting duct cells\",\n      \"pmids\": [\"16641100\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Phosphosites and responsible kinase not defined\", \"Functional effect of phosphorylation on RGL3 activity unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RGL3's RalGEF activity, its RBD-independent inhibition of Elk-1 transcription, and its profilin II-actin axis integrate into a single coherent function in normal and transformed cells remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of full-length RGL3\", \"Endogenous substrate and effector hierarchy not established\", \"Physiological context outside cancer cell lines undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RIT1\", \"HRAS\", \"NRAS\", \"MRAS\", \"RAP1\", \"RRAS\", \"PFN2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}