{"gene":"RASGRP3","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2000,"finding":"CalDAG-GEFIII (RasGRP3) functions as a guanine nucleotide exchange factor (GEF) with broad substrate specificity, directly catalyzing GDP-to-GTP exchange on Ha-Ras, R-Ras, and Rap1 in vitro and in cells, and activating ERK/MAPK signaling.","method":"In vitro GEF assay (GTP/GDP ratio measurement), transfection in 293T cells, PC12 neuronal differentiation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro GEF reconstitution with multiple substrates, confirmed in cells","pmids":["10835426"],"is_preprint":false},{"year":2001,"finding":"RasGRP3 binds phorbol esters via its C1 domain in an anionic phospholipid-dependent manner, and phorbol ester or diacylglycerol (DAG) binding causes RasGRP3 translocation to the plasma membrane/perinuclear area and activates its Ras exchange activity in intact cells.","method":"Phorbol ester binding assay, GFP-RasGRP3 fluorescence microscopy, RasGTP pull-down, ERK phosphorylation assay in HEK-293 cells","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1-2 — direct binding assay plus live-cell translocation and downstream signaling, multiple orthogonal methods","pmids":["11221888"],"is_preprint":false},{"year":2003,"finding":"RasGRP3 is phosphorylated upon BCR stimulation coincident with Ras activation; PKC inhibition attenuates both RasGRP3 phosphorylation and Ras activation. PKC-theta and PKC-beta2 phosphorylate RasGRP3 in vitro, and a dominant-active PKC-theta enhances Ras-ERK signaling via RasGRP3 when co-expressed in HEK-293 cells.","method":"In vitro kinase assay with PKC-theta and PKC-beta2, co-expression with dominant-active PKC-theta, PKC inhibitor studies, membrane fractionation, Ras activation assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro phosphorylation confirmed, epistasis via inhibitors, replicated in two cell types","pmids":["12730099"],"is_preprint":false},{"year":2004,"finding":"PKC phosphorylates RasGRP3 specifically on Thr133; the Thr133Ala substitution abolishes PKC-dependent phosphorylation in vitro and severely impairs RasGRP3-mediated Ras activation in vivo after BCR stimulation. PKC activity (conventional PKCs) is required for Thr133 phosphorylation and full Ras-ERK activation.","method":"Mass spectrometry to identify phosphorylation site, in vitro kinase assay, site-directed mutagenesis (Thr133Ala), antiphospho-peptide antibody, PKC inhibitors, BCR stimulation of B cells","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 — site identified by mass spectrometry, mutagenesis confirms functional requirement, replicated across two labs (PMID 15657177 and 15545601)","pmids":["15657177","15545601"],"is_preprint":false},{"year":2004,"finding":"PKCdelta physically associates with RasGRP3 upon PMA treatment (co-immunoprecipitation, colocalization in perinuclear region) and phosphorylates RasGRP3 in vitro; a PKCdelta kinase-dead mutant blocks the PMA-induced mobility shift of RasGRP3.","method":"Co-immunoprecipitation, in vitro kinase assay, immunofluorescence colocalization, dominant-negative PKCdelta, rottlerin inhibitor","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1-2 — reciprocal co-IP plus in vitro kinase assay plus dominant-negative, multiple orthogonal methods","pmids":["15213298"],"is_preprint":false},{"year":2004,"finding":"RasGRP3 activates Rap2B, leading to Rap2B-dependent translocation of PLC-epsilon to the plasma membrane and PLC/Ca2+ signaling downstream of the EGF receptor. EGF induces tyrosine phosphorylation of RasGRP3 by c-Src, and c-Src inhibition blocks both Rap2B activation and PLC stimulation.","method":"Dominant-negative Rap2B expression, clostridial toxin inactivation, co-expression of RasGRP3, GTP-loading assay for Rap2B, c-Src inhibition, Ca2+ signaling measurement in HEK-293 cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — epistasis by dominant-negative, pharmacological inhibition, and GTP-loading assays, multiple orthogonal approaches","pmids":["15143162"],"is_preprint":false},{"year":2004,"finding":"RasGRP3 is expressed in embryonic blood vessels and is specifically required for the aberrant endothelial morphogenesis induced by phorbol ester (PMA); RasGRP3 loss-of-function makes vessels refractory to PMA-induced dysmorphogenesis. RasGRP3 expression is upregulated by VEGF stimulation of endothelial cells.","method":"ES cell gene trap screen, in situ hybridization, loss-of-function mouse model, ES cell-derived vascular morphogenesis assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — genetic loss-of-function with specific vascular phenotype, replicated in mouse model and ES-derived vessels","pmids":["15572660"],"is_preprint":false},{"year":2005,"finding":"Both RasGRP1 and RasGRP3 contribute to BCR-induced Ras and ERK activation in B cells; RasGRP3 alone maintains basal Ras-GTP levels in resting B cells. Loss of RasGRP3 causes isotype-specific antibody deficiencies and hypogammaglobulinemia. BCR-induced B cell proliferation is RasGRP1- and RasGRP3-dependent.","method":"Single and double null mutant mice (Rasgrp1-/-, Rasgrp3-/-, double KO), Ras-GTP pull-down, ERK phosphorylation, B cell proliferation assays, serum immunoglobulin measurement","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with double KO mice, multiple orthogonal readouts","pmids":["16301621"],"is_preprint":false},{"year":2005,"finding":"Fluorescent phorbol esters induce translocation of RasGRP3 to intracellular membranes (primarily perinuclear/intracellular), and RasGRP3 colocalizes with the fluorescent phorbol ester. The lipophilicity of the phorbol ester determines kinetics and pattern of RasGRP3 redistribution.","method":"Fluorescent phorbol ester live-cell imaging, GFP-RasGRP3 fusion protein colocalization in CHO cells","journal":"Molecular cancer therapeutics","confidence":"Medium","confidence_rationale":"Tier 3 — direct localization experiment with functional implication, single lab","pmids":["15657361"],"is_preprint":false},{"year":2005,"finding":"RasGRP3 mediates phorbol ester-induced exocytosis in a PKC-independent manner; RasGRP3 is expressed in endocrine tissues and its effects on exocytosis are not blocked by MEK inhibitor but are partially sensitive to PKC inhibitor.","method":"Exocytosis assay in endocrine cells, PKC inhibitor, MEK inhibitor, ERK phosphorylation readout","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, pharmacological dissection of mechanism, partial follow-up","pmids":["15737652"],"is_preprint":false},{"year":2006,"finding":"RasGRP3 interacts with dynein light chain 1 (DLC1) through its C-terminal 127 amino acids; this interaction was confirmed in vitro and by co-immunoprecipitation. Deletion of the C-terminal domain abolishes DLC1 interaction and dramatically alters RasGRP3 subcellular localization (strong reticular/perinuclear/nuclear distribution).","method":"Yeast two-hybrid screen, in vitro pull-down, co-immunoprecipitation, subcellular localization of truncation mutant (fluorescence microscopy)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — yeast two-hybrid confirmed by in vitro pulldown and co-IP, domain mapping by mutagenesis, localization consequence demonstrated","pmids":["17012239"],"is_preprint":false},{"year":2010,"finding":"RasGRP3 is required for Ras-GTP formation, AKT and ERK1/2 phosphorylation, cell proliferation, migration, and anchorage-independent growth in prostate cancer cells. RasGRP3 overexpression in LNCaP cells elevates Ras-GTP, stimulates proliferation, and confers resistance to PMA-induced apoptosis.","method":"siRNA knockdown and overexpression, Ras-GTP pull-down, AKT/ERK phosphorylation, proliferation assay, migration assay, mouse xenograft","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — reciprocal gain/loss-of-function with multiple cellular readouts and in vivo xenograft","pmids":["20876802"],"is_preprint":false},{"year":2011,"finding":"RasGRP3 is required for Ras-GTP formation, Akt phosphorylation, c-Met expression, and cell proliferation/transformation in human melanoma cells. Overexpression of RasGRP3 in primary melanocytes alters morphology and induces tumorigenicity in mouse xenografts.","method":"siRNA knockdown, overexpression in primary melanocytes, Ras-GTP pull-down, Akt phosphorylation, soft agar colony formation, mouse xenograft","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — reciprocal gain/loss-of-function, multiple downstream readouts, in vivo validation","pmids":["21602881"],"is_preprint":false},{"year":2011,"finding":"RasGRP3 in endothelial cells promotes Ras-ERK signaling and endothelin-1-stimulated cell migration; Rasgrp3 loss-of-function attenuates Ras-ERK signaling and abolishes endothelin-1-induced migration, and embryos lacking Rasgrp3 are dramatically protected from diabetes-induced vascular developmental defects.","method":"Loss-of-function mouse model (Rasgrp3 null), endothelial cell migration assay, Ras-ERK signaling measurement, diabetic mouse model, primary endothelial cells with activated RasGRP3","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 — genetic loss-of-function in mouse with in vivo developmental phenotype and complementary cell-based mechanistic studies","pmids":["21474816"],"is_preprint":false},{"year":2014,"finding":"RasGRP3 activates Rap1 upon low-level TLR stimulation in macrophages, which limits production of pro-inflammatory cytokines (especially IL-6). CRISPR-Cas9 deletion of RasGRP3 in RAW264.7 cells inhibits TLR3/4/9-induced Rap1 activation while enhancing ERK1/2 activation and IL-6 production.","method":"CRISPR-Cas9 knockout in RAW264.7, Rap1 activity assay, ERK1/2 phosphorylation, cytokine ELISA, DSS-colitis and collagen-induced arthritis mouse models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — clean CRISPR KO with defined molecular phenotype (Rap1 activation), in vivo validation, multiple orthogonal methods","pmids":["25118589"],"is_preprint":false},{"year":2015,"finding":"RasGRP3 interacts with Arp3 (actin-related protein), as identified by pull-down/mass spectrometry and confirmed by co-immunoprecipitation and immunofluorescence. PMA-induced translocation of RasGRP3 increases its association with Arp3. Arp3 silencing partially reduces RasGRP3-mediated glioma cell migration.","method":"Pull-down assay with mass spectrometry, co-immunoprecipitation, immunofluorescence colocalization, siRNA knockdown of Arp3, migration assay","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2-3 — interaction confirmed by orthogonal methods, functional link via Arp3 knockdown, single lab","pmids":["25682201"],"is_preprint":false},{"year":2017,"finding":"In GNAQ-mutant uveal melanoma, RasGRP3 is phosphorylated and activated by PKC-delta and PKC-epsilon, enabling Ras-MAPK pathway activation. RasGRP3 activation also occurs through PKC-independent DAG-mediated membrane recruitment. RasGRP3 knockdown suppresses MAPK activation, identifying RasGRP3 as the mechanistic link between Gαq signaling and MAPK in uveal melanoma.","method":"Knockdown (siRNA/shRNA), PKC isoform-specific studies, Ras-GTP assay, ERK phosphorylation, membrane fractionation, uveal melanoma cell lines and patient samples","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, PKC isoform identification, replicated in multiple UM cell lines","pmids":["28486107"],"is_preprint":false},{"year":2018,"finding":"In a GNA11Q209L mouse model, RasGRP3 is specifically required for GNAQ/GNA11-driven Ras activation and tumorigenesis; integrative transcriptome analysis identified RasGRP3 as selectively expressed in Gq-driven melanomas, and its loss suppresses tumor formation.","method":"Transgenic GNA11Q209L mouse model, Bap1 conditional KO, integrative transcriptome analysis, RasGRP3 knockdown in human UM cell lines, Ras activation assay, in vivo tumor models","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — genetic mouse model plus human cell line validation, integrative transcriptomics, loss-of-function with defined phenotype","pmids":["29490280"],"is_preprint":false},{"year":2018,"finding":"Alpha-arylidene DAG-lactones can selectively bind the RasGRP3 C1 domain with up to 73-fold selectivity over PKCα and 45-fold over PKCε in vitro, and selectively activate Ras (via RasGRP3) over PKCδ phosphorylation in intact cells.","method":"In vitro C1-domain binding assay (competitive displacement), intact cell Ras activation assay, PKCδ phosphorylation assay","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 1-3 — in vitro binding and cell-based selectivity, but pharmacological tool study without mutagenesis","pmids":["29860841"],"is_preprint":false},{"year":2023,"finding":"In NPM1-mutant AML, cytoplasmic NPM1-mA binds E3 ubiquitin ligase MID1, blocking MID1-mediated degradation of RasGRP3 and thus stabilizing RasGRP3 protein. Stabilized RasGRP3 activates the EGFR-STAT3 axis to promote AML cell proliferation and autophagy.","method":"Co-immunoprecipitation, Western blot, cycloheximide chase assay, CCK8, EdU staining, immunofluorescence","journal":"Journal of leukocyte biology","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP for interaction, cycloheximide chase for stability, downstream signaling confirmed, single lab","pmids":["36826998"],"is_preprint":false},{"year":2023,"finding":"AC092894.1 lncRNA acts as a scaffold mediating USP3-dependent de-ubiquitination of AR, which then drives transcription of RASGRP3 to sustain MAPK signaling in colorectal cancer cells; loss of this axis promotes oxaliplatin resistance.","method":"RNA pull-down, RIP, co-immunoprecipitation, gain/loss-of-function experiments, in vivo mouse model","journal":"BMC medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple biochemical methods for scaffold mechanism, in vivo validation, single lab","pmids":["37013584"],"is_preprint":false},{"year":2026,"finding":"RasGRP3 promotes RAP1B GTP-loading (exchange factor activity) in endothelial cells, thereby inhibiting NF-κB pathway activation and reducing pro-inflammatory cytokine production and monocyte adhesion. UHRF1 (E3 ubiquitin ligase) binds RasGRP3 and promotes its ubiquitination and degradation; UHRF1 knockdown increases RasGRP3 protein levels.","method":"RasGRP3 overexpression in endothelial cells and ApoE-/- mice, RAP1 activity assay, NF-κB pathway assay, cytokine ELISA, THP-1 adhesion assay, co-immunoprecipitation for UHRF1-RasGRP3 interaction","journal":"Inflammation","confidence":"Medium","confidence_rationale":"Tier 2-3 — RAP1B activation confirmed, ubiquitination by UHRF1 identified by co-IP, in vivo atherosclerosis model, single lab","pmids":["41689678"],"is_preprint":false}],"current_model":"RasGRP3 is a DAG/phorbol ester-regulated guanine nucleotide exchange factor (GEF) that activates Ras, R-Ras, and Rap1; it is recruited to membranes via its C1 domain upon DAG/phorbol ester binding and is further activated by PKC-mediated phosphorylation on Thr133, integrating DAG signals from receptors (BCR, TCR, EGF receptor, Gαq-coupled GPCRs) into Ras-MAPK and Rap1 effector outputs in B cells, endothelial cells, macrophages, and cancer cells, and interacts with binding partners including dynein light chain 1 (regulating localization), Arp3 (regulating migration), and is subject to ubiquitin-mediated degradation regulated by UHRF1 and MID1."},"narrative":{"teleology":[{"year":2000,"claim":"Establishing that RasGRP3 is a bona fide GEF with broad Ras-family substrate specificity resolved whether the CalDAG-GEFIII gene product had direct catalytic exchange activity and defined H-Ras, R-Ras, and Rap1 as its substrates.","evidence":"In vitro GEF assay measuring GDP/GTP exchange on purified GTPases, confirmed by transfection in 293T cells and PC12 differentiation","pmids":["10835426"],"confidence":"High","gaps":["Relative substrate preference under physiological DAG concentrations unknown","No structural basis for multi-substrate recognition"]},{"year":2001,"claim":"Demonstrating that the C1 domain binds DAG/phorbol esters and drives membrane translocation established the activation mechanism linking receptor-generated DAG to RasGRP3 catalytic output.","evidence":"Phorbol ester binding assay, GFP-RasGRP3 live-cell imaging, Ras-GTP pull-down in HEK-293 cells","pmids":["11221888"],"confidence":"High","gaps":["Contribution of EF-hand and PT domains to membrane targeting not fully dissected","Whether C1-domain lipid selectivity differs from PKC C1 domains was untested"]},{"year":2004,"claim":"Identification of Thr133 as the PKC phosphorylation site required for full RasGRP3 activation revealed a two-signal activation model: DAG recruits RasGRP3 to membranes, and PKC phosphorylation (by PKCθ, PKCβ2, or PKCδ) amplifies its GEF output.","evidence":"Mass spectrometry, Thr133Ala mutagenesis, in vitro kinase assays with multiple PKC isoforms, BCR stimulation in B cells, co-IP of PKCδ–RasGRP3","pmids":["15657177","15545601","15213298"],"confidence":"High","gaps":["Structural mechanism by which Thr133 phosphorylation enhances catalysis is unknown","Relative contributions of individual PKC isoforms in different cell types not resolved"]},{"year":2004,"claim":"Showing that RasGRP3 activates Rap2B downstream of EGF/c-Src to drive PLCε translocation extended the signaling repertoire beyond Ras-ERK and linked RasGRP3 to calcium signaling cascades.","evidence":"Dominant-negative Rap2B, c-Src inhibition, Rap2B GTP-loading assay, Ca²⁺ measurement in HEK-293 cells","pmids":["15143162"],"confidence":"High","gaps":["Whether this Rap2B–PLCε axis operates in primary cell types is untested","Tyrosine phosphorylation site on RasGRP3 by c-Src not mapped"]},{"year":2005,"claim":"Genetic studies in Rasgrp1/Rasgrp3 single- and double-knockout mice established that RasGRP3 maintains basal Ras-GTP in resting B cells, cooperates with RasGRP1 for BCR-induced ERK activation, and is required for normal immunoglobulin production.","evidence":"Single and double null mice, Ras-GTP pull-down, B cell proliferation, serum immunoglobulin measurement","pmids":["16301621"],"confidence":"High","gaps":["Which immunoglobulin class-switch step is RasGRP3-dependent is not defined","Whether B cell developmental stages are differentially affected is unclear"]},{"year":2004,"claim":"Loss-of-function in the embryonic vasculature demonstrated a non-immune role for RasGRP3: it is required for phorbol ester–induced endothelial dysmorphogenesis and is upregulated by VEGF, establishing RasGRP3 as a vascular DAG effector.","evidence":"Gene-trap mouse model, in situ hybridization, ES cell–derived vascular morphogenesis assay","pmids":["15572660"],"confidence":"High","gaps":["Downstream GTPase substrate (Ras vs. Rap1) mediating vascular phenotype not identified","Relationship to VEGF receptor signaling pathway not fully mapped"]},{"year":2006,"claim":"Discovery that dynein light chain 1 (DLC1) binds the C-terminal domain of RasGRP3 and controls its subcellular distribution revealed a cytoskeletal-dependent localization mechanism for this GEF.","evidence":"Yeast two-hybrid, in vitro pull-down, co-IP, fluorescence microscopy of truncation mutant","pmids":["17012239"],"confidence":"High","gaps":["Functional consequence of DLC1 interaction on Ras/Rap activation not tested","Whether dynein motor activity is required for RasGRP3 transport unknown"]},{"year":2011,"claim":"Gain- and loss-of-function studies in prostate cancer and melanoma cells showed that RasGRP3 overexpression is sufficient to drive Ras-AKT/ERK signaling, proliferation, and tumorigenicity, positioning it as an oncogenic driver in non-immune cancers.","evidence":"siRNA knockdown and overexpression, Ras-GTP pull-down, soft agar assay, mouse xenograft in prostate cancer and melanoma models","pmids":["20876802","21602881"],"confidence":"High","gaps":["Mechanism of RasGRP3 overexpression in these cancers (amplification, transcriptional, post-translational) not established","Whether RasGRP3 signals through Ras or Rap in these contexts is unresolved"]},{"year":2014,"claim":"CRISPR knockout in macrophages revealed that RasGRP3 preferentially activates Rap1 (not Ras) upon TLR stimulation and thereby dampens inflammatory cytokine production, establishing a substrate-selective anti-inflammatory role.","evidence":"CRISPR-Cas9 KO in RAW264.7, Rap1 activity assay, cytokine ELISA, DSS-colitis and collagen-induced arthritis mouse models","pmids":["25118589"],"confidence":"High","gaps":["Mechanism determining Rap1 vs. Ras substrate selectivity in macrophages unknown","Cell-type–specific transcriptional regulation of RasGRP3 in myeloid lineage not characterized"]},{"year":2017,"claim":"Identification of RasGRP3 as the obligate link between oncogenic GNAQ/GNA11 and MAPK in uveal melanoma solved a long-standing question about how constitutive Gαq signals reach Ras, revealing dual activation by PKC phosphorylation and DAG-mediated membrane recruitment.","evidence":"siRNA/shRNA knockdown, PKC isoform–specific studies, Ras-GTP and ERK assays in multiple UM cell lines, patient tumor analysis; confirmed by GNA11Q209L transgenic mouse model","pmids":["28486107","29490280"],"confidence":"High","gaps":["Whether pharmacological targeting of RasGRP3 C1 domain can suppress UM growth in vivo is untested","Structural basis for RasGRP3 selectivity by DAG-lactone agonists incompletely understood"]},{"year":2023,"claim":"Discovery that MID1 and UHRF1 are E3 ubiquitin ligases targeting RasGRP3 for proteasomal degradation established post-translational stability control as a key regulatory layer, with pathological stabilization occurring in NPM1-mutant AML.","evidence":"Co-IP, cycloheximide chase, Western blot for NPM1-mA/MID1/RasGRP3 axis in AML cells; co-IP and ubiquitination assay for UHRF1–RasGRP3 in endothelial cells and ApoE−/− mice","pmids":["36826998","41689678"],"confidence":"Medium","gaps":["Ubiquitination sites on RasGRP3 not mapped","Relative contributions of MID1 vs. UHRF1 in different tissues unknown","Findings from single labs, independent replication pending"]},{"year":null,"claim":"A structural understanding of how DAG binding, Thr133 phosphorylation, and substrate selectivity are integrated within the RasGRP3 catalytic cycle, and the in vivo therapeutic potential of C1-domain–selective ligands, remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure of RasGRP3","In vivo efficacy of RasGRP3-selective DAG-lactones not demonstrated","Mechanism of Ras vs. Rap substrate selection in different cell types undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,5,14,21]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[1,8,18]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,8,16]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,10]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,3,5,11,12,16,17]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[11,12,16,17]}],"complexes":[],"partners":["DYNLL1","ACTR3","PRKCD","PRKCQ","PRKCB","MID1","UHRF1","RAP2B"],"other_free_text":[]},"mechanistic_narrative":"RasGRP3 is a diacylglycerol (DAG)/phorbol ester–regulated guanine nucleotide exchange factor (GEF) that integrates lipid second-messenger signals into Ras-family GTPase activation across immune, endothelial, and epithelial cell types. It catalyzes GDP-to-GTP exchange on H-Ras, R-Ras, Rap1, and Rap2B, and is recruited to membranes via its C1 domain upon DAG or phorbol ester binding; full catalytic activation additionally requires PKC-mediated phosphorylation on Thr133, with PKCδ, PKCε, PKCθ, and PKCβ2 each capable of this modification [PMID:10835426, PMID:11221888, PMID:15657177, PMID:15213298, PMID:28486107]. In B cells, RasGRP3 cooperates with RasGRP1 to drive BCR-induced Ras-ERK signaling and proliferation, and Rasgrp3-null mice display hypogammaglobulinemia; in macrophages, RasGRP3 instead preferentially activates Rap1 upon TLR stimulation to restrain pro-inflammatory cytokine output [PMID:16301621, PMID:25118589]. RasGRP3 protein stability is controlled by ubiquitin-dependent degradation mediated by the E3 ligases UHRF1 and MID1, and in GNAQ/GNA11-mutant uveal melanoma RasGRP3 serves as the obligate link between constitutive Gαq signaling and MAPK pathway activation [PMID:28486107, PMID:29490280, PMID:41689678, PMID:36826998]."},"prefetch_data":{"uniprot":{"accession":"Q8IV61","full_name":"Ras guanyl-releasing protein 3","aliases":["Calcium and DAG-regulated guanine nucleotide exchange factor III","Guanine nucleotide exchange factor for Rap1"],"length_aa":690,"mass_kda":78.3,"function":"Guanine nucleotide exchange factor (GEF) for Ras and Rap1","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q8IV61/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RASGRP3","classification":"Not Classified","n_dependent_lines":10,"n_total_lines":1208,"dependency_fraction":0.008278145695364239},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RASGRP3","total_profiled":1310},"omim":[{"mim_id":"609531","title":"RAS GUANYL NUCLEOTIDE-RELEASING PROTEIN 3; RASGRP3","url":"https://www.omim.org/entry/609531"},{"mim_id":"607320","title":"RAS GUANYL NUCLEOTIDE-RELEASING PROTEIN 4; RASGRP4","url":"https://www.omim.org/entry/607320"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Golgi apparatus","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":47.4},{"tissue":"skeletal muscle","ntpm":39.3}],"url":"https://www.proteinatlas.org/search/RASGRP3"},"hgnc":{"alias_symbol":["KIAA0846","GRP3","CalDAG-GEFIII"],"prev_symbol":[]},"alphafold":{"accession":"Q8IV61","domains":[{"cath_id":"1.20.870.10","chopping":"10-127","consensus_level":"high","plddt":89.889,"start":10,"end":127},{"cath_id":"1.10.840.10","chopping":"154-384_402-406","consensus_level":"high","plddt":87.6806,"start":154,"end":406},{"cath_id":"1.10.238.10","chopping":"413-488","consensus_level":"medium","plddt":69.0263,"start":413,"end":488},{"cath_id":"3.30.60.20","chopping":"492-548","consensus_level":"medium","plddt":78.956,"start":492,"end":548}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IV61","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IV61-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8IV61-F1-predicted_aligned_error_v6.png","plddt_mean":69.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RASGRP3","jax_strain_url":"https://www.jax.org/strain/search?query=RASGRP3"},"sequence":{"accession":"Q8IV61","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8IV61.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8IV61/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8IV61"}},"corpus_meta":[{"pmid":"28486107","id":"PMC_28486107","title":"RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma.","date":"2017","source":"Cancer cell","url":"https://pubmed.ncbi.nlm.nih.gov/28486107","citation_count":129,"is_preprint":false},{"pmid":"10835426","id":"PMC_10835426","title":"CalDAG-GEFIII activation of Ras, R-ras, and Rap1.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10835426","citation_count":124,"is_preprint":false},{"pmid":"16301621","id":"PMC_16301621","title":"RasGRP1 and RasGRP3 regulate B cell proliferation by facilitating B cell receptor-Ras signaling.","date":"2005","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/16301621","citation_count":111,"is_preprint":false},{"pmid":"12730099","id":"PMC_12730099","title":"Integration of DAG signaling systems mediated by PKC-dependent phosphorylation of RasGRP3.","date":"2003","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/12730099","citation_count":104,"is_preprint":false},{"pmid":"11221888","id":"PMC_11221888","title":"Phorbol esters modulate the Ras exchange factor RasGRP3.","date":"2001","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/11221888","citation_count":90,"is_preprint":false},{"pmid":"15657177","id":"PMC_15657177","title":"Phosphorylation of RasGRP3 on threonine 133 provides a mechanistic link between PKC and Ras signaling systems in B cells.","date":"2005","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/15657177","citation_count":86,"is_preprint":false},{"pmid":"29490280","id":"PMC_29490280","title":"GNA11 Q209L Mouse Model Reveals RasGRP3 as an Essential Signaling Node in Uveal Melanoma.","date":"2018","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/29490280","citation_count":69,"is_preprint":false},{"pmid":"15545601","id":"PMC_15545601","title":"Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation.","date":"2004","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/15545601","citation_count":65,"is_preprint":false},{"pmid":"26748394","id":"PMC_26748394","title":"GRP-3 and KAPP, encoding interactors of WAK1, negatively affect defense responses induced by oligogalacturonides and local response to wounding.","date":"2016","source":"Journal of experimental botany","url":"https://pubmed.ncbi.nlm.nih.gov/26748394","citation_count":64,"is_preprint":false},{"pmid":"25118589","id":"PMC_25118589","title":"RasGRP3 limits Toll-like receptor-triggered inflammatory response in macrophages by activating Rap1 small GTPase.","date":"2014","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/25118589","citation_count":50,"is_preprint":false},{"pmid":"15143162","id":"PMC_15143162","title":"Rap2B-dependent stimulation of 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\"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro GEF reconstitution with multiple substrates, confirmed in cells\",\n      \"pmids\": [\"10835426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"RasGRP3 binds phorbol esters via its C1 domain in an anionic phospholipid-dependent manner, and phorbol ester or diacylglycerol (DAG) binding causes RasGRP3 translocation to the plasma membrane/perinuclear area and activates its Ras exchange activity in intact cells.\",\n      \"method\": \"Phorbol ester binding assay, GFP-RasGRP3 fluorescence microscopy, RasGTP pull-down, ERK phosphorylation assay in HEK-293 cells\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct binding assay plus live-cell translocation and downstream signaling, multiple orthogonal methods\",\n      \"pmids\": [\"11221888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"RasGRP3 is phosphorylated upon BCR stimulation coincident with Ras activation; PKC inhibition attenuates both RasGRP3 phosphorylation and Ras activation. PKC-theta and PKC-beta2 phosphorylate RasGRP3 in vitro, and a dominant-active PKC-theta enhances Ras-ERK signaling via RasGRP3 when co-expressed in HEK-293 cells.\",\n      \"method\": \"In vitro kinase assay with PKC-theta and PKC-beta2, co-expression with dominant-active PKC-theta, PKC inhibitor studies, membrane fractionation, Ras activation assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro phosphorylation confirmed, epistasis via inhibitors, replicated in two cell types\",\n      \"pmids\": [\"12730099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PKC phosphorylates RasGRP3 specifically on Thr133; the Thr133Ala substitution abolishes PKC-dependent phosphorylation in vitro and severely impairs RasGRP3-mediated Ras activation in vivo after BCR stimulation. PKC activity (conventional PKCs) is required for Thr133 phosphorylation and full Ras-ERK activation.\",\n      \"method\": \"Mass spectrometry to identify phosphorylation site, in vitro kinase assay, site-directed mutagenesis (Thr133Ala), antiphospho-peptide antibody, PKC inhibitors, BCR stimulation of B cells\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — site identified by mass spectrometry, mutagenesis confirms functional requirement, replicated across two labs (PMID 15657177 and 15545601)\",\n      \"pmids\": [\"15657177\", \"15545601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PKCdelta physically associates with RasGRP3 upon PMA treatment (co-immunoprecipitation, colocalization in perinuclear region) and phosphorylates RasGRP3 in vitro; a PKCdelta kinase-dead mutant blocks the PMA-induced mobility shift of RasGRP3.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, immunofluorescence colocalization, dominant-negative PKCdelta, rottlerin inhibitor\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reciprocal co-IP plus in vitro kinase assay plus dominant-negative, multiple orthogonal methods\",\n      \"pmids\": [\"15213298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RasGRP3 activates Rap2B, leading to Rap2B-dependent translocation of PLC-epsilon to the plasma membrane and PLC/Ca2+ signaling downstream of the EGF receptor. EGF induces tyrosine phosphorylation of RasGRP3 by c-Src, and c-Src inhibition blocks both Rap2B activation and PLC stimulation.\",\n      \"method\": \"Dominant-negative Rap2B expression, clostridial toxin inactivation, co-expression of RasGRP3, GTP-loading assay for Rap2B, c-Src inhibition, Ca2+ signaling measurement in HEK-293 cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis by dominant-negative, pharmacological inhibition, and GTP-loading assays, multiple orthogonal approaches\",\n      \"pmids\": [\"15143162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RasGRP3 is expressed in embryonic blood vessels and is specifically required for the aberrant endothelial morphogenesis induced by phorbol ester (PMA); RasGRP3 loss-of-function makes vessels refractory to PMA-induced dysmorphogenesis. RasGRP3 expression is upregulated by VEGF stimulation of endothelial cells.\",\n      \"method\": \"ES cell gene trap screen, in situ hybridization, loss-of-function mouse model, ES cell-derived vascular morphogenesis assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic loss-of-function with specific vascular phenotype, replicated in mouse model and ES-derived vessels\",\n      \"pmids\": [\"15572660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Both RasGRP1 and RasGRP3 contribute to BCR-induced Ras and ERK activation in B cells; RasGRP3 alone maintains basal Ras-GTP levels in resting B cells. Loss of RasGRP3 causes isotype-specific antibody deficiencies and hypogammaglobulinemia. BCR-induced B cell proliferation is RasGRP1- and RasGRP3-dependent.\",\n      \"method\": \"Single and double null mutant mice (Rasgrp1-/-, Rasgrp3-/-, double KO), Ras-GTP pull-down, ERK phosphorylation, B cell proliferation assays, serum immunoglobulin measurement\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with double KO mice, multiple orthogonal readouts\",\n      \"pmids\": [\"16301621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Fluorescent phorbol esters induce translocation of RasGRP3 to intracellular membranes (primarily perinuclear/intracellular), and RasGRP3 colocalizes with the fluorescent phorbol ester. The lipophilicity of the phorbol ester determines kinetics and pattern of RasGRP3 redistribution.\",\n      \"method\": \"Fluorescent phorbol ester live-cell imaging, GFP-RasGRP3 fusion protein colocalization in CHO cells\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — direct localization experiment with functional implication, single lab\",\n      \"pmids\": [\"15657361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RasGRP3 mediates phorbol ester-induced exocytosis in a PKC-independent manner; RasGRP3 is expressed in endocrine tissues and its effects on exocytosis are not blocked by MEK inhibitor but are partially sensitive to PKC inhibitor.\",\n      \"method\": \"Exocytosis assay in endocrine cells, PKC inhibitor, MEK inhibitor, ERK phosphorylation readout\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, pharmacological dissection of mechanism, partial follow-up\",\n      \"pmids\": [\"15737652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RasGRP3 interacts with dynein light chain 1 (DLC1) through its C-terminal 127 amino acids; this interaction was confirmed in vitro and by co-immunoprecipitation. Deletion of the C-terminal domain abolishes DLC1 interaction and dramatically alters RasGRP3 subcellular localization (strong reticular/perinuclear/nuclear distribution).\",\n      \"method\": \"Yeast two-hybrid screen, in vitro pull-down, co-immunoprecipitation, subcellular localization of truncation mutant (fluorescence microscopy)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid confirmed by in vitro pulldown and co-IP, domain mapping by mutagenesis, localization consequence demonstrated\",\n      \"pmids\": [\"17012239\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RasGRP3 is required for Ras-GTP formation, AKT and ERK1/2 phosphorylation, cell proliferation, migration, and anchorage-independent growth in prostate cancer cells. RasGRP3 overexpression in LNCaP cells elevates Ras-GTP, stimulates proliferation, and confers resistance to PMA-induced apoptosis.\",\n      \"method\": \"siRNA knockdown and overexpression, Ras-GTP pull-down, AKT/ERK phosphorylation, proliferation assay, migration assay, mouse xenograft\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal gain/loss-of-function with multiple cellular readouts and in vivo xenograft\",\n      \"pmids\": [\"20876802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RasGRP3 is required for Ras-GTP formation, Akt phosphorylation, c-Met expression, and cell proliferation/transformation in human melanoma cells. Overexpression of RasGRP3 in primary melanocytes alters morphology and induces tumorigenicity in mouse xenografts.\",\n      \"method\": \"siRNA knockdown, overexpression in primary melanocytes, Ras-GTP pull-down, Akt phosphorylation, soft agar colony formation, mouse xenograft\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal gain/loss-of-function, multiple downstream readouts, in vivo validation\",\n      \"pmids\": [\"21602881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RasGRP3 in endothelial cells promotes Ras-ERK signaling and endothelin-1-stimulated cell migration; Rasgrp3 loss-of-function attenuates Ras-ERK signaling and abolishes endothelin-1-induced migration, and embryos lacking Rasgrp3 are dramatically protected from diabetes-induced vascular developmental defects.\",\n      \"method\": \"Loss-of-function mouse model (Rasgrp3 null), endothelial cell migration assay, Ras-ERK signaling measurement, diabetic mouse model, primary endothelial cells with activated RasGRP3\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic loss-of-function in mouse with in vivo developmental phenotype and complementary cell-based mechanistic studies\",\n      \"pmids\": [\"21474816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RasGRP3 activates Rap1 upon low-level TLR stimulation in macrophages, which limits production of pro-inflammatory cytokines (especially IL-6). CRISPR-Cas9 deletion of RasGRP3 in RAW264.7 cells inhibits TLR3/4/9-induced Rap1 activation while enhancing ERK1/2 activation and IL-6 production.\",\n      \"method\": \"CRISPR-Cas9 knockout in RAW264.7, Rap1 activity assay, ERK1/2 phosphorylation, cytokine ELISA, DSS-colitis and collagen-induced arthritis mouse models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean CRISPR KO with defined molecular phenotype (Rap1 activation), in vivo validation, multiple orthogonal methods\",\n      \"pmids\": [\"25118589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RasGRP3 interacts with Arp3 (actin-related protein), as identified by pull-down/mass spectrometry and confirmed by co-immunoprecipitation and immunofluorescence. PMA-induced translocation of RasGRP3 increases its association with Arp3. Arp3 silencing partially reduces RasGRP3-mediated glioma cell migration.\",\n      \"method\": \"Pull-down assay with mass spectrometry, co-immunoprecipitation, immunofluorescence colocalization, siRNA knockdown of Arp3, migration assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — interaction confirmed by orthogonal methods, functional link via Arp3 knockdown, single lab\",\n      \"pmids\": [\"25682201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In GNAQ-mutant uveal melanoma, RasGRP3 is phosphorylated and activated by PKC-delta and PKC-epsilon, enabling Ras-MAPK pathway activation. RasGRP3 activation also occurs through PKC-independent DAG-mediated membrane recruitment. RasGRP3 knockdown suppresses MAPK activation, identifying RasGRP3 as the mechanistic link between Gαq signaling and MAPK in uveal melanoma.\",\n      \"method\": \"Knockdown (siRNA/shRNA), PKC isoform-specific studies, Ras-GTP assay, ERK phosphorylation, membrane fractionation, uveal melanoma cell lines and patient samples\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, PKC isoform identification, replicated in multiple UM cell lines\",\n      \"pmids\": [\"28486107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In a GNA11Q209L mouse model, RasGRP3 is specifically required for GNAQ/GNA11-driven Ras activation and tumorigenesis; integrative transcriptome analysis identified RasGRP3 as selectively expressed in Gq-driven melanomas, and its loss suppresses tumor formation.\",\n      \"method\": \"Transgenic GNA11Q209L mouse model, Bap1 conditional KO, integrative transcriptome analysis, RasGRP3 knockdown in human UM cell lines, Ras activation assay, in vivo tumor models\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic mouse model plus human cell line validation, integrative transcriptomics, loss-of-function with defined phenotype\",\n      \"pmids\": [\"29490280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Alpha-arylidene DAG-lactones can selectively bind the RasGRP3 C1 domain with up to 73-fold selectivity over PKCα and 45-fold over PKCε in vitro, and selectively activate Ras (via RasGRP3) over PKCδ phosphorylation in intact cells.\",\n      \"method\": \"In vitro C1-domain binding assay (competitive displacement), intact cell Ras activation assay, PKCδ phosphorylation assay\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-3 — in vitro binding and cell-based selectivity, but pharmacological tool study without mutagenesis\",\n      \"pmids\": [\"29860841\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In NPM1-mutant AML, cytoplasmic NPM1-mA binds E3 ubiquitin ligase MID1, blocking MID1-mediated degradation of RasGRP3 and thus stabilizing RasGRP3 protein. Stabilized RasGRP3 activates the EGFR-STAT3 axis to promote AML cell proliferation and autophagy.\",\n      \"method\": \"Co-immunoprecipitation, Western blot, cycloheximide chase assay, CCK8, EdU staining, immunofluorescence\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP for interaction, cycloheximide chase for stability, downstream signaling confirmed, single lab\",\n      \"pmids\": [\"36826998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AC092894.1 lncRNA acts as a scaffold mediating USP3-dependent de-ubiquitination of AR, which then drives transcription of RASGRP3 to sustain MAPK signaling in colorectal cancer cells; loss of this axis promotes oxaliplatin resistance.\",\n      \"method\": \"RNA pull-down, RIP, co-immunoprecipitation, gain/loss-of-function experiments, in vivo mouse model\",\n      \"journal\": \"BMC medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple biochemical methods for scaffold mechanism, in vivo validation, single lab\",\n      \"pmids\": [\"37013584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RasGRP3 promotes RAP1B GTP-loading (exchange factor activity) in endothelial cells, thereby inhibiting NF-κB pathway activation and reducing pro-inflammatory cytokine production and monocyte adhesion. UHRF1 (E3 ubiquitin ligase) binds RasGRP3 and promotes its ubiquitination and degradation; UHRF1 knockdown increases RasGRP3 protein levels.\",\n      \"method\": \"RasGRP3 overexpression in endothelial cells and ApoE-/- mice, RAP1 activity assay, NF-κB pathway assay, cytokine ELISA, THP-1 adhesion assay, co-immunoprecipitation for UHRF1-RasGRP3 interaction\",\n      \"journal\": \"Inflammation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — RAP1B activation confirmed, ubiquitination by UHRF1 identified by co-IP, in vivo atherosclerosis model, single lab\",\n      \"pmids\": [\"41689678\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RasGRP3 is a DAG/phorbol ester-regulated guanine nucleotide exchange factor (GEF) that activates Ras, R-Ras, and Rap1; it is recruited to membranes via its C1 domain upon DAG/phorbol ester binding and is further activated by PKC-mediated phosphorylation on Thr133, integrating DAG signals from receptors (BCR, TCR, EGF receptor, Gαq-coupled GPCRs) into Ras-MAPK and Rap1 effector outputs in B cells, endothelial cells, macrophages, and cancer cells, and interacts with binding partners including dynein light chain 1 (regulating localization), Arp3 (regulating migration), and is subject to ubiquitin-mediated degradation regulated by UHRF1 and MID1.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RasGRP3 is a diacylglycerol (DAG)/phorbol ester–regulated guanine nucleotide exchange factor (GEF) that integrates lipid second-messenger signals into Ras-family GTPase activation across immune, endothelial, and epithelial cell types. It catalyzes GDP-to-GTP exchange on H-Ras, R-Ras, Rap1, and Rap2B, and is recruited to membranes via its C1 domain upon DAG or phorbol ester binding; full catalytic activation additionally requires PKC-mediated phosphorylation on Thr133, with PKCδ, PKCε, PKCθ, and PKCβ2 each capable of this modification [PMID:10835426, PMID:11221888, PMID:15657177, PMID:15213298, PMID:28486107]. In B cells, RasGRP3 cooperates with RasGRP1 to drive BCR-induced Ras-ERK signaling and proliferation, and Rasgrp3-null mice display hypogammaglobulinemia; in macrophages, RasGRP3 instead preferentially activates Rap1 upon TLR stimulation to restrain pro-inflammatory cytokine output [PMID:16301621, PMID:25118589]. RasGRP3 protein stability is controlled by ubiquitin-dependent degradation mediated by the E3 ligases UHRF1 and MID1, and in GNAQ/GNA11-mutant uveal melanoma RasGRP3 serves as the obligate link between constitutive Gαq signaling and MAPK pathway activation [PMID:28486107, PMID:29490280, PMID:41689678, PMID:36826998].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing that RasGRP3 is a bona fide GEF with broad Ras-family substrate specificity resolved whether the CalDAG-GEFIII gene product had direct catalytic exchange activity and defined H-Ras, R-Ras, and Rap1 as its substrates.\",\n      \"evidence\": \"In vitro GEF assay measuring GDP/GTP exchange on purified GTPases, confirmed by transfection in 293T cells and PC12 differentiation\",\n      \"pmids\": [\"10835426\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative substrate preference under physiological DAG concentrations unknown\", \"No structural basis for multi-substrate recognition\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that the C1 domain binds DAG/phorbol esters and drives membrane translocation established the activation mechanism linking receptor-generated DAG to RasGRP3 catalytic output.\",\n      \"evidence\": \"Phorbol ester binding assay, GFP-RasGRP3 live-cell imaging, Ras-GTP pull-down in HEK-293 cells\",\n      \"pmids\": [\"11221888\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Contribution of EF-hand and PT domains to membrane targeting not fully dissected\", \"Whether C1-domain lipid selectivity differs from PKC C1 domains was untested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of Thr133 as the PKC phosphorylation site required for full RasGRP3 activation revealed a two-signal activation model: DAG recruits RasGRP3 to membranes, and PKC phosphorylation (by PKCθ, PKCβ2, or PKCδ) amplifies its GEF output.\",\n      \"evidence\": \"Mass spectrometry, Thr133Ala mutagenesis, in vitro kinase assays with multiple PKC isoforms, BCR stimulation in B cells, co-IP of PKCδ–RasGRP3\",\n      \"pmids\": [\"15657177\", \"15545601\", \"15213298\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism by which Thr133 phosphorylation enhances catalysis is unknown\", \"Relative contributions of individual PKC isoforms in different cell types not resolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showing that RasGRP3 activates Rap2B downstream of EGF/c-Src to drive PLCε translocation extended the signaling repertoire beyond Ras-ERK and linked RasGRP3 to calcium signaling cascades.\",\n      \"evidence\": \"Dominant-negative Rap2B, c-Src inhibition, Rap2B GTP-loading assay, Ca²⁺ measurement in HEK-293 cells\",\n      \"pmids\": [\"15143162\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this Rap2B–PLCε axis operates in primary cell types is untested\", \"Tyrosine phosphorylation site on RasGRP3 by c-Src not mapped\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Genetic studies in Rasgrp1/Rasgrp3 single- and double-knockout mice established that RasGRP3 maintains basal Ras-GTP in resting B cells, cooperates with RasGRP1 for BCR-induced ERK activation, and is required for normal immunoglobulin production.\",\n      \"evidence\": \"Single and double null mice, Ras-GTP pull-down, B cell proliferation, serum immunoglobulin measurement\",\n      \"pmids\": [\"16301621\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which immunoglobulin class-switch step is RasGRP3-dependent is not defined\", \"Whether B cell developmental stages are differentially affected is unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Loss-of-function in the embryonic vasculature demonstrated a non-immune role for RasGRP3: it is required for phorbol ester–induced endothelial dysmorphogenesis and is upregulated by VEGF, establishing RasGRP3 as a vascular DAG effector.\",\n      \"evidence\": \"Gene-trap mouse model, in situ hybridization, ES cell–derived vascular morphogenesis assay\",\n      \"pmids\": [\"15572660\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream GTPase substrate (Ras vs. Rap1) mediating vascular phenotype not identified\", \"Relationship to VEGF receptor signaling pathway not fully mapped\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Discovery that dynein light chain 1 (DLC1) binds the C-terminal domain of RasGRP3 and controls its subcellular distribution revealed a cytoskeletal-dependent localization mechanism for this GEF.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro pull-down, co-IP, fluorescence microscopy of truncation mutant\",\n      \"pmids\": [\"17012239\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of DLC1 interaction on Ras/Rap activation not tested\", \"Whether dynein motor activity is required for RasGRP3 transport unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Gain- and loss-of-function studies in prostate cancer and melanoma cells showed that RasGRP3 overexpression is sufficient to drive Ras-AKT/ERK signaling, proliferation, and tumorigenicity, positioning it as an oncogenic driver in non-immune cancers.\",\n      \"evidence\": \"siRNA knockdown and overexpression, Ras-GTP pull-down, soft agar assay, mouse xenograft in prostate cancer and melanoma models\",\n      \"pmids\": [\"20876802\", \"21602881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of RasGRP3 overexpression in these cancers (amplification, transcriptional, post-translational) not established\", \"Whether RasGRP3 signals through Ras or Rap in these contexts is unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"CRISPR knockout in macrophages revealed that RasGRP3 preferentially activates Rap1 (not Ras) upon TLR stimulation and thereby dampens inflammatory cytokine production, establishing a substrate-selective anti-inflammatory role.\",\n      \"evidence\": \"CRISPR-Cas9 KO in RAW264.7, Rap1 activity assay, cytokine ELISA, DSS-colitis and collagen-induced arthritis mouse models\",\n      \"pmids\": [\"25118589\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism determining Rap1 vs. Ras substrate selectivity in macrophages unknown\", \"Cell-type–specific transcriptional regulation of RasGRP3 in myeloid lineage not characterized\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identification of RasGRP3 as the obligate link between oncogenic GNAQ/GNA11 and MAPK in uveal melanoma solved a long-standing question about how constitutive Gαq signals reach Ras, revealing dual activation by PKC phosphorylation and DAG-mediated membrane recruitment.\",\n      \"evidence\": \"siRNA/shRNA knockdown, PKC isoform–specific studies, Ras-GTP and ERK assays in multiple UM cell lines, patient tumor analysis; confirmed by GNA11Q209L transgenic mouse model\",\n      \"pmids\": [\"28486107\", \"29490280\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether pharmacological targeting of RasGRP3 C1 domain can suppress UM growth in vivo is untested\", \"Structural basis for RasGRP3 selectivity by DAG-lactone agonists incompletely understood\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Discovery that MID1 and UHRF1 are E3 ubiquitin ligases targeting RasGRP3 for proteasomal degradation established post-translational stability control as a key regulatory layer, with pathological stabilization occurring in NPM1-mutant AML.\",\n      \"evidence\": \"Co-IP, cycloheximide chase, Western blot for NPM1-mA/MID1/RasGRP3 axis in AML cells; co-IP and ubiquitination assay for UHRF1–RasGRP3 in endothelial cells and ApoE−/− mice\",\n      \"pmids\": [\"36826998\", \"41689678\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination sites on RasGRP3 not mapped\", \"Relative contributions of MID1 vs. UHRF1 in different tissues unknown\", \"Findings from single labs, independent replication pending\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A structural understanding of how DAG binding, Thr133 phosphorylation, and substrate selectivity are integrated within the RasGRP3 catalytic cycle, and the in vivo therapeutic potential of C1-domain–selective ligands, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal or cryo-EM structure of RasGRP3\", \"In vivo efficacy of RasGRP3-selective DAG-lactones not demonstrated\", \"Mechanism of Ras vs. Rap substrate selection in different cell types undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5, 14, 21]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [1, 8, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 8, 16]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 11, 12, 16, 17]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [11, 12, 16, 17]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"DYNLL1\",\n      \"ACTR3\",\n      \"PRKCD\",\n      \"PRKCQ\",\n      \"PRKCB\",\n      \"MID1\",\n      \"UHRF1\",\n      \"RAP2B\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}