{"gene":"ARHGEF28","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2000,"finding":"The isolated tandem DH/PH domain of p190RhoGEF activates RhoA in vitro (GDP release and protein binding assays), but not Rac1 or Cdc42, establishing RhoA-specific GEF activity. Full-length p190RhoGEF fails to activate RhoA in vitro, suggesting autoinhibition requiring unknown binding partners to unmask exchange activity in vivo. p190RhoGEF directly interacts with microtubules via its C-terminal region adjacent to the DH/PH domain, shown by in vitro and in vivo binding experiments.","method":"In vitro GDP release assay, protein binding assay, immunofluorescence, in vitro and in vivo microtubule binding experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay with domain dissection, microtubule binding confirmed both in vitro and in vivo, multiple orthogonal methods in one study","pmids":["11058585"],"is_preprint":false},{"year":2003,"finding":"FAK directly interacts with p190RhoGEF in neuronal cells and brain tissue extracts. The FAK C-terminal focal adhesion targeting (FAT) domain binds the C-terminal coiled-coil domain of p190RhoGEF, identified by two-hybrid assay and deletion mutagenesis. A FAK FAT domain mutation (Leu-1034 to Ser) disrupts this interaction. FAK activity promotes p190RhoGEF tyrosine phosphorylation and RhoA GTP loading downstream of laminin/integrin and IGF-1 receptor stimulation.","method":"Co-immunoprecipitation, co-localization, yeast two-hybrid, deletion mutagenesis, dominant-negative inhibition (FRNK), RhoA GTP-loading assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, two-hybrid with mutagenesis, functional GTP-loading assay, multiple orthogonal methods","pmids":["12702722"],"is_preprint":false},{"year":2001,"finding":"14-3-3η and 14-3-3ε are binding partners of p190RhoGEF, identified by yeast two-hybrid screen and confirmed biochemically by co-immunoprecipitation and co-localization. A phosphorylation-independent binding site (I1370QAIQNL) in p190RhoGEF was mapped; deletion of this site abolishes 14-3-3η interaction in vitro and prevents 14-3-3η-mediated alteration of p190RhoGEF cytoplasmic aggregation in cells.","method":"Yeast two-hybrid, co-immunoprecipitation, co-localization with fluorescent fusion proteins, deletion mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by biochemical co-IP and colocalization, site mapped by deletion mutagenesis, single lab with multiple orthogonal methods","pmids":["11533041"],"is_preprint":false},{"year":2001,"finding":"p190RhoGEF binds directly and specifically to the 68-nucleotide destabilizing element in the 3' UTR of NF-L (neurofilament light) mRNA via its C-terminal domain (clone 39), demonstrated by Northwestern blot, gel-shift, and cross-linkage assays. Expression of p190RhoGEF in stably transfected neuronal cells increased the half-life of wild-type NF-L mRNA but not of a mutant lacking the destabilizing element, establishing a functional role in NF-L mRNA stability.","method":"GST-fusion pulldown, Northwestern blot, gel-shift assay, RNA cross-linkage, stable transfection with mRNA half-life measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstituted RNA binding with multiple biochemical methods plus functional mRNA stability assay with appropriate controls (wild-type vs. mutant mRNA)","pmids":["11435431"],"is_preprint":false},{"year":2002,"finding":"BC1 RNA (a neuronally expressed non-coding RNA) binds to the same C-terminal site of p190RhoGEF as NF-L mRNA and competes with NF-L mRNA for p190RhoGEF binding, identified by affinity chromatography and cross-competition experiments using a GST-p190RhoGEF C-terminal fusion protein.","method":"GST-fusion affinity chromatography, gel-shift, cross-competition binding assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro reconstituted competition assay with identified binding site, single lab","pmids":["12215442"],"is_preprint":false},{"year":2003,"finding":"Anti-apoptotic activity of p190RhoGEF is localized to two cytoplasmic retention sequences (CRS-1 and CRS-2) in its C-terminal region that overlap with the JIP-1 and 14-3-3 binding sites. Deleting both CRS sequences abolishes cytoplasmic retention and anti-apoptotic activity of EGFP-tagged p190RhoGEF in Neuro 2a cells; restoring either CRS-1 or CRS-2 rescues both properties.","method":"Transfection of EGFP-tagged deletion constructs, apoptosis assay, fluorescence microscopy in Neuro 2a cells","journal":"Brain research. Molecular brain research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — domain deletion mapping with functional apoptosis readout, single lab, cell-based assay without in vitro reconstitution","pmids":["14499478"],"is_preprint":false},{"year":2003,"finding":"p190RhoGEF expression is induced by CD40 stimulation in WEHI 231 B cells. Overexpression of p190RhoGEF mimics CD40-stimulated cellular structure changes and NF-κB activation through RhoA; these effects are blocked by dominant-negative RhoA (T19N) or dominant-negative p190RhoGEF (Y1003A).","method":"2D gel electrophoresis identification, overexpression with dominant-negative constructs, NF-κB reporter assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — dominant-negative epistasis, single lab, multiple constructs tested","pmids":["12496377"],"is_preprint":false},{"year":2008,"finding":"delta-Catenin interacts with p190RhoGEF in the cytoplasm at low cell density, reducing RhoA activity. At high cell density, E-cadherin outcompetes p190RhoGEF for delta-catenin binding, shifting delta-catenin to the plasma membrane and restoring RhoA activity. Ectopic E-cadherin expression in mouse embryonic fibroblasts decreased delta-catenin's effect on RhoA activity reduction.","method":"Co-immunoprecipitation, immunofluorescence, RhoA activity assay, ectopic E-cadherin expression","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and functional RhoA assay, competition shown by cell density experiment and ectopic E-cadherin, single lab","pmids":["18930028"],"is_preprint":false},{"year":2010,"finding":"Human RGNEF (the human homologue of p190RhoGEF) directly interacts with human NFL mRNA in vitro by gel-shift assay. In tissue lysates, RGNEF-NFL mRNA interaction was detected by IP-RT-PCR only in ALS patient samples, not in neuropathologically normal controls.","method":"Gel-shift assay (in vitro), IP-RT-PCR (in tissue lysates)","journal":"Amyotrophic lateral sclerosis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro RNA binding confirmed by gel shift plus in-tissue IP-RT-PCR, single lab","pmids":["19488899"],"is_preprint":false},{"year":2011,"finding":"Rgnef forms a complex with FAK in human colon carcinoma cells. Upon gastrin stimulation, Rgnef-FAK interaction is required for FAK translocation to focal adhesions, paxillin tyrosine phosphorylation, cell motility, and invadopodia formation. Overexpression of the Rgnef C-terminal region (aa 1279–1582) disrupts endogenous Rgnef-FAK interaction and blocks these events; a version lacking the FAK binding site (aa 1302–1582) does not. Rgnef-C-expressing cells form smaller, less invasive tumors in vivo.","method":"shRNA knockdown, co-immunoprecipitation, dominant-negative C-terminal fragment competition, phosphorylation assays, orthotopic tumor implantation","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — shRNA KD, dominant-negative competition with defined binding-site deletion, in vivo tumor model, multiple orthogonal readouts, single lab","pmids":["21224360"],"is_preprint":false},{"year":2012,"finding":"Genetic knockout of Rgnef in mouse embryo fibroblasts (Rgnef-/- MEFs) significantly inhibits haptotaxis migration, wound closure motility, focal adhesion number, and RhoA GTPase activation after fibronectin-integrin stimulation. These phenotypes are rescued by epitope-tagged Rgnef re-expression, establishing Rgnef as essential for RhoA regulation downstream of integrins.","method":"Conditional knockout (floxed Rgnef × CMV-Cre), primary MEF isolation, haptotaxis assay, wound closure assay, focal adhesion quantification, RhoA GTP-loading assay, rescue by re-expression","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined phenotypes and rescue by re-expression, multiple orthogonal readouts, clean loss-of-function","pmids":["22649559"],"is_preprint":false},{"year":2013,"finding":"Rgnef plays a non-canonical, upstream scaffolding role in promoting FAK localization to peripheral adhesions and FAK-Y397 activation upon fibronectin binding, independent of its GEF catalytic activity. A PH domain mutation in Rgnef blocks adhesion formation, FAK localization, and FAK/paxillin phosphorylation without disrupting the Rgnef-FAK interaction. A GEF-inactive Rgnef mutant rescues FAK-Y397 phosphorylation and adhesion localization but not paxillin-Y118 phosphorylation, indicating paxillin-pY118 requires Rgnef GEF activity through a distinct mechanism.","method":"Rgnef-null MEFs, site-directed mutagenesis (PH domain and GEF-inactive mutants), immunofluorescence, phosphotyrosine immunoblotting, rescue experiments","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic null MEFs with domain-specific mutant rescue, dissection of GEF-dependent vs. GEF-independent functions, multiple orthogonal methods","pmids":["24006257"],"is_preprint":false},{"year":2015,"finding":"Rgnef is a new effector for Gα13 downstream of gastrin and the CCK2 receptor in DLD-1 colon carcinoma cells. Rgnef co-immunoprecipitates with activated Gα13Q226L but not Gα12Q229L; the Rgnef C-terminal region (aa 1279–1582) is sufficient for this interaction and its exogenous expression blocks Gα13-stimulated SRE activity. Point mutations in the Rgnef C-terminal region disrupt Gα13 association but not Gαq association. Gα13 depletion reduces gastrin-induced FAK-pY397 and paxillin-pY31.","method":"Co-immunoprecipitation, shRNA depletion of Gα13, SRE-luciferase reporter assay, point mutagenesis of Rgnef C-terminus, RhoA GTP-binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, mutagenesis defining binding site, functional SRE assay, shRNA epistasis, single lab with multiple orthogonal methods","pmids":["25922072"],"is_preprint":false},{"year":2017,"finding":"Crystal structures reveal that activated Rac1·GTP and RhoA·GTP use their effector-binding surfaces to associate with the same hydrophobic surface on the p190RhoGEF PH domain. Both activated RhoA and Rac1 stimulate nucleotide exchange on RhoA·GDP by p190RhoGEF in vitro, localizing it to its substrate. This demonstrates a positive feedback (activated RhoA) and a cross-talk mechanism (activated Rac1 directly stimulates RhoA activation through p190RhoGEF).","method":"X-ray crystallography, in vitro nucleotide exchange assay","journal":"Journal of structural biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of PH domain complexes with Rac1·GTP and RhoA·GTP plus in vitro exchange assay confirming functional consequence","pmids":["29196061"],"is_preprint":false},{"year":2017,"finding":"RGNEF expression is upregulated in murine spinal motor neurons following distal sciatic nerve injury. Under cellular stress (sodium arsenite or sorbitol), RGNEF expression confers a survival benefit in HEK293T cells; the NH2-terminus domain is essential for this protective effect. Under stress, RGNEF associates with Staufen1-positive granules but not TIA-1-positive stress granules.","method":"In vivo nerve injury model, in vitro stress assay with deletion constructs, immunofluorescence co-localization","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — deletion construct mapping in cell-based survival assay plus colocalization, single lab","pmids":["28495450"],"is_preprint":false},{"year":2018,"finding":"A 23-amino acid bipartite nuclear localization signal (NLS) within the Pleckstrin Homology (PH) domain of RGNEF controls its nuclear localization; deletion or mutation of this region abolishes nuclear localization. Within this NLS, an overlapping nuclear export signal (NES) promotes nuclear export in an exportin-1-dependent manner (confirmed by Leptomycin B treatment). The PH domain alone is sufficient to translocate a 160 kDa fusion protein to the nucleus.","method":"Deletion and point mutagenesis of NLS/NES, fluorescence microscopy of fusion proteins, Leptomycin B inhibition","journal":"European journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — domain mutagenesis with functional localization readout and pharmacological confirmation, single lab","pmids":["30482479"],"is_preprint":false},{"year":2019,"finding":"Rgnef is essential for ovarian tumor spheroid formation in vitro and tumor growth in vivo using transgenic and transplantable Rgnef knockout mouse models. Rgnef supports an NF-κB-mediated antioxidant gene signature (including Gpx4, Nqo1, Gsta4); antioxidant treatment rescues growth of Rgnef-knockout spheroids, and Rgnef re-expression facilitates NF-κB-dependent tumorsphere survival.","method":"Rgnef knockout mouse model, spheroid formation assay, RNA-sequencing, antioxidant rescue experiment, NF-κB reporter/pathway analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with rescue, RNA-seq pathway identification, pharmacological rescue, single lab","pmids":["31308489"],"is_preprint":false},{"year":2024,"finding":"An N-terminal fragment of RGNEF (NF242) directly interacts with the RNA recognition motifs (RRMs) of TDP-43, competing with RNA binding. The IPT/TIG domain of NF242 is essential for this interaction. In a Drosophila ALS model overexpressing TDP-43, genetic expression of NF242 suppressed neuropathological phenotypes (increased lifespan, abolished motor defects, prevented neurodegeneration). Intracerebroventricular injection of AAV9/NF242 in a murine TDP-43 model (rNLS8) improved lifespan and motor phenotype and decreased neuroinflammation markers.","method":"Direct protein-protein interaction assay, domain deletion (IPT/TIG), Drosophila genetic epistasis model, murine AAV9 intracerebroventricular injection","journal":"Brain : a journal of neurology","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding with domain mutagenesis confirmed functionally in two independent in vivo models (fly and mouse), multiple orthogonal methods","pmids":["38739752"],"is_preprint":false},{"year":2024,"finding":"RGNEF and TDP-43 act predominantly in an antagonistic manner to regulate expression of axon guidance genes in neuronal cells. Mechanistically, both factors affect the processivity of long intron removal (splicing), explaining their mode of transcriptomic action upon depletion.","method":"Comparative transcriptomics (RNA-seq) of TDP-43- and RGNEF-depleted neuronal cells, long intron processivity analysis","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — transcriptomic comparison of KD cells with mechanistic intron-processivity analysis, single lab","pmids":["39360635"],"is_preprint":false},{"year":2026,"finding":"Rgnef promotes osteoclastogenesis and attenuates osteoblastogenesis through activation of RhoA and Rac1, leading to enhanced NF-κB, MAPK, and AKT signaling. Rgnef-deficient mice show increased bone mass due to reduced osteolysis and increased osteogenesis, while Rgnef-overexpressing mice show the opposite. Rgnef-deficient mice are protected from bone loss in LPS-induced inflammation and ovariectomy models.","method":"Rgnef-deficient and transgenic overexpressing mice, osteoclast/osteoblast differentiation assays, RhoA/Rac1 activity assay, NF-κB/MAPK/AKT pathway analysis, in vivo bone loss models","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO and transgenic OE with mechanistic pathway analysis, single lab, pathway placement by GTPase activity assay","pmids":["41571890"],"is_preprint":false}],"current_model":"ARHGEF28/RGNEF/p190RhoGEF is a 190 kDa bifunctional protein that acts as a RhoA-specific guanine nucleotide exchange factor (DH/PH domain) and RNA-binding protein; its PH domain binds activated RhoA·GTP and Rac1·GTP (crystal structure confirmed) to localize it to substrate RhoA·GDP, enabling both positive feedback and Rac→Rho cross-talk, while its C-terminal coiled-coil domain directly binds FAK (FAT domain), Gα13, 14-3-3, delta-catenin, and NF-L/BC1 RNAs; downstream of integrins and GPCRs, a Rgnef–FAK scaffold (requiring the Rgnef PH domain) promotes FAK-Y397 autophosphorylation and peripheral adhesion formation in a GEF-activity-independent manner, whereas Rgnef GEF activity separately drives RhoA-dependent paxillin-Y118 phosphorylation, focal adhesion maturation, and cell motility, as established by genetic knockout, mutagenesis, structural, and in vitro reconstitution studies."},"narrative":{"mechanistic_narrative":"ARHGEF28 (RGNEF/p190RhoGEF) is a bifunctional 190 kDa protein that couples RhoA-specific guanine nucleotide exchange to RNA binding, integrating cytoskeletal signaling at focal adhesions with post-transcriptional control in neurons [PMID:11058585, PMID:11435431]. Its tandem DH/PH module catalyzes RhoA-specific GDP release in vitro while sparing Rac1 and Cdc42, and the full-length protein is autoinhibited until binding partners unmask activity [PMID:11058585]; crystallography shows that activated RhoA·GTP and Rac1·GTP dock onto the same hydrophobic surface of its PH domain to recruit the enzyme to substrate RhoA·GDP, generating both positive feedback and Rac→Rho cross-talk [PMID:29196061]. Downstream of integrins and GPCRs, ARHGEF28 forms a scaffold with FAK through its C-terminal coiled-coil binding the FAK FAT domain [PMID:12702722, PMID:21224360], and genetic knockout establishes it as essential for RhoA activation, focal adhesion formation, and migration after fibronectin stimulation [PMID:22649559]; this scaffolding role promotes FAK-Y397 autophosphorylation and peripheral adhesion assembly in a PH-domain-dependent but GEF-activity-independent manner, whereas its GEF activity separately drives paxillin-Y118 phosphorylation [PMID:24006257]. It is an effector of Gα13 downstream of gastrin/CCK2 receptor signaling [PMID:25922072] and supports NF-κB-dependent transcriptional programs in tumor and bone contexts [PMID:31308489, PMID:41571890]. As an RNA-binding protein, its C-terminal domain binds the destabilizing element of NF-L mRNA to extend its half-life, a site also engaged by BC1 RNA [PMID:11435431, PMID:12215442]. The protein additionally engages TDP-43 RNA-recognition motifs through an N-terminal IPT/TIG-containing fragment, and ARHGEF28 and TDP-43 act antagonistically to regulate splicing of long introns in axon-guidance genes [PMID:38739752, PMID:39360635].","teleology":[{"year":2000,"claim":"Established the catalytic identity and substrate specificity of the protein, defining it as a RhoA-selective GEF and revealing an autoinhibited full-length state.","evidence":"In vitro GDP-release and protein-binding assays with domain dissection plus microtubule binding in vitro and in vivo","pmids":["11058585"],"confidence":"High","gaps":["Identity of the partner(s) that relieve autoinhibition in vivo not defined","Functional role of microtubule binding not characterized"]},{"year":2001,"claim":"Identified the protein as a sequence-specific RNA-binding factor, linking it to neurofilament mRNA stability and revealing a function distinct from its GEF activity.","evidence":"GST-pulldown, Northwestern, gel-shift, RNA cross-linkage, and mRNA half-life measurement in stable neuronal transfectants","pmids":["11435431"],"confidence":"High","gaps":["Mechanism by which binding stabilizes NF-L mRNA unknown","Connection between RNA-binding and GEF functions unresolved"]},{"year":2001,"claim":"Mapped phosphorylation-independent 14-3-3 binding and showed it regulates the protein's cytoplasmic aggregation, hinting at scaffold/localization control.","evidence":"Yeast two-hybrid, co-IP, co-localization, and deletion mutagenesis mapping the I1370QAIQNL site","pmids":["11533041"],"confidence":"High","gaps":["Functional consequence of 14-3-3 binding for GEF or RNA activity not established"]},{"year":2002,"claim":"Showed the noncoding BC1 RNA competes for the same C-terminal RNA-binding site as NF-L, implying regulatory competition among RNA ligands.","evidence":"GST-fusion affinity chromatography and cross-competition gel-shift assays","pmids":["12215442"],"confidence":"Medium","gaps":["In vitro only; cellular relevance of competition not demonstrated","Single lab"]},{"year":2003,"claim":"Defined the direct FAK–ARHGEF28 interaction and placed the protein downstream of integrin and growth-factor receptor signaling driving RhoA activation.","evidence":"Reciprocal co-IP, yeast two-hybrid with FAT domain mutagenesis, and RhoA GTP-loading assay in neuronal cells/brain extract","pmids":["12702722"],"confidence":"High","gaps":["Whether interaction governs GEF activity directly not shown","Tyrosine phosphorylation sites on the GEF not mapped"]},{"year":2003,"claim":"Localized anti-apoptotic activity to cytoplasmic retention sequences overlapping JIP-1/14-3-3 sites, linking subcellular retention to survival function.","evidence":"EGFP-tagged deletion constructs with apoptosis and microscopy readouts in Neuro 2a cells","pmids":["14499478"],"confidence":"Medium","gaps":["No in vitro reconstitution","Mechanism of anti-apoptotic effect undefined","Single lab"]},{"year":2003,"claim":"Connected the protein to immune-receptor signaling, showing CD40-induced expression drives RhoA-dependent NF-κB activation in B cells.","evidence":"2D-gel identification, overexpression with dominant-negative RhoA and GEF constructs, NF-κB reporter assay","pmids":["12496377"],"confidence":"Medium","gaps":["Direct biochemical link from GEF to NF-κB not shown","Single lab"]},{"year":2008,"claim":"Revealed density-dependent competition between ARHGEF28 and E-cadherin for delta-catenin, modulating RhoA activity with cell contact.","evidence":"Co-IP, immunofluorescence, RhoA activity assay, and ectopic E-cadherin expression in MEFs","pmids":["18930028"],"confidence":"Medium","gaps":["Direct vs indirect delta-catenin binding not fully resolved","Single lab"]},{"year":2010,"claim":"Extended the RNA-binding function to human RGNEF and tied NFL mRNA interaction to ALS pathology.","evidence":"In vitro gel-shift and IP-RT-PCR in ALS patient vs control tissue lysates","pmids":["19488899"],"confidence":"Medium","gaps":["Disease-specific interaction correlative, not causal","Single lab"]},{"year":2011,"claim":"Demonstrated the Rgnef–FAK complex is required for FAK adhesion targeting, paxillin phosphorylation, invasion, and tumor growth, establishing oncogenic relevance.","evidence":"shRNA knockdown, dominant-negative C-terminal competition with binding-site deletion, phosphorylation assays, orthotopic tumor implantation","pmids":["21224360"],"confidence":"High","gaps":["Did not separate GEF-dependent from scaffold-dependent contributions"]},{"year":2012,"claim":"Genetic knockout established the protein as essential for integrin-stimulated RhoA activation, focal adhesion formation, and migration.","evidence":"Conditional Rgnef knockout MEFs with haptotaxis, wound closure, focal adhesion quantification, RhoA assay, and rescue by re-expression","pmids":["22649559"],"confidence":"High","gaps":["Did not dissect which molecular function (GEF vs scaffold) drives each phenotype"]},{"year":2013,"claim":"Separated the scaffold and catalytic functions, showing PH-dependent GEF-independent FAK-Y397 activation versus GEF-dependent paxillin-Y118 phosphorylation.","evidence":"Rgnef-null MEFs with PH-domain and GEF-inactive mutant rescue, immunofluorescence, phosphotyrosine immunoblotting","pmids":["24006257"],"confidence":"High","gaps":["How the PH domain promotes FAK activation mechanistically not resolved"]},{"year":2015,"claim":"Identified the protein as a selective Gα13 effector downstream of gastrin/CCK2 receptor, linking GPCR signaling to FAK/paxillin phosphorylation.","evidence":"Reciprocal co-IP with constitutively active Gα mutants, point mutagenesis, SRE-luciferase reporter, shRNA epistasis, RhoA assay","pmids":["25922072"],"confidence":"High","gaps":["Structural basis of Gα13 selectivity not defined"]},{"year":2017,"claim":"Provided structural mechanism for GEF recruitment, showing both RhoA·GTP and Rac1·GTP bind the PH domain to localize the enzyme, enabling feedback and cross-talk.","evidence":"X-ray crystallography of PH-domain complexes plus in vitro nucleotide exchange assays","pmids":["29196061"],"confidence":"High","gaps":["Cellular validation of Rac→Rho cross-talk through this surface not shown"]},{"year":2017,"claim":"Linked the protein to stress survival and injury responses in neurons and to Staufen1 RNA granules rather than canonical stress granules.","evidence":"In vivo sciatic nerve injury model, stress survival assays with deletion constructs, immunofluorescence co-localization","pmids":["28495450"],"confidence":"Medium","gaps":["Mechanism of N-terminal protective effect undefined","Single lab"]},{"year":2018,"claim":"Defined an NLS/NES within the PH domain controlling nucleocytoplasmic shuttling via exportin-1, providing a basis for nuclear functions.","evidence":"NLS/NES mutagenesis, fusion-protein microscopy, Leptomycin B inhibition","pmids":["30482479"],"confidence":"Medium","gaps":["Nuclear function of the shuttling not established","Single lab"]},{"year":2019,"claim":"Showed the protein is required for ovarian tumor growth by sustaining an NF-κB-driven antioxidant gene program.","evidence":"Rgnef knockout mouse models, spheroid assays, RNA-seq, antioxidant rescue, NF-κB pathway analysis","pmids":["31308489"],"confidence":"Medium","gaps":["Direct link from GEF activity to NF-κB antioxidant signature not biochemically defined","Single lab"]},{"year":2024,"claim":"Established a direct ARHGEF28–TDP-43 interaction whose N-terminal fragment is neuroprotective in two ALS models, defining a therapeutic mechanism.","evidence":"Direct protein interaction with IPT/TIG domain mapping, Drosophila genetic epistasis, AAV9/NF242 delivery in rNLS8 mice","pmids":["38739752"],"confidence":"High","gaps":["Whether endogenous full-length protein performs this function not addressed"]},{"year":2024,"claim":"Revealed that ARHGEF28 and TDP-43 antagonistically regulate long-intron splicing of axon-guidance genes, defining a shared post-transcriptional mechanism.","evidence":"Comparative RNA-seq of TDP-43- and RGNEF-depleted neuronal cells with long-intron processivity analysis","pmids":["39360635"],"confidence":"Medium","gaps":["Direct splicing-factor activity vs indirect effect not distinguished","Single lab"]},{"year":2026,"claim":"Demonstrated an in vivo role in bone remodeling, with Rgnef driving osteoclastogenesis via RhoA/Rac1 and NF-κB/MAPK/AKT signaling.","evidence":"Rgnef-deficient and transgenic mice, differentiation assays, GTPase activity assays, in vivo bone loss models","pmids":["41571890"],"confidence":"Medium","gaps":["Direct effectors linking GTPase activation to bone-cell transcription not defined","Single lab"]},{"year":null,"claim":"How the dual GEF and RNA-binding/splicing activities are coordinated within a single protein, and what governs the switch between cytoskeletal and post-transcriptional roles, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking GEF, scaffold, and RNA-regulatory functions","Endogenous regulation of nucleocytoplasmic shuttling and its functional output undefined","Whether RNA binding and exchange activity are mutually exclusive not tested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[3,4,8,17]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,9,11]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[12]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,5,7]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[15]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[12,13,19]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[10,11]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[3,18]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,16,17]}],"complexes":["Rgnef-FAK scaffold complex"],"partners":["PTK2/FAK","GNA13","YWHAH","YWHAE","CTNND2","TARDBP","RHOA","RAC1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8N1W1","full_name":"Rho guanine nucleotide exchange factor 28","aliases":["190 kDa guanine nucleotide exchange factor","p190-RhoGEF","p190RhoGEF","Rho guanine nucleotide exchange factor"],"length_aa":1705,"mass_kda":191.9,"function":"Functions as a RHOA-specific guanine nucleotide exchange factor regulating signaling pathways downstream of integrins and growth factor receptors. Functions in axonal branching, synapse formation and dendritic morphogenesis. Also functions in focal adhesion formation, cell motility and B-lymphocytes activation. May regulate NEFL expression and aggregation and play a role in apoptosis (By similarity)","subcellular_location":"Cytoplasm; Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q8N1W1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ARHGEF28","classification":"Not Classified","n_dependent_lines":21,"n_total_lines":1208,"dependency_fraction":0.0173841059602649},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ARHGEF28","total_profiled":1310},"omim":[{"mim_id":"612790","title":"RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR 28; ARHGEF28","url":"https://www.omim.org/entry/612790"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Microtubules","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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Full-length p190RhoGEF fails to activate RhoA in vitro, suggesting autoinhibition requiring unknown binding partners to unmask exchange activity in vivo. p190RhoGEF directly interacts with microtubules via its C-terminal region adjacent to the DH/PH domain, shown by in vitro and in vivo binding experiments.\",\n      \"method\": \"In vitro GDP release assay, protein binding assay, immunofluorescence, in vitro and in vivo microtubule binding experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay with domain dissection, microtubule binding confirmed both in vitro and in vivo, multiple orthogonal methods in one study\",\n      \"pmids\": [\"11058585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"FAK directly interacts with p190RhoGEF in neuronal cells and brain tissue extracts. The FAK C-terminal focal adhesion targeting (FAT) domain binds the C-terminal coiled-coil domain of p190RhoGEF, identified by two-hybrid assay and deletion mutagenesis. A FAK FAT domain mutation (Leu-1034 to Ser) disrupts this interaction. FAK activity promotes p190RhoGEF tyrosine phosphorylation and RhoA GTP loading downstream of laminin/integrin and IGF-1 receptor stimulation.\",\n      \"method\": \"Co-immunoprecipitation, co-localization, yeast two-hybrid, deletion mutagenesis, dominant-negative inhibition (FRNK), RhoA GTP-loading assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, two-hybrid with mutagenesis, functional GTP-loading assay, multiple orthogonal methods\",\n      \"pmids\": [\"12702722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"14-3-3η and 14-3-3ε are binding partners of p190RhoGEF, identified by yeast two-hybrid screen and confirmed biochemically by co-immunoprecipitation and co-localization. A phosphorylation-independent binding site (I1370QAIQNL) in p190RhoGEF was mapped; deletion of this site abolishes 14-3-3η interaction in vitro and prevents 14-3-3η-mediated alteration of p190RhoGEF cytoplasmic aggregation in cells.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, co-localization with fluorescent fusion proteins, deletion mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by biochemical co-IP and colocalization, site mapped by deletion mutagenesis, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"11533041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"p190RhoGEF binds directly and specifically to the 68-nucleotide destabilizing element in the 3' UTR of NF-L (neurofilament light) mRNA via its C-terminal domain (clone 39), demonstrated by Northwestern blot, gel-shift, and cross-linkage assays. Expression of p190RhoGEF in stably transfected neuronal cells increased the half-life of wild-type NF-L mRNA but not of a mutant lacking the destabilizing element, establishing a functional role in NF-L mRNA stability.\",\n      \"method\": \"GST-fusion pulldown, Northwestern blot, gel-shift assay, RNA cross-linkage, stable transfection with mRNA half-life measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstituted RNA binding with multiple biochemical methods plus functional mRNA stability assay with appropriate controls (wild-type vs. mutant mRNA)\",\n      \"pmids\": [\"11435431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"BC1 RNA (a neuronally expressed non-coding RNA) binds to the same C-terminal site of p190RhoGEF as NF-L mRNA and competes with NF-L mRNA for p190RhoGEF binding, identified by affinity chromatography and cross-competition experiments using a GST-p190RhoGEF C-terminal fusion protein.\",\n      \"method\": \"GST-fusion affinity chromatography, gel-shift, cross-competition binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro reconstituted competition assay with identified binding site, single lab\",\n      \"pmids\": [\"12215442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Anti-apoptotic activity of p190RhoGEF is localized to two cytoplasmic retention sequences (CRS-1 and CRS-2) in its C-terminal region that overlap with the JIP-1 and 14-3-3 binding sites. Deleting both CRS sequences abolishes cytoplasmic retention and anti-apoptotic activity of EGFP-tagged p190RhoGEF in Neuro 2a cells; restoring either CRS-1 or CRS-2 rescues both properties.\",\n      \"method\": \"Transfection of EGFP-tagged deletion constructs, apoptosis assay, fluorescence microscopy in Neuro 2a cells\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — domain deletion mapping with functional apoptosis readout, single lab, cell-based assay without in vitro reconstitution\",\n      \"pmids\": [\"14499478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"p190RhoGEF expression is induced by CD40 stimulation in WEHI 231 B cells. Overexpression of p190RhoGEF mimics CD40-stimulated cellular structure changes and NF-κB activation through RhoA; these effects are blocked by dominant-negative RhoA (T19N) or dominant-negative p190RhoGEF (Y1003A).\",\n      \"method\": \"2D gel electrophoresis identification, overexpression with dominant-negative constructs, NF-κB reporter assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — dominant-negative epistasis, single lab, multiple constructs tested\",\n      \"pmids\": [\"12496377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"delta-Catenin interacts with p190RhoGEF in the cytoplasm at low cell density, reducing RhoA activity. At high cell density, E-cadherin outcompetes p190RhoGEF for delta-catenin binding, shifting delta-catenin to the plasma membrane and restoring RhoA activity. Ectopic E-cadherin expression in mouse embryonic fibroblasts decreased delta-catenin's effect on RhoA activity reduction.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, RhoA activity assay, ectopic E-cadherin expression\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and functional RhoA assay, competition shown by cell density experiment and ectopic E-cadherin, single lab\",\n      \"pmids\": [\"18930028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Human RGNEF (the human homologue of p190RhoGEF) directly interacts with human NFL mRNA in vitro by gel-shift assay. In tissue lysates, RGNEF-NFL mRNA interaction was detected by IP-RT-PCR only in ALS patient samples, not in neuropathologically normal controls.\",\n      \"method\": \"Gel-shift assay (in vitro), IP-RT-PCR (in tissue lysates)\",\n      \"journal\": \"Amyotrophic lateral sclerosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro RNA binding confirmed by gel shift plus in-tissue IP-RT-PCR, single lab\",\n      \"pmids\": [\"19488899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Rgnef forms a complex with FAK in human colon carcinoma cells. Upon gastrin stimulation, Rgnef-FAK interaction is required for FAK translocation to focal adhesions, paxillin tyrosine phosphorylation, cell motility, and invadopodia formation. Overexpression of the Rgnef C-terminal region (aa 1279–1582) disrupts endogenous Rgnef-FAK interaction and blocks these events; a version lacking the FAK binding site (aa 1302–1582) does not. Rgnef-C-expressing cells form smaller, less invasive tumors in vivo.\",\n      \"method\": \"shRNA knockdown, co-immunoprecipitation, dominant-negative C-terminal fragment competition, phosphorylation assays, orthotopic tumor implantation\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — shRNA KD, dominant-negative competition with defined binding-site deletion, in vivo tumor model, multiple orthogonal readouts, single lab\",\n      \"pmids\": [\"21224360\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Genetic knockout of Rgnef in mouse embryo fibroblasts (Rgnef-/- MEFs) significantly inhibits haptotaxis migration, wound closure motility, focal adhesion number, and RhoA GTPase activation after fibronectin-integrin stimulation. These phenotypes are rescued by epitope-tagged Rgnef re-expression, establishing Rgnef as essential for RhoA regulation downstream of integrins.\",\n      \"method\": \"Conditional knockout (floxed Rgnef × CMV-Cre), primary MEF isolation, haptotaxis assay, wound closure assay, focal adhesion quantification, RhoA GTP-loading assay, rescue by re-expression\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined phenotypes and rescue by re-expression, multiple orthogonal readouts, clean loss-of-function\",\n      \"pmids\": [\"22649559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Rgnef plays a non-canonical, upstream scaffolding role in promoting FAK localization to peripheral adhesions and FAK-Y397 activation upon fibronectin binding, independent of its GEF catalytic activity. A PH domain mutation in Rgnef blocks adhesion formation, FAK localization, and FAK/paxillin phosphorylation without disrupting the Rgnef-FAK interaction. A GEF-inactive Rgnef mutant rescues FAK-Y397 phosphorylation and adhesion localization but not paxillin-Y118 phosphorylation, indicating paxillin-pY118 requires Rgnef GEF activity through a distinct mechanism.\",\n      \"method\": \"Rgnef-null MEFs, site-directed mutagenesis (PH domain and GEF-inactive mutants), immunofluorescence, phosphotyrosine immunoblotting, rescue experiments\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic null MEFs with domain-specific mutant rescue, dissection of GEF-dependent vs. GEF-independent functions, multiple orthogonal methods\",\n      \"pmids\": [\"24006257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Rgnef is a new effector for Gα13 downstream of gastrin and the CCK2 receptor in DLD-1 colon carcinoma cells. Rgnef co-immunoprecipitates with activated Gα13Q226L but not Gα12Q229L; the Rgnef C-terminal region (aa 1279–1582) is sufficient for this interaction and its exogenous expression blocks Gα13-stimulated SRE activity. Point mutations in the Rgnef C-terminal region disrupt Gα13 association but not Gαq association. Gα13 depletion reduces gastrin-induced FAK-pY397 and paxillin-pY31.\",\n      \"method\": \"Co-immunoprecipitation, shRNA depletion of Gα13, SRE-luciferase reporter assay, point mutagenesis of Rgnef C-terminus, RhoA GTP-binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, mutagenesis defining binding site, functional SRE assay, shRNA epistasis, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25922072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structures reveal that activated Rac1·GTP and RhoA·GTP use their effector-binding surfaces to associate with the same hydrophobic surface on the p190RhoGEF PH domain. Both activated RhoA and Rac1 stimulate nucleotide exchange on RhoA·GDP by p190RhoGEF in vitro, localizing it to its substrate. This demonstrates a positive feedback (activated RhoA) and a cross-talk mechanism (activated Rac1 directly stimulates RhoA activation through p190RhoGEF).\",\n      \"method\": \"X-ray crystallography, in vitro nucleotide exchange assay\",\n      \"journal\": \"Journal of structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of PH domain complexes with Rac1·GTP and RhoA·GTP plus in vitro exchange assay confirming functional consequence\",\n      \"pmids\": [\"29196061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RGNEF expression is upregulated in murine spinal motor neurons following distal sciatic nerve injury. Under cellular stress (sodium arsenite or sorbitol), RGNEF expression confers a survival benefit in HEK293T cells; the NH2-terminus domain is essential for this protective effect. Under stress, RGNEF associates with Staufen1-positive granules but not TIA-1-positive stress granules.\",\n      \"method\": \"In vivo nerve injury model, in vitro stress assay with deletion constructs, immunofluorescence co-localization\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — deletion construct mapping in cell-based survival assay plus colocalization, single lab\",\n      \"pmids\": [\"28495450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A 23-amino acid bipartite nuclear localization signal (NLS) within the Pleckstrin Homology (PH) domain of RGNEF controls its nuclear localization; deletion or mutation of this region abolishes nuclear localization. Within this NLS, an overlapping nuclear export signal (NES) promotes nuclear export in an exportin-1-dependent manner (confirmed by Leptomycin B treatment). The PH domain alone is sufficient to translocate a 160 kDa fusion protein to the nucleus.\",\n      \"method\": \"Deletion and point mutagenesis of NLS/NES, fluorescence microscopy of fusion proteins, Leptomycin B inhibition\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — domain mutagenesis with functional localization readout and pharmacological confirmation, single lab\",\n      \"pmids\": [\"30482479\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Rgnef is essential for ovarian tumor spheroid formation in vitro and tumor growth in vivo using transgenic and transplantable Rgnef knockout mouse models. Rgnef supports an NF-κB-mediated antioxidant gene signature (including Gpx4, Nqo1, Gsta4); antioxidant treatment rescues growth of Rgnef-knockout spheroids, and Rgnef re-expression facilitates NF-κB-dependent tumorsphere survival.\",\n      \"method\": \"Rgnef knockout mouse model, spheroid formation assay, RNA-sequencing, antioxidant rescue experiment, NF-κB reporter/pathway analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with rescue, RNA-seq pathway identification, pharmacological rescue, single lab\",\n      \"pmids\": [\"31308489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"An N-terminal fragment of RGNEF (NF242) directly interacts with the RNA recognition motifs (RRMs) of TDP-43, competing with RNA binding. The IPT/TIG domain of NF242 is essential for this interaction. In a Drosophila ALS model overexpressing TDP-43, genetic expression of NF242 suppressed neuropathological phenotypes (increased lifespan, abolished motor defects, prevented neurodegeneration). Intracerebroventricular injection of AAV9/NF242 in a murine TDP-43 model (rNLS8) improved lifespan and motor phenotype and decreased neuroinflammation markers.\",\n      \"method\": \"Direct protein-protein interaction assay, domain deletion (IPT/TIG), Drosophila genetic epistasis model, murine AAV9 intracerebroventricular injection\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding with domain mutagenesis confirmed functionally in two independent in vivo models (fly and mouse), multiple orthogonal methods\",\n      \"pmids\": [\"38739752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RGNEF and TDP-43 act predominantly in an antagonistic manner to regulate expression of axon guidance genes in neuronal cells. Mechanistically, both factors affect the processivity of long intron removal (splicing), explaining their mode of transcriptomic action upon depletion.\",\n      \"method\": \"Comparative transcriptomics (RNA-seq) of TDP-43- and RGNEF-depleted neuronal cells, long intron processivity analysis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — transcriptomic comparison of KD cells with mechanistic intron-processivity analysis, single lab\",\n      \"pmids\": [\"39360635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Rgnef promotes osteoclastogenesis and attenuates osteoblastogenesis through activation of RhoA and Rac1, leading to enhanced NF-κB, MAPK, and AKT signaling. Rgnef-deficient mice show increased bone mass due to reduced osteolysis and increased osteogenesis, while Rgnef-overexpressing mice show the opposite. Rgnef-deficient mice are protected from bone loss in LPS-induced inflammation and ovariectomy models.\",\n      \"method\": \"Rgnef-deficient and transgenic overexpressing mice, osteoclast/osteoblast differentiation assays, RhoA/Rac1 activity assay, NF-κB/MAPK/AKT pathway analysis, in vivo bone loss models\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO and transgenic OE with mechanistic pathway analysis, single lab, pathway placement by GTPase activity assay\",\n      \"pmids\": [\"41571890\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ARHGEF28/RGNEF/p190RhoGEF is a 190 kDa bifunctional protein that acts as a RhoA-specific guanine nucleotide exchange factor (DH/PH domain) and RNA-binding protein; its PH domain binds activated RhoA·GTP and Rac1·GTP (crystal structure confirmed) to localize it to substrate RhoA·GDP, enabling both positive feedback and Rac→Rho cross-talk, while its C-terminal coiled-coil domain directly binds FAK (FAT domain), Gα13, 14-3-3, delta-catenin, and NF-L/BC1 RNAs; downstream of integrins and GPCRs, a Rgnef–FAK scaffold (requiring the Rgnef PH domain) promotes FAK-Y397 autophosphorylation and peripheral adhesion formation in a GEF-activity-independent manner, whereas Rgnef GEF activity separately drives RhoA-dependent paxillin-Y118 phosphorylation, focal adhesion maturation, and cell motility, as established by genetic knockout, mutagenesis, structural, and in vitro reconstitution studies.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ARHGEF28 (RGNEF/p190RhoGEF) is a bifunctional 190 kDa protein that couples RhoA-specific guanine nucleotide exchange to RNA binding, integrating cytoskeletal signaling at focal adhesions with post-transcriptional control in neurons [#0, #3]. Its tandem DH/PH module catalyzes RhoA-specific GDP release in vitro while sparing Rac1 and Cdc42, and the full-length protein is autoinhibited until binding partners unmask activity [#0]; crystallography shows that activated RhoA\\u00b7GTP and Rac1\\u00b7GTP dock onto the same hydrophobic surface of its PH domain to recruit the enzyme to substrate RhoA\\u00b7GDP, generating both positive feedback and Rac\\u2192Rho cross-talk [#13]. Downstream of integrins and GPCRs, ARHGEF28 forms a scaffold with FAK through its C-terminal coiled-coil binding the FAK FAT domain [#1, #9], and genetic knockout establishes it as essential for RhoA activation, focal adhesion formation, and migration after fibronectin stimulation [#10]; this scaffolding role promotes FAK-Y397 autophosphorylation and peripheral adhesion assembly in a PH-domain-dependent but GEF-activity-independent manner, whereas its GEF activity separately drives paxillin-Y118 phosphorylation [#11]. It is an effector of G\\u03b113 downstream of gastrin/CCK2 receptor signaling [#12] and supports NF-\\u03baB-dependent transcriptional programs in tumor and bone contexts [#16, #19]. As an RNA-binding protein, its C-terminal domain binds the destabilizing element of NF-L mRNA to extend its half-life, a site also engaged by BC1 RNA [#3, #4]. The protein additionally engages TDP-43 RNA-recognition motifs through an N-terminal IPT/TIG-containing fragment, and ARHGEF28 and TDP-43 act antagonistically to regulate splicing of long introns in axon-guidance genes [#17, #18].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established the catalytic identity and substrate specificity of the protein, defining it as a RhoA-selective GEF and revealing an autoinhibited full-length state.\",\n      \"evidence\": \"In vitro GDP-release and protein-binding assays with domain dissection plus microtubule binding in vitro and in vivo\",\n      \"pmids\": [\"11058585\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the partner(s) that relieve autoinhibition in vivo not defined\", \"Functional role of microtubule binding not characterized\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified the protein as a sequence-specific RNA-binding factor, linking it to neurofilament mRNA stability and revealing a function distinct from its GEF activity.\",\n      \"evidence\": \"GST-pulldown, Northwestern, gel-shift, RNA cross-linkage, and mRNA half-life measurement in stable neuronal transfectants\",\n      \"pmids\": [\"11435431\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which binding stabilizes NF-L mRNA unknown\", \"Connection between RNA-binding and GEF functions unresolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Mapped phosphorylation-independent 14-3-3 binding and showed it regulates the protein's cytoplasmic aggregation, hinting at scaffold/localization control.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, co-localization, and deletion mutagenesis mapping the I1370QAIQNL site\",\n      \"pmids\": [\"11533041\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of 14-3-3 binding for GEF or RNA activity not established\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showed the noncoding BC1 RNA competes for the same C-terminal RNA-binding site as NF-L, implying regulatory competition among RNA ligands.\",\n      \"evidence\": \"GST-fusion affinity chromatography and cross-competition gel-shift assays\",\n      \"pmids\": [\"12215442\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro only; cellular relevance of competition not demonstrated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the direct FAK\\u2013ARHGEF28 interaction and placed the protein downstream of integrin and growth-factor receptor signaling driving RhoA activation.\",\n      \"evidence\": \"Reciprocal co-IP, yeast two-hybrid with FAT domain mutagenesis, and RhoA GTP-loading assay in neuronal cells/brain extract\",\n      \"pmids\": [\"12702722\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether interaction governs GEF activity directly not shown\", \"Tyrosine phosphorylation sites on the GEF not mapped\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Localized anti-apoptotic activity to cytoplasmic retention sequences overlapping JIP-1/14-3-3 sites, linking subcellular retention to survival function.\",\n      \"evidence\": \"EGFP-tagged deletion constructs with apoptosis and microscopy readouts in Neuro 2a cells\",\n      \"pmids\": [\"14499478\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution\", \"Mechanism of anti-apoptotic effect undefined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Connected the protein to immune-receptor signaling, showing CD40-induced expression drives RhoA-dependent NF-\\u03baB activation in B cells.\",\n      \"evidence\": \"2D-gel identification, overexpression with dominant-negative RhoA and GEF constructs, NF-\\u03baB reporter assay\",\n      \"pmids\": [\"12496377\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical link from GEF to NF-\\u03baB not shown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed density-dependent competition between ARHGEF28 and E-cadherin for delta-catenin, modulating RhoA activity with cell contact.\",\n      \"evidence\": \"Co-IP, immunofluorescence, RhoA activity assay, and ectopic E-cadherin expression in MEFs\",\n      \"pmids\": [\"18930028\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect delta-catenin binding not fully resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Extended the RNA-binding function to human RGNEF and tied NFL mRNA interaction to ALS pathology.\",\n      \"evidence\": \"In vitro gel-shift and IP-RT-PCR in ALS patient vs control tissue lysates\",\n      \"pmids\": [\"19488899\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Disease-specific interaction correlative, not causal\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated the Rgnef\\u2013FAK complex is required for FAK adhesion targeting, paxillin phosphorylation, invasion, and tumor growth, establishing oncogenic relevance.\",\n      \"evidence\": \"shRNA knockdown, dominant-negative C-terminal competition with binding-site deletion, phosphorylation assays, orthotopic tumor implantation\",\n      \"pmids\": [\"21224360\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate GEF-dependent from scaffold-dependent contributions\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Genetic knockout established the protein as essential for integrin-stimulated RhoA activation, focal adhesion formation, and migration.\",\n      \"evidence\": \"Conditional Rgnef knockout MEFs with haptotaxis, wound closure, focal adhesion quantification, RhoA assay, and rescue by re-expression\",\n      \"pmids\": [\"22649559\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not dissect which molecular function (GEF vs scaffold) drives each phenotype\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Separated the scaffold and catalytic functions, showing PH-dependent GEF-independent FAK-Y397 activation versus GEF-dependent paxillin-Y118 phosphorylation.\",\n      \"evidence\": \"Rgnef-null MEFs with PH-domain and GEF-inactive mutant rescue, immunofluorescence, phosphotyrosine immunoblotting\",\n      \"pmids\": [\"24006257\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the PH domain promotes FAK activation mechanistically not resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified the protein as a selective G\\u03b113 effector downstream of gastrin/CCK2 receptor, linking GPCR signaling to FAK/paxillin phosphorylation.\",\n      \"evidence\": \"Reciprocal co-IP with constitutively active G\\u03b1 mutants, point mutagenesis, SRE-luciferase reporter, shRNA epistasis, RhoA assay\",\n      \"pmids\": [\"25922072\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of G\\u03b113 selectivity not defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided structural mechanism for GEF recruitment, showing both RhoA\\u00b7GTP and Rac1\\u00b7GTP bind the PH domain to localize the enzyme, enabling feedback and cross-talk.\",\n      \"evidence\": \"X-ray crystallography of PH-domain complexes plus in vitro nucleotide exchange assays\",\n      \"pmids\": [\"29196061\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular validation of Rac\\u2192Rho cross-talk through this surface not shown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Linked the protein to stress survival and injury responses in neurons and to Staufen1 RNA granules rather than canonical stress granules.\",\n      \"evidence\": \"In vivo sciatic nerve injury model, stress survival assays with deletion constructs, immunofluorescence co-localization\",\n      \"pmids\": [\"28495450\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of N-terminal protective effect undefined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined an NLS/NES within the PH domain controlling nucleocytoplasmic shuttling via exportin-1, providing a basis for nuclear functions.\",\n      \"evidence\": \"NLS/NES mutagenesis, fusion-protein microscopy, Leptomycin B inhibition\",\n      \"pmids\": [\"30482479\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nuclear function of the shuttling not established\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed the protein is required for ovarian tumor growth by sustaining an NF-\\u03baB-driven antioxidant gene program.\",\n      \"evidence\": \"Rgnef knockout mouse models, spheroid assays, RNA-seq, antioxidant rescue, NF-\\u03baB pathway analysis\",\n      \"pmids\": [\"31308489\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link from GEF activity to NF-\\u03baB antioxidant signature not biochemically defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a direct ARHGEF28\\u2013TDP-43 interaction whose N-terminal fragment is neuroprotective in two ALS models, defining a therapeutic mechanism.\",\n      \"evidence\": \"Direct protein interaction with IPT/TIG domain mapping, Drosophila genetic epistasis, AAV9/NF242 delivery in rNLS8 mice\",\n      \"pmids\": [\"38739752\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether endogenous full-length protein performs this function not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed that ARHGEF28 and TDP-43 antagonistically regulate long-intron splicing of axon-guidance genes, defining a shared post-transcriptional mechanism.\",\n      \"evidence\": \"Comparative RNA-seq of TDP-43- and RGNEF-depleted neuronal cells with long-intron processivity analysis\",\n      \"pmids\": [\"39360635\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct splicing-factor activity vs indirect effect not distinguished\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstrated an in vivo role in bone remodeling, with Rgnef driving osteoclastogenesis via RhoA/Rac1 and NF-\\u03baB/MAPK/AKT signaling.\",\n      \"evidence\": \"Rgnef-deficient and transgenic mice, differentiation assays, GTPase activity assays, in vivo bone loss models\",\n      \"pmids\": [\"41571890\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct effectors linking GTPase activation to bone-cell transcription not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the dual GEF and RNA-binding/splicing activities are coordinated within a single protein, and what governs the switch between cytoskeletal and post-transcriptional roles, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking GEF, scaffold, and RNA-regulatory functions\", \"Endogenous regulation of nucleocytoplasmic shuttling and its functional output undefined\", \"Whether RNA binding and exchange activity are mutually exclusive not tested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005088\", \"supporting_discovery_ids\": [0, 13]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [3, 4, 8, 17]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 9, 11]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 5, 7]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [12, 13, 19]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [10, 11]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [3, 18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 16, 17]}\n    ],\n    \"complexes\": [\n      \"Rgnef-FAK scaffold complex\"\n    ],\n    \"partners\": [\n      \"PTK2/FAK\",\n      \"GNA13\",\n      \"YWHAH\",\n      \"YWHAE\",\n      \"CTNND2\",\n      \"TARDBP\",\n      \"RHOA\",\n      \"RAC1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}