{"gene":"PLEKHG5","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2005,"finding":"Tech (PLEKHG5) selectively binds to and activates RhoA (but not Rac1 or Cdc42) via its DH domain in vitro, and a constitutively active Tech construct decreases dendritic process number in cortical neurons; this effect is blocked by a DH-domain point mutation abolishing RhoA activation or by dominant-negative RhoA.","method":"In vitro GEF assay with prototypical Rho subfamily members; point mutagenesis of DH domain; dominant-negative RhoA co-expression; primary cortical neuron morphology readout","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro GEF activity assay plus mutagenesis plus cellular rescue in a single focused study","pmids":["15686487"],"is_preprint":false},{"year":2007,"finding":"Wild-type PLEKHG5 activates the NF-κB signaling pathway in transfected HEK293 and MCF10A cells; a disease-causing missense mutation (p.Phe647Ser) alters protein stability and intracellular localization, severely impairing NF-κB transduction and causing protein aggregates in NSC34 motor neurons.","method":"Transient transfection of wild-type and mutant PLEKHG5 in HEK293, MCF10A, and NSC34 cells; NF-κB reporter assays; immunofluorescence localization; aggregate detection","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell lines and orthogonal assays (reporter, localization, aggregate) in a single study; replicated functionally in later studies","pmids":["17564964"],"is_preprint":false},{"year":2008,"finding":"Syx (PLEKHG5) forms a ternary complex with Amot and Patj/Mupp1 via its C-terminal PDZ-binding motif; this complex spatially controls RhoA GTPase activity at lamellipodia of migrating endothelial cells as shown by FRET analysis.","method":"Peptide pull-down, yeast two-hybrid screening, FRET analysis of RhoA activity in lamellipodia, morpholino knockdown in zebrafish","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Y2H, pulldown, FRET, in vivo knockdown) across labs; replicated by subsequent studies","pmids":["18824598"],"is_preprint":false},{"year":2008,"finding":"Syx (PLEKHG5) is required for angiogenic sprouting in vivo; morpholino knockdown in zebrafish specifically impairs vascular sprouting without affecting vasculogenesis or angioblast differentiation, and this defect is partially rescued by mouse Syx mRNA; Syx knockdown in vitro impairs VEGF-A-induced endothelial cell migration.","method":"Morpholino knockdown in zebrafish; mRNA rescue; in vitro endothelial migration assay","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo loss-of-function with rescue, replicated in vitro, consistent with independent zebrafish knockdown data in PMID:18824598","pmids":["18757825"],"is_preprint":false},{"year":2008,"finding":"Tech (PLEKHG5) binds to MUPP1 PDZ domains 10 and 13 via its C-terminal PDZ ligand motif; endogenous Tech co-precipitates with MUPP1 (but not PSD-95) from hippocampal/cortical extracts, and the two proteins co-localize at peri-synaptic puncta in cortical neurons.","method":"Yeast two-hybrid; co-transfection/co-IP in HEK293; endogenous co-IP from rat brain extracts; PDZ-domain mutagenesis; immunofluorescence co-localization","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal endogenous Co-IP plus mutagenesis plus co-localization in a single study","pmids":["18537874"],"is_preprint":false},{"year":2010,"finding":"Rnd3 (RhoE) directly interacts with Syx (PLEKHG5) via a Raf1-like Ras-binding domain (RBD) in Syx, identified by affinity purification/mass spectrometry; this interaction does not involve the Syx DH domain. A Rnd3-binding-defective Syx mutant (E164A/R165D) is more potent in rescuing zebrafish axis defects than wild-type, indicating Rnd3 negatively regulates Syx GEF activity in vivo.","method":"Two-step affinity purification/mass spectrometry; co-IP; RBD point mutagenesis; zebrafish morpholino knockdown and mRNA rescue","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — affinity purification/MS identification plus mutagenesis plus in vivo functional validation in a single study","pmids":["20811643"],"is_preprint":false},{"year":2012,"finding":"Syx (PLEKHG5) is recruited to endothelial cell junctions by members of the Crumbs polarity complex and promotes junction integrity by activating the RhoA downstream effector Diaphanous; VEGF causes PKD1-mediated phosphorylation of Syx at Ser806, reducing its association with junctional anchors and promoting junction disassembly; Ang1 maintains Syx at junctions to stabilize them. syx−/− mice display defective junctions, vascular leakiness, edema, and impaired heart function.","method":"Co-IP; subcellular fractionation/localization; PKD1 kinase assay; phosphomutant constructs; syx knockout mouse; vascular permeability and cardiac function assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods including knockout mouse, phosphomutants, and functional vascular readouts; replicated across conditions","pmids":["23253477"],"is_preprint":false},{"year":2013,"finding":"14-3-3 proteins interact with Syx (PLEKHG5) at both N- and C-terminal regions in a phosphorylation-dependent manner; PKD-mediated phosphorylation at Ser92 and additional phosphorylation at Ser938 are critical for 14-3-3 association. 14-3-3 binding inhibits Syx GEF activity; phosphorylation-deficient, 14-3-3-uncoupled Syx shows increased junctional targeting and GEF activity, strengthening the circumferential junctional actin ring in MDCK cells.","method":"Co-IP; in vitro kinase assay; phosphomutant constructs; GEF activity assay; immunofluorescence of junctional actin","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro GEF assay plus phosphomutant rescue plus junctional actin readout in a single study","pmids":["23335514"],"is_preprint":false},{"year":2013,"finding":"Syx (PLEKHG5) is required for polarity of migrating brain and breast tumor cells; this function is mediated by selective activation of the RhoA effector Dia1, leading to microtubule reorganization, downregulation of focal adhesions and actin stress fibers, and activation of cofilin-mediated actin reorganization. Syx recruitment to the membrane suppresses ROCK activity while activating Dia1.","method":"siRNA knockdown; live-cell migration assay; Dia1 and ROCK activity measurements; microtubule and focal adhesion immunofluorescence","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotypes and pathway effector measurements, single lab","pmids":["24126053"],"is_preprint":false},{"year":2015,"finding":"VEGF-A/NRP1 signaling induces formation of a GIPC1–Syx (PLEKHG5) complex; this complex activates RhoA and promotes cell proliferation in DJM-1 skin cancer cells. Knockdown of GIPC1 or Syx reduces active RhoA and proliferation; constitutively active RhoA rescues proliferation in siVEGF-A cells. A cell-penetrating oligopeptide targeting GIPC1/Syx complex formation inhibits RhoA activation.","method":"Co-IP; siRNA knockdown; RhoA-GTP pull-down; proliferation assays; constitutively active RhoA rescue; inhibitory peptide","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus multiple functional assays in a single lab","pmids":["26209534"],"is_preprint":false},{"year":2017,"finding":"Plekhg5 acts as a guanine exchange factor for Rab26, a small GTPase that directs synaptic vesicles to preautophagosomal structures; Plekhg5 gene knockout in mice causes late-onset motor neuron disease with degeneration of axon terminals. Cultured Plekhg5-depleted motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, rescued by constitutively active Rab26.","method":"Plekhg5 knockout mouse; cultured motoneuron knockdown; GEF assay for Rab26; autophagy reporters; axon growth measurements; constitutively active Rab26 rescue","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro GEF assay for Rab26, knockout mouse with defined phenotype, rescue with constitutively active GTPase; multiple orthogonal methods","pmids":["29084947"],"is_preprint":false},{"year":2017,"finding":"Plekhg5 (PLEKHG5) regulates cell polarity, directional migration, adhesion, podosome organization, and bone resorption in macrophages and osteoclasts; depletion causes abnormal localization of mDia1, LIMK1, cofilin, EB1, and vinculin, with upregulation of mDia1 and LIMK1 protein levels.","method":"siRNA knockdown in macrophages and osteoclasts; migration assay; podosome and bone resorption assays; immunofluorescence of Rho effectors","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA loss-of-function with multiple cellular phenotypes and effector localization, single lab","pmids":["28847484"],"is_preprint":false},{"year":2018,"finding":"Plekhg5 RhoGEF activity is required for apical constriction of bottle cells during Xenopus gastrulation; Plekhg5 protein localizes to the apical cell cortex via its pleckstrin homology (PH) domain, and GEF activity enhances this apical recruitment. Plekhg5 induces apical actomyosin accumulation and cell elongation; knockdown inhibits activin-induced bottle cell formation and blastopore lip formation in a Rho-dependent manner.","method":"Morpholino knockdown in Xenopus embryos; GEF-activity-deficient mutants; PH domain truncations; F-actin/myosin immunofluorescence; ectopic bottle cell induction; Rho inhibitor treatment","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — loss-of-function plus domain dissection plus GEF-dead mutants plus Rho-dependency test in a single study","pmids":["30446627"],"is_preprint":false},{"year":2018,"finding":"NRP-1 forms a complex with GIPC1 and Syx (PLEKHG5) to activate RhoA/ROCK-dependent p38 MAPK activity, enhancing epidermal cancer stem cell (ECS) spheroid formation, invasion, migration, and angiogenic potential; constitutively active RhoA or p38 in NRP1-knockout cells restores the ECS cell phenotype.","method":"Co-IP; siRNA/shRNA knockdown; RhoA-GTP pull-down; p38 activity assay; spheroid and invasion assays; constitutively active rescue","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus RhoA/p38 activity assays plus rescue, single lab","pmids":["30456845"],"is_preprint":false},{"year":2020,"finding":"PLEKHG5 knockout in U251-MG glioblastoma cells (via CRISPR/Cas9) impairs autophagy (accumulation of autolysosomes, decreased LAMP-1), reduces RhoA activity, alters morphology, and reduces filopodia; rescue by constitutively active RAB26 (RAB26QL) restores RhoA levels, autophagy, and cellular fitness, and RAB26 overexpression activates MGMT expression.","method":"CRISPR/Cas9 knockout; mRFP-GFP-LC3 autophagy reporter; RAB26QL lentiviral rescue; RhoA activity assay; LAMP-1 immunofluorescence","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO plus autophagy reporter plus RAB26 rescue in a single lab","pmids":["33318498"],"is_preprint":false},{"year":2020,"finding":"Absence of Plekhg5 in mice results in myelin infoldings in peripheral nerves and impaired Schwann cell autophagy, with a reduced number of CD4+ and CD8+ T-cells in sciatic nerves; RNAseq identified a transcriptional signature of impaired immune response in Plekhg5-deficient peripheral nerves.","method":"Plekhg5 knockout mouse; electron microscopy of peripheral nerves; T-cell immunofluorescence/flow cytometry; RNAseq of sciatic nerves","journal":"Frontiers in cellular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout mouse with histological, cellular, and transcriptomic readouts in a single study","pmids":["32733205"],"is_preprint":false},{"year":2022,"finding":"Human Syx (PLEKHG5) can be expressed, purified, and shown to be folded by circular dichroism; it actively binds RhoA as determined by co-elution in size exclusion chromatography. Molecular dynamics simulations on a physiologically realistic membrane reveal novel allosteric interactions between the PH domain and the membrane-embedded region of RhoA, supporting PH domain allosteric regulation of DH-domain GEF activity.","method":"Recombinant protein expression/purification; circular dichroism spectroscopy; size exclusion chromatography co-elution; homology modeling; molecular dynamics simulation","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — biophysical characterization and computational modeling in a single study; no mutagenesis validation of allosteric site","pmids":["35639414"],"is_preprint":false},{"year":2023,"finding":"The Syx–RhoA–Dia1 signaling axis promotes GBM cell cycle progression; Syx depletion causes prolonged mitosis, increased DNA damage, G2/M arrest, and apoptosis. These effects are phenocopied by Dia1 depletion and are mediated, at least in part, by increased phosphorylation, cytoplasmic retention, and reduced activity of YAP/TAZ transcriptional coactivators. Targeting Syx cooperates with radiation and temozolomide to decrease GBM cell viability.","method":"siRNA knockdown; orthotopic GBM xenografts; cell cycle analysis; DNA damage markers; YAP/TAZ phosphorylation and localization; combination drug/radiation assays","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function in vitro and in vivo with multiple mechanistic readouts, single lab","pmids":["37427593"],"is_preprint":false},{"year":2023,"finding":"HDAC2 directly interacts with PLEKHG5 and deacetylates lysine residues within its PH domain, maintaining PLEKHG5 protein stability. HDAC2 knockout or selective inhibition reduces PLEKHG5 protein levels and sensitizes HCC cells to sorafenib; overexpression of PLEKHG5 in HDAC2-KO cells restores sorafenib resistance. PLEKHG5 overexpression activates Rac1/AKT/NF-κB signaling.","method":"Co-IP; HDAC2 KO; selective HDAC2 inhibitor; ubiquitination/acetylation assays; in vitro and in vivo HCC drug sensitivity assays","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus KO rescue plus in vivo xenograft, single lab","pmids":["37248230"],"is_preprint":false},{"year":2023,"finding":"Plekhg5 regulates both medioapical and junctional actomyosin dynamics during apical constriction of Xenopus bottle cells; knockdown of plekhg5 disrupts medioapical and junctional actomyosin activity and apical constriction without affecting initial F-actin dynamics. Correlation of apical constriction with medioapical actomyosin localization is stronger than with junctional actomyosin.","method":"Morpholino knockdown in Xenopus embryos; live imaging; quantitative image analysis of actomyosin signals; F-actin dynamics measurements","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — morpholino knockdown with live imaging and quantitative actomyosin analysis, single lab","pmids":["37043306"],"is_preprint":false},{"year":2024,"finding":"Plekhg5 apical cortex localization requires N-terminal sequences and intact GEF activity; C-terminal sequences prevent basolateral mis-localization. Plekhg5 self-associates via its PH domain, and this self-association functionally rescues in trans two mutants lacking the N-terminal region or GEF activity. A disease-associated PH domain point mutation abolishes self-association and fails to induce apical constriction.","method":"Deletion and point mutant constructs in Xenopus; subcellular localization imaging; co-IP for self-association; in trans rescue assay; apical constriction functional readout","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — domain dissection, mutagenesis, Co-IP for self-association, and functional rescue in a single rigorous study","pmids":["39196644"],"is_preprint":false},{"year":2024,"finding":"Plekhg5 drives unconventional secretion (UPS) of Sod1 by sequestering Sod1 into autophagosomal carriers that fuse with secretory lysosome-related organelles (LROs); exocytosis of LROs requires Plekhg5-mediated activation of Rab26. Plekhg5 deletion in mice causes Sod1 accumulation in LROs at swollen presynaptic sites. In SOD1-G93A/Plekhg5-deleted mice, reduced secretion of toxic SOD1 accelerated disease onset but prolonged survival via attenuated microglial activation. Human iPSC-derived motoneurons with reduced PLEKHG5 show impaired ALS-linked SOD1 secretion.","method":"Plekhg5 knockout mouse; SOD1G93A/Plekhg5 double-mutant mouse; autophagosome/LRO marker co-localization; Rab26 GEF assay; secretion assay; iPSC-derived motoneurons; microglial activation markers","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple in vivo models, GEF assay, iPSC-derived human neurons, orthogonal secretion and organelle assays in a single study","pmids":["39366938"],"is_preprint":false},{"year":2025,"finding":"RND3 directly interacts with PLEKHG5 (shown by Co-IP) and upregulates PLEKHG5 expression; RND3 overexpression in ectopic endometrial stromal cells enhances autophagy and suppresses oxidative stress in a PLEKHG5-dependent manner, inhibiting EMS progression in vitro and in vivo.","method":"Co-IP; siRNA knockdown; autophagy markers; oxidative stress assays; mouse EMS model","journal":"FASEB journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP plus knockdown in a single lab; RND3-PLEKHG5 interaction previously reported mechanistically in PMID:20811643 with stronger evidence","pmids":["41137701"],"is_preprint":false}],"current_model":"PLEKHG5 (Syx/Tech) is a DH-PH domain RhoGEF that primarily activates RhoA (and also Rab26) in neurons, endothelial cells, and epithelial contexts: it forms scaffolded complexes with Amot/Patj/MUPP1 and GIPC1/NRP1 to spatially control RhoA activity at lamellipodia and cell junctions, drives junction integrity via Diaphanous/Dia1 and is removed from junctions by PKD1-phosphorylation/14-3-3-mediated inhibition; it acts as a GEF for Rab26 in motor neuron axon terminals to direct autophagy of synaptic vesicles and unconventional secretion of Sod1 via lysosome-related organelles; it localizes apically via its PH domain (which also mediates self-association required for activity) to activate RhoA-dependent actomyosin and drive apical constriction during gastrulation; and it activates NF-κB signaling, with disease-causing mutations impairing stability, localization, and these downstream activities to cause motor neuron disease and peripheral neuropathy."},"narrative":{"mechanistic_narrative":"PLEKHG5 (Syx/Tech) is a DH-PH domain guanine nucleotide exchange factor that selectively activates RhoA to control actomyosin organization, cell polarity, junction integrity, and migration across neuronal, endothelial, epithelial, and tumor contexts [PMID:15686487, PMID:18824598, PMID:23253477]. Its output is spatially confined through scaffolded complexes: a C-terminal PDZ-binding motif tethers it to Amot–Patj/MUPP1 to position RhoA activity at endothelial lamellipodia [PMID:18824598, PMID:18537874], and Crumbs-complex recruitment to cell junctions drives integrity via the RhoA effector Diaphanous/Dia1 [PMID:23253477, PMID:24126053]. This activity is switched off by phosphorylation-dependent inhibition: VEGF/PKD1-mediated phosphorylation (Ser806, Ser92, Ser938) couples PLEKHG5 to 14-3-3 proteins, removing it from junctions and suppressing GEF activity, whereas phospho-deficient PLEKHG5 shows enhanced junctional targeting and actin-ring strengthening [PMID:23253477, PMID:23335514]; binding of Rnd3 to a Raf1-like RBD provides an additional layer of negative regulation independent of the DH domain [PMID:20811643]. Membrane and apical targeting occur through the PH domain, which also mediates self-association required for GEF activity and allosterically regulates the DH domain against RhoA [PMID:35639414, PMID:39196644]. In motor neurons PLEKHG5 also acts as a GEF for Rab26 to direct autophagy of synaptic vesicles and unconventional secretion of Sod1 via lysosome-related organelles, and its loss causes late-onset motor neuron degeneration and peripheral nerve pathology in mice [PMID:29084947, PMID:39366938, PMID:32733205]. PLEKHG5 mutations cause inherited motor neuron disease and peripheral neuropathy, with disease alleles impairing protein stability, localization, NF-κB signaling, and PH-domain self-association [PMID:17564964, PMID:39196644]. Recurrently across cancers, NRP1/GIPC1- and VEGF-A-driven PLEKHG5–RhoA signaling promotes proliferation, invasion, and cell-cycle progression [PMID:26209534, PMID:30456845, PMID:37427593].","teleology":[{"year":2005,"claim":"Establishing PLEKHG5 as a RhoA-specific GEF defined its core biochemical activity and linked it to neuronal morphology.","evidence":"In vitro GEF assay across Rho subfamily members with DH-domain point mutagenesis and dominant-negative RhoA in cortical neurons","pmids":["15686487"],"confidence":"High","gaps":["Did not address regulation of GEF activity","No endogenous localization or partners defined"]},{"year":2007,"claim":"Linking PLEKHG5 to NF-κB activation and to a disease-causing missense mutation tied its molecular activity to motor neuron disease.","evidence":"Wild-type vs p.Phe647Ser mutant transfection in HEK293/MCF10A/NSC34 with NF-κB reporters, localization, and aggregate detection","pmids":["17564964"],"confidence":"Medium","gaps":["Mechanism connecting GEF activity to NF-κB not resolved","Aggregation cause vs consequence unclear"]},{"year":2008,"claim":"Identification of Amot–Patj/MUPP1 scaffolds and a role in angiogenic sprouting explained how RhoA activity is spatially confined and gave PLEKHG5 a developmental vascular function.","evidence":"Y2H, peptide pull-down, FRET of RhoA in lamellipodia, endogenous Co-IP from brain, and zebrafish morpholino knockdown with rescue","pmids":["18824598","18537874","18757825"],"confidence":"High","gaps":["How recruitment is dynamically regulated not yet shown","Effector downstream of localized RhoA not identified at this stage"]},{"year":2010,"claim":"Discovery of Rnd3 binding to a Raf1-like RBD revealed DH-independent negative regulation of PLEKHG5 GEF activity in vivo.","evidence":"Affinity purification/MS, RBD point mutagenesis (E164A/R165D), and zebrafish axis rescue assays","pmids":["20811643"],"confidence":"High","gaps":["Structural basis of RBD-mediated inhibition not defined","Physiological signals controlling Rnd3 binding unknown"]},{"year":2013,"claim":"Phosphoregulation via PKD1/14-3-3 and selective Dia1 (not ROCK) engagement defined a complete on/off switch and effector branch governing junction stability and directional migration.","evidence":"Co-IP, in vitro kinase and GEF assays, phosphomutant constructs, junctional actin imaging, and siRNA migration assays in MDCK and tumor cells","pmids":["23335514","24126053"],"confidence":"High","gaps":["Stoichiometry and kinetics of multi-site phosphorylation not resolved","How Dia1 is selected over ROCK mechanistically unclear"]},{"year":2012,"claim":"A syx knockout mouse demonstrated that junctional RhoA-Diaphanous signaling controls vascular barrier integrity in vivo, with VEGF/PKD1 and Ang1 setting junction disassembly versus stability.","evidence":"syx-/- mouse with vascular permeability and cardiac function assays, PKD1 kinase assay, Ser806 phosphomutants, and junctional fractionation","pmids":["23253477"],"confidence":"High","gaps":["Cell-type-specific contributions in vivo not dissected","Link between cardiac phenotype and direct PLEKHG5 function incomplete"]},{"year":2017,"claim":"Identification of PLEKHG5 as a Rab26 GEF redefined it as a regulator of synaptic-vesicle autophagy whose loss causes motor neuron degeneration, expanding its substrate range beyond RhoA.","evidence":"Plekhg5 knockout mouse, cultured motoneuron knockdown, Rab26 GEF assay, autophagy reporters, and constitutively active Rab26 rescue","pmids":["29084947"],"confidence":"High","gaps":["Determinants of RhoA vs Rab26 substrate choice unknown","Spatial control of Rab26 activation in axon terminals not resolved"]},{"year":2018,"claim":"PH-domain-dependent apical cortex targeting coupling GEF activity to apical actomyosin established PLEKHG5 as a driver of apical constriction during gastrulation.","evidence":"Xenopus morpholino knockdown, GEF-dead mutants, PH-domain truncations, actomyosin imaging, and Rho inhibitor tests","pmids":["30446627"],"confidence":"High","gaps":["PH-domain lipid/membrane ligand not identified at this stage","How GEF activity feeds back on apical recruitment mechanistically unclear"]},{"year":2015,"claim":"NRP1/GIPC1 complex formation showed PLEKHG5 functions downstream of VEGF-A receptors to drive RhoA-dependent proliferation and invasion in cancer.","evidence":"Co-IP, siRNA knockdown, RhoA-GTP pull-down, proliferation/spheroid/invasion assays, and constitutively active RhoA/p38 rescue in skin cancer and ECS cells","pmids":["26209534","30456845"],"confidence":"Medium","gaps":["Direct vs indirect NRP1 binding not fully resolved","Whether GIPC1 alters localization or activity is unclear"]},{"year":2020,"claim":"Knockout phenotypes in glioblastoma and peripheral nerve linked PLEKHG5 loss to defective autophagy, reduced RhoA, myelin pathology, and altered immune signature, broadening its physiological footprint.","evidence":"CRISPR knockout with autophagy reporters and RAB26QL rescue in U251 cells; Plekhg5 knockout mouse with nerve EM, T-cell profiling, and RNAseq","pmids":["33318498","32733205"],"confidence":"Medium","gaps":["Mechanism connecting RhoA and Rab26 outputs to autophagy not unified","Cell-autonomous vs non-autonomous immune effects undefined"]},{"year":2022,"claim":"Biophysical and computational characterization of purified PLEKHG5 supported PH-domain allosteric regulation of DH-domain GEF activity toward membrane-embedded RhoA.","evidence":"Recombinant expression, circular dichroism, size-exclusion co-elution with RhoA, homology modeling, and molecular dynamics on a realistic membrane","pmids":["35639414"],"confidence":"Medium","gaps":["Allosteric site not validated by mutagenesis","No experimental structure of the full protein"]},{"year":2024,"claim":"PH-domain self-association required for GEF activity and apical localization, with a disease mutation abolishing it, mechanistically connected a structural feature to function and pathology.","evidence":"Xenopus deletion/point mutants, self-association Co-IP, in trans rescue, and apical constriction readouts","pmids":["39196644","39366938"],"confidence":"High","gaps":["Oligomeric stoichiometry not defined","Whether self-association is regulated by signaling unknown"]},{"year":2025,"claim":"An RND3–PLEKHG5 axis was reported to modulate autophagy and oxidative stress in endometriosis, extending Rnd3 regulation to a new disease context.","evidence":"Co-IP, siRNA knockdown, autophagy and oxidative stress markers, and a mouse EMS model","pmids":["41137701"],"confidence":"Low","gaps":["Single Co-IP without reciprocal structural mapping in this context","Reported transcriptional upregulation of PLEKHG5 by RND3 mechanism unexplained","Direct vs indirect effects on autophagy not separated"]},{"year":null,"claim":"How PLEKHG5 selects between its RhoA and Rab26 substrates, and how its scaffolds, phosphorylation, and PH-domain self-association are integrated to choose between junction control, migration, apical constriction, and synaptic-vesicle autophagy in a given cell, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model of RhoA vs Rab26 substrate switching","No full-length experimental structure","Context-specific regulatory inputs incompletely mapped"]}],"mechanism_profile":{"molecular_activity":[],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,6,8,12,20]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[8,11,19]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,6,9]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[10,14,21]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,12,19]}],"complexes":["Amot–Patj/MUPP1 ternary complex","NRP1–GIPC1–Syx complex"],"partners":["RHOA","RAB26","AMOT","MPDZ","GIPC1","NRP1","RND3","YWHAB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O94827","full_name":"Pleckstrin homology domain-containing family G member 5","aliases":["Guanine nucleotide exchange factor 720","GEF720"],"length_aa":1006,"mass_kda":111.2,"function":"Functions as a guanine exchange factor (GEF) for RAB26 and thus regulates autophagy of synaptic vesicles in axon terminal of motoneurons (By similarity). Involved in the control of neuronal cell differentiation (PubMed:11704860). Plays a role in angiogenesis through regulation of endothelial cells chemotaxis. 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The CPMP, \"High Tech\" and Multi-State procedures.","date":"1989","source":"Biotherapy (Dordrecht, Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/2701848","citation_count":1,"is_preprint":false},{"pmid":"39741357","id":"PMC_39741357","title":"Molecular epidemiological analysis of Influenza viruses in Influenza-like illness cases: a retrospective study in Chongqing Hi-Tech Zone, China (2021-2024).","date":"2024","source":"Virology journal","url":"https://pubmed.ncbi.nlm.nih.gov/39741357","citation_count":1,"is_preprint":false},{"pmid":"36469612","id":"PMC_36469612","title":"A Simple and Low-Tech Heat-Shock Method to Increase Genome Editing Efficiency in Plants.","date":"2022","source":"Current protocols","url":"https://pubmed.ncbi.nlm.nih.gov/36469612","citation_count":1,"is_preprint":false},{"pmid":"40218664","id":"PMC_40218664","title":"Wearable Arduino-Based Electronic Interactive Tattoo: A New Type of High-Tech Humanized Emotional Expression for Electronic Skin.","date":"2025","source":"Sensors (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/40218664","citation_count":1,"is_preprint":false},{"pmid":"20827064","id":"PMC_20827064","title":"High-tech/high-touch team-centered care provides best outcomes for wound prevention in critically ill patients.","date":"2010","source":"Critical care nursing quarterly","url":"https://pubmed.ncbi.nlm.nih.gov/20827064","citation_count":1,"is_preprint":false},{"pmid":"12349328","id":"PMC_12349328","title":"A plethora of hi-tech vaccines -- genetic, edible, sugar glass, and more.","date":"1999","source":"CVI forum","url":"https://pubmed.ncbi.nlm.nih.gov/12349328","citation_count":1,"is_preprint":false},{"pmid":"41137701","id":"PMC_41137701","title":"RND3 Inhibits Endometriosis Progression by Regulating Autophagy and Oxidative Stress Through PLEKHG5.","date":"2025","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/41137701","citation_count":0,"is_preprint":false},{"pmid":"40236144","id":"PMC_40236144","title":"CDH-3/Cadherin, YAP-1/YAP and EGL-44/TEAD promote SYX-2/Syntaxin and EFF-1 fusogen-mediated phagosome closure.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40236144","citation_count":0,"is_preprint":false},{"pmid":"40632948","id":"PMC_40632948","title":"Melanoma 3.0T-Tech Innovations, New Targeted Therapies, and T-Cell Breakthroughs.","date":"2025","source":"American Society of Clinical Oncology educational book. American Society of Clinical Oncology. Annual Meeting","url":"https://pubmed.ncbi.nlm.nih.gov/40632948","citation_count":0,"is_preprint":false},{"pmid":"8379664","id":"PMC_8379664","title":"High-tech breakthrough DNA scanner for reading sequence and detecting gene mutation. A powerful 1 lb, 20 micron resolution, 16-bit personal scanner (PS) that scans 17\" x 14\" X-ray film in 48 s, with laser, UV and white light sources.","date":"1993","source":"Applied biochemistry and biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/8379664","citation_count":0,"is_preprint":false},{"pmid":"30477326","id":"PMC_30477326","title":"Tech news: stem cells for modeling and curing disease.","date":"2018","source":"BioTechniques","url":"https://pubmed.ncbi.nlm.nih.gov/30477326","citation_count":0,"is_preprint":false},{"pmid":"41615019","id":"PMC_41615019","title":"RNA interference tech takes aim at poultry mites.","date":"2026","source":"The Veterinary record","url":"https://pubmed.ncbi.nlm.nih.gov/41615019","citation_count":0,"is_preprint":false},{"pmid":"40751618","id":"PMC_40751618","title":"Tech strain: the musculoskeletal impact of electronic devices on young adults: A cross-sectional study.","date":"2025","source":"JPMA. The Journal of the Pakistan Medical Association","url":"https://pubmed.ncbi.nlm.nih.gov/40751618","citation_count":0,"is_preprint":false},{"pmid":"40680623","id":"PMC_40680623","title":"Catalyzing clean tech Innovation: The role of intellectual property protection in China's new energy sector.","date":"2025","source":"Journal of environmental management","url":"https://pubmed.ncbi.nlm.nih.gov/40680623","citation_count":0,"is_preprint":false},{"pmid":"42073847","id":"PMC_42073847","title":"Coaching Leadership and Employees' Bootlegging Innovation Behavior in Chinese High-Tech Enterprises.","date":"2026","source":"Behavioral sciences (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/42073847","citation_count":0,"is_preprint":false},{"pmid":"19048066","id":"PMC_19048066","title":"A high-tech infusion for science.","date":"2008","source":"Disease models & mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/19048066","citation_count":0,"is_preprint":false},{"pmid":"40190623","id":"PMC_40190623","title":"Comparative analysis of the toxic effects on the mouse lung of 4 weeks exposure to the heated tobacco product Ploom TECH+ and 3R4F reference cigarettes.","date":"2024","source":"Journal of toxicologic pathology","url":"https://pubmed.ncbi.nlm.nih.gov/40190623","citation_count":0,"is_preprint":false},{"pmid":"41568019","id":"PMC_41568019","title":"Gene Flooding: Proposal to Flood Invasive Populations With Inbred Individuals as a Form of Low-Tech Genetic Control.","date":"2026","source":"Ecology and evolution","url":"https://pubmed.ncbi.nlm.nih.gov/41568019","citation_count":0,"is_preprint":false},{"pmid":"11112087","id":"PMC_11112087","title":"A pharmacokinetic study of JOMO-tech in rats.","date":"2000","source":"European journal of drug metabolism and pharmacokinetics","url":"https://pubmed.ncbi.nlm.nih.gov/11112087","citation_count":0,"is_preprint":false},{"pmid":"17520874","id":"PMC_17520874","title":"VA Tech disaster response shows value of drills, planning.","date":"2007","source":"Healthcare benchmarks and quality improvement","url":"https://pubmed.ncbi.nlm.nih.gov/17520874","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.03.19.25324254","title":"DNA methylation patterns associated with prior tuberculosis infection in people with HIV: a pilot cross-sectional study","date":"2025-03-20","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.19.25324254","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":39476,"output_tokens":6419,"usd":0.107356,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15016,"output_tokens":4473,"usd":0.093452,"stage2_stop_reason":"end_turn"},"total_usd":0.200808,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"Tech (PLEKHG5) selectively binds to and activates RhoA (but not Rac1 or Cdc42) via its DH domain in vitro, and a constitutively active Tech construct decreases dendritic process number in cortical neurons; this effect is blocked by a DH-domain point mutation abolishing RhoA activation or by dominant-negative RhoA.\",\n      \"method\": \"In vitro GEF assay with prototypical Rho subfamily members; point mutagenesis of DH domain; dominant-negative RhoA co-expression; primary cortical neuron morphology readout\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro GEF activity assay plus mutagenesis plus cellular rescue in a single focused study\",\n      \"pmids\": [\"15686487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Wild-type PLEKHG5 activates the NF-κB signaling pathway in transfected HEK293 and MCF10A cells; a disease-causing missense mutation (p.Phe647Ser) alters protein stability and intracellular localization, severely impairing NF-κB transduction and causing protein aggregates in NSC34 motor neurons.\",\n      \"method\": \"Transient transfection of wild-type and mutant PLEKHG5 in HEK293, MCF10A, and NSC34 cells; NF-κB reporter assays; immunofluorescence localization; aggregate detection\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell lines and orthogonal assays (reporter, localization, aggregate) in a single study; replicated functionally in later studies\",\n      \"pmids\": [\"17564964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Syx (PLEKHG5) forms a ternary complex with Amot and Patj/Mupp1 via its C-terminal PDZ-binding motif; this complex spatially controls RhoA GTPase activity at lamellipodia of migrating endothelial cells as shown by FRET analysis.\",\n      \"method\": \"Peptide pull-down, yeast two-hybrid screening, FRET analysis of RhoA activity in lamellipodia, morpholino knockdown in zebrafish\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Y2H, pulldown, FRET, in vivo knockdown) across labs; replicated by subsequent studies\",\n      \"pmids\": [\"18824598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Syx (PLEKHG5) is required for angiogenic sprouting in vivo; morpholino knockdown in zebrafish specifically impairs vascular sprouting without affecting vasculogenesis or angioblast differentiation, and this defect is partially rescued by mouse Syx mRNA; Syx knockdown in vitro impairs VEGF-A-induced endothelial cell migration.\",\n      \"method\": \"Morpholino knockdown in zebrafish; mRNA rescue; in vitro endothelial migration assay\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo loss-of-function with rescue, replicated in vitro, consistent with independent zebrafish knockdown data in PMID:18824598\",\n      \"pmids\": [\"18757825\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Tech (PLEKHG5) binds to MUPP1 PDZ domains 10 and 13 via its C-terminal PDZ ligand motif; endogenous Tech co-precipitates with MUPP1 (but not PSD-95) from hippocampal/cortical extracts, and the two proteins co-localize at peri-synaptic puncta in cortical neurons.\",\n      \"method\": \"Yeast two-hybrid; co-transfection/co-IP in HEK293; endogenous co-IP from rat brain extracts; PDZ-domain mutagenesis; immunofluorescence co-localization\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal endogenous Co-IP plus mutagenesis plus co-localization in a single study\",\n      \"pmids\": [\"18537874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Rnd3 (RhoE) directly interacts with Syx (PLEKHG5) via a Raf1-like Ras-binding domain (RBD) in Syx, identified by affinity purification/mass spectrometry; this interaction does not involve the Syx DH domain. A Rnd3-binding-defective Syx mutant (E164A/R165D) is more potent in rescuing zebrafish axis defects than wild-type, indicating Rnd3 negatively regulates Syx GEF activity in vivo.\",\n      \"method\": \"Two-step affinity purification/mass spectrometry; co-IP; RBD point mutagenesis; zebrafish morpholino knockdown and mRNA rescue\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — affinity purification/MS identification plus mutagenesis plus in vivo functional validation in a single study\",\n      \"pmids\": [\"20811643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Syx (PLEKHG5) is recruited to endothelial cell junctions by members of the Crumbs polarity complex and promotes junction integrity by activating the RhoA downstream effector Diaphanous; VEGF causes PKD1-mediated phosphorylation of Syx at Ser806, reducing its association with junctional anchors and promoting junction disassembly; Ang1 maintains Syx at junctions to stabilize them. syx−/− mice display defective junctions, vascular leakiness, edema, and impaired heart function.\",\n      \"method\": \"Co-IP; subcellular fractionation/localization; PKD1 kinase assay; phosphomutant constructs; syx knockout mouse; vascular permeability and cardiac function assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods including knockout mouse, phosphomutants, and functional vascular readouts; replicated across conditions\",\n      \"pmids\": [\"23253477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"14-3-3 proteins interact with Syx (PLEKHG5) at both N- and C-terminal regions in a phosphorylation-dependent manner; PKD-mediated phosphorylation at Ser92 and additional phosphorylation at Ser938 are critical for 14-3-3 association. 14-3-3 binding inhibits Syx GEF activity; phosphorylation-deficient, 14-3-3-uncoupled Syx shows increased junctional targeting and GEF activity, strengthening the circumferential junctional actin ring in MDCK cells.\",\n      \"method\": \"Co-IP; in vitro kinase assay; phosphomutant constructs; GEF activity assay; immunofluorescence of junctional actin\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro GEF assay plus phosphomutant rescue plus junctional actin readout in a single study\",\n      \"pmids\": [\"23335514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Syx (PLEKHG5) is required for polarity of migrating brain and breast tumor cells; this function is mediated by selective activation of the RhoA effector Dia1, leading to microtubule reorganization, downregulation of focal adhesions and actin stress fibers, and activation of cofilin-mediated actin reorganization. Syx recruitment to the membrane suppresses ROCK activity while activating Dia1.\",\n      \"method\": \"siRNA knockdown; live-cell migration assay; Dia1 and ROCK activity measurements; microtubule and focal adhesion immunofluorescence\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotypes and pathway effector measurements, single lab\",\n      \"pmids\": [\"24126053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"VEGF-A/NRP1 signaling induces formation of a GIPC1–Syx (PLEKHG5) complex; this complex activates RhoA and promotes cell proliferation in DJM-1 skin cancer cells. Knockdown of GIPC1 or Syx reduces active RhoA and proliferation; constitutively active RhoA rescues proliferation in siVEGF-A cells. A cell-penetrating oligopeptide targeting GIPC1/Syx complex formation inhibits RhoA activation.\",\n      \"method\": \"Co-IP; siRNA knockdown; RhoA-GTP pull-down; proliferation assays; constitutively active RhoA rescue; inhibitory peptide\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus multiple functional assays in a single lab\",\n      \"pmids\": [\"26209534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Plekhg5 acts as a guanine exchange factor for Rab26, a small GTPase that directs synaptic vesicles to preautophagosomal structures; Plekhg5 gene knockout in mice causes late-onset motor neuron disease with degeneration of axon terminals. Cultured Plekhg5-depleted motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, rescued by constitutively active Rab26.\",\n      \"method\": \"Plekhg5 knockout mouse; cultured motoneuron knockdown; GEF assay for Rab26; autophagy reporters; axon growth measurements; constitutively active Rab26 rescue\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro GEF assay for Rab26, knockout mouse with defined phenotype, rescue with constitutively active GTPase; multiple orthogonal methods\",\n      \"pmids\": [\"29084947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Plekhg5 (PLEKHG5) regulates cell polarity, directional migration, adhesion, podosome organization, and bone resorption in macrophages and osteoclasts; depletion causes abnormal localization of mDia1, LIMK1, cofilin, EB1, and vinculin, with upregulation of mDia1 and LIMK1 protein levels.\",\n      \"method\": \"siRNA knockdown in macrophages and osteoclasts; migration assay; podosome and bone resorption assays; immunofluorescence of Rho effectors\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA loss-of-function with multiple cellular phenotypes and effector localization, single lab\",\n      \"pmids\": [\"28847484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Plekhg5 RhoGEF activity is required for apical constriction of bottle cells during Xenopus gastrulation; Plekhg5 protein localizes to the apical cell cortex via its pleckstrin homology (PH) domain, and GEF activity enhances this apical recruitment. Plekhg5 induces apical actomyosin accumulation and cell elongation; knockdown inhibits activin-induced bottle cell formation and blastopore lip formation in a Rho-dependent manner.\",\n      \"method\": \"Morpholino knockdown in Xenopus embryos; GEF-activity-deficient mutants; PH domain truncations; F-actin/myosin immunofluorescence; ectopic bottle cell induction; Rho inhibitor treatment\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — loss-of-function plus domain dissection plus GEF-dead mutants plus Rho-dependency test in a single study\",\n      \"pmids\": [\"30446627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NRP-1 forms a complex with GIPC1 and Syx (PLEKHG5) to activate RhoA/ROCK-dependent p38 MAPK activity, enhancing epidermal cancer stem cell (ECS) spheroid formation, invasion, migration, and angiogenic potential; constitutively active RhoA or p38 in NRP1-knockout cells restores the ECS cell phenotype.\",\n      \"method\": \"Co-IP; siRNA/shRNA knockdown; RhoA-GTP pull-down; p38 activity assay; spheroid and invasion assays; constitutively active rescue\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus RhoA/p38 activity assays plus rescue, single lab\",\n      \"pmids\": [\"30456845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PLEKHG5 knockout in U251-MG glioblastoma cells (via CRISPR/Cas9) impairs autophagy (accumulation of autolysosomes, decreased LAMP-1), reduces RhoA activity, alters morphology, and reduces filopodia; rescue by constitutively active RAB26 (RAB26QL) restores RhoA levels, autophagy, and cellular fitness, and RAB26 overexpression activates MGMT expression.\",\n      \"method\": \"CRISPR/Cas9 knockout; mRFP-GFP-LC3 autophagy reporter; RAB26QL lentiviral rescue; RhoA activity assay; LAMP-1 immunofluorescence\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO plus autophagy reporter plus RAB26 rescue in a single lab\",\n      \"pmids\": [\"33318498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Absence of Plekhg5 in mice results in myelin infoldings in peripheral nerves and impaired Schwann cell autophagy, with a reduced number of CD4+ and CD8+ T-cells in sciatic nerves; RNAseq identified a transcriptional signature of impaired immune response in Plekhg5-deficient peripheral nerves.\",\n      \"method\": \"Plekhg5 knockout mouse; electron microscopy of peripheral nerves; T-cell immunofluorescence/flow cytometry; RNAseq of sciatic nerves\",\n      \"journal\": \"Frontiers in cellular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse with histological, cellular, and transcriptomic readouts in a single study\",\n      \"pmids\": [\"32733205\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Human Syx (PLEKHG5) can be expressed, purified, and shown to be folded by circular dichroism; it actively binds RhoA as determined by co-elution in size exclusion chromatography. Molecular dynamics simulations on a physiologically realistic membrane reveal novel allosteric interactions between the PH domain and the membrane-embedded region of RhoA, supporting PH domain allosteric regulation of DH-domain GEF activity.\",\n      \"method\": \"Recombinant protein expression/purification; circular dichroism spectroscopy; size exclusion chromatography co-elution; homology modeling; molecular dynamics simulation\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — biophysical characterization and computational modeling in a single study; no mutagenesis validation of allosteric site\",\n      \"pmids\": [\"35639414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The Syx–RhoA–Dia1 signaling axis promotes GBM cell cycle progression; Syx depletion causes prolonged mitosis, increased DNA damage, G2/M arrest, and apoptosis. These effects are phenocopied by Dia1 depletion and are mediated, at least in part, by increased phosphorylation, cytoplasmic retention, and reduced activity of YAP/TAZ transcriptional coactivators. Targeting Syx cooperates with radiation and temozolomide to decrease GBM cell viability.\",\n      \"method\": \"siRNA knockdown; orthotopic GBM xenografts; cell cycle analysis; DNA damage markers; YAP/TAZ phosphorylation and localization; combination drug/radiation assays\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in vitro and in vivo with multiple mechanistic readouts, single lab\",\n      \"pmids\": [\"37427593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HDAC2 directly interacts with PLEKHG5 and deacetylates lysine residues within its PH domain, maintaining PLEKHG5 protein stability. HDAC2 knockout or selective inhibition reduces PLEKHG5 protein levels and sensitizes HCC cells to sorafenib; overexpression of PLEKHG5 in HDAC2-KO cells restores sorafenib resistance. PLEKHG5 overexpression activates Rac1/AKT/NF-κB signaling.\",\n      \"method\": \"Co-IP; HDAC2 KO; selective HDAC2 inhibitor; ubiquitination/acetylation assays; in vitro and in vivo HCC drug sensitivity assays\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus KO rescue plus in vivo xenograft, single lab\",\n      \"pmids\": [\"37248230\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Plekhg5 regulates both medioapical and junctional actomyosin dynamics during apical constriction of Xenopus bottle cells; knockdown of plekhg5 disrupts medioapical and junctional actomyosin activity and apical constriction without affecting initial F-actin dynamics. Correlation of apical constriction with medioapical actomyosin localization is stronger than with junctional actomyosin.\",\n      \"method\": \"Morpholino knockdown in Xenopus embryos; live imaging; quantitative image analysis of actomyosin signals; F-actin dynamics measurements\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — morpholino knockdown with live imaging and quantitative actomyosin analysis, single lab\",\n      \"pmids\": [\"37043306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Plekhg5 apical cortex localization requires N-terminal sequences and intact GEF activity; C-terminal sequences prevent basolateral mis-localization. Plekhg5 self-associates via its PH domain, and this self-association functionally rescues in trans two mutants lacking the N-terminal region or GEF activity. A disease-associated PH domain point mutation abolishes self-association and fails to induce apical constriction.\",\n      \"method\": \"Deletion and point mutant constructs in Xenopus; subcellular localization imaging; co-IP for self-association; in trans rescue assay; apical constriction functional readout\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — domain dissection, mutagenesis, Co-IP for self-association, and functional rescue in a single rigorous study\",\n      \"pmids\": [\"39196644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Plekhg5 drives unconventional secretion (UPS) of Sod1 by sequestering Sod1 into autophagosomal carriers that fuse with secretory lysosome-related organelles (LROs); exocytosis of LROs requires Plekhg5-mediated activation of Rab26. Plekhg5 deletion in mice causes Sod1 accumulation in LROs at swollen presynaptic sites. In SOD1-G93A/Plekhg5-deleted mice, reduced secretion of toxic SOD1 accelerated disease onset but prolonged survival via attenuated microglial activation. Human iPSC-derived motoneurons with reduced PLEKHG5 show impaired ALS-linked SOD1 secretion.\",\n      \"method\": \"Plekhg5 knockout mouse; SOD1G93A/Plekhg5 double-mutant mouse; autophagosome/LRO marker co-localization; Rab26 GEF assay; secretion assay; iPSC-derived motoneurons; microglial activation markers\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple in vivo models, GEF assay, iPSC-derived human neurons, orthogonal secretion and organelle assays in a single study\",\n      \"pmids\": [\"39366938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RND3 directly interacts with PLEKHG5 (shown by Co-IP) and upregulates PLEKHG5 expression; RND3 overexpression in ectopic endometrial stromal cells enhances autophagy and suppresses oxidative stress in a PLEKHG5-dependent manner, inhibiting EMS progression in vitro and in vivo.\",\n      \"method\": \"Co-IP; siRNA knockdown; autophagy markers; oxidative stress assays; mouse EMS model\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus knockdown in a single lab; RND3-PLEKHG5 interaction previously reported mechanistically in PMID:20811643 with stronger evidence\",\n      \"pmids\": [\"41137701\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLEKHG5 (Syx/Tech) is a DH-PH domain RhoGEF that primarily activates RhoA (and also Rab26) in neurons, endothelial cells, and epithelial contexts: it forms scaffolded complexes with Amot/Patj/MUPP1 and GIPC1/NRP1 to spatially control RhoA activity at lamellipodia and cell junctions, drives junction integrity via Diaphanous/Dia1 and is removed from junctions by PKD1-phosphorylation/14-3-3-mediated inhibition; it acts as a GEF for Rab26 in motor neuron axon terminals to direct autophagy of synaptic vesicles and unconventional secretion of Sod1 via lysosome-related organelles; it localizes apically via its PH domain (which also mediates self-association required for activity) to activate RhoA-dependent actomyosin and drive apical constriction during gastrulation; and it activates NF-κB signaling, with disease-causing mutations impairing stability, localization, and these downstream activities to cause motor neuron disease and peripheral neuropathy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PLEKHG5 (Syx/Tech) is a DH-PH domain guanine nucleotide exchange factor that selectively activates RhoA to control actomyosin organization, cell polarity, junction integrity, and migration across neuronal, endothelial, epithelial, and tumor contexts [#0, #2, #6]. Its output is spatially confined through scaffolded complexes: a C-terminal PDZ-binding motif tethers it to Amot–Patj/MUPP1 to position RhoA activity at endothelial lamellipodia [#2, #4], and Crumbs-complex recruitment to cell junctions drives integrity via the RhoA effector Diaphanous/Dia1 [#6, #8]. This activity is switched off by phosphorylation-dependent inhibition: VEGF/PKD1-mediated phosphorylation (Ser806, Ser92, Ser938) couples PLEKHG5 to 14-3-3 proteins, removing it from junctions and suppressing GEF activity, whereas phospho-deficient PLEKHG5 shows enhanced junctional targeting and actin-ring strengthening [#6, #7]; binding of Rnd3 to a Raf1-like RBD provides an additional layer of negative regulation independent of the DH domain [#5]. Membrane and apical targeting occur through the PH domain, which also mediates self-association required for GEF activity and allosterically regulates the DH domain against RhoA [#16, #20]. In motor neurons PLEKHG5 also acts as a GEF for Rab26 to direct autophagy of synaptic vesicles and unconventional secretion of Sod1 via lysosome-related organelles, and its loss causes late-onset motor neuron degeneration and peripheral nerve pathology in mice [#10, #21, #15]. PLEKHG5 mutations cause inherited motor neuron disease and peripheral neuropathy, with disease alleles impairing protein stability, localization, NF-κB signaling, and PH-domain self-association [#1, #20]. Recurrently across cancers, NRP1/GIPC1- and VEGF-A-driven PLEKHG5–RhoA signaling promotes proliferation, invasion, and cell-cycle progression [#9, #13, #17].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing PLEKHG5 as a RhoA-specific GEF defined its core biochemical activity and linked it to neuronal morphology.\",\n      \"evidence\": \"In vitro GEF assay across Rho subfamily members with DH-domain point mutagenesis and dominant-negative RhoA in cortical neurons\",\n      \"pmids\": [\"15686487\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address regulation of GEF activity\", \"No endogenous localization or partners defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Linking PLEKHG5 to NF-κB activation and to a disease-causing missense mutation tied its molecular activity to motor neuron disease.\",\n      \"evidence\": \"Wild-type vs p.Phe647Ser mutant transfection in HEK293/MCF10A/NSC34 with NF-κB reporters, localization, and aggregate detection\",\n      \"pmids\": [\"17564964\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting GEF activity to NF-κB not resolved\", \"Aggregation cause vs consequence unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identification of Amot–Patj/MUPP1 scaffolds and a role in angiogenic sprouting explained how RhoA activity is spatially confined and gave PLEKHG5 a developmental vascular function.\",\n      \"evidence\": \"Y2H, peptide pull-down, FRET of RhoA in lamellipodia, endogenous Co-IP from brain, and zebrafish morpholino knockdown with rescue\",\n      \"pmids\": [\"18824598\", \"18537874\", \"18757825\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How recruitment is dynamically regulated not yet shown\", \"Effector downstream of localized RhoA not identified at this stage\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Discovery of Rnd3 binding to a Raf1-like RBD revealed DH-independent negative regulation of PLEKHG5 GEF activity in vivo.\",\n      \"evidence\": \"Affinity purification/MS, RBD point mutagenesis (E164A/R165D), and zebrafish axis rescue assays\",\n      \"pmids\": [\"20811643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of RBD-mediated inhibition not defined\", \"Physiological signals controlling Rnd3 binding unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Phosphoregulation via PKD1/14-3-3 and selective Dia1 (not ROCK) engagement defined a complete on/off switch and effector branch governing junction stability and directional migration.\",\n      \"evidence\": \"Co-IP, in vitro kinase and GEF assays, phosphomutant constructs, junctional actin imaging, and siRNA migration assays in MDCK and tumor cells\",\n      \"pmids\": [\"23335514\", \"24126053\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and kinetics of multi-site phosphorylation not resolved\", \"How Dia1 is selected over ROCK mechanistically unclear\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"A syx knockout mouse demonstrated that junctional RhoA-Diaphanous signaling controls vascular barrier integrity in vivo, with VEGF/PKD1 and Ang1 setting junction disassembly versus stability.\",\n      \"evidence\": \"syx-/- mouse with vascular permeability and cardiac function assays, PKD1 kinase assay, Ser806 phosphomutants, and junctional fractionation\",\n      \"pmids\": [\"23253477\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-type-specific contributions in vivo not dissected\", \"Link between cardiac phenotype and direct PLEKHG5 function incomplete\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identification of PLEKHG5 as a Rab26 GEF redefined it as a regulator of synaptic-vesicle autophagy whose loss causes motor neuron degeneration, expanding its substrate range beyond RhoA.\",\n      \"evidence\": \"Plekhg5 knockout mouse, cultured motoneuron knockdown, Rab26 GEF assay, autophagy reporters, and constitutively active Rab26 rescue\",\n      \"pmids\": [\"29084947\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of RhoA vs Rab26 substrate choice unknown\", \"Spatial control of Rab26 activation in axon terminals not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"PH-domain-dependent apical cortex targeting coupling GEF activity to apical actomyosin established PLEKHG5 as a driver of apical constriction during gastrulation.\",\n      \"evidence\": \"Xenopus morpholino knockdown, GEF-dead mutants, PH-domain truncations, actomyosin imaging, and Rho inhibitor tests\",\n      \"pmids\": [\"30446627\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PH-domain lipid/membrane ligand not identified at this stage\", \"How GEF activity feeds back on apical recruitment mechanistically unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"NRP1/GIPC1 complex formation showed PLEKHG5 functions downstream of VEGF-A receptors to drive RhoA-dependent proliferation and invasion in cancer.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, RhoA-GTP pull-down, proliferation/spheroid/invasion assays, and constitutively active RhoA/p38 rescue in skin cancer and ECS cells\",\n      \"pmids\": [\"26209534\", \"30456845\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect NRP1 binding not fully resolved\", \"Whether GIPC1 alters localization or activity is unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Knockout phenotypes in glioblastoma and peripheral nerve linked PLEKHG5 loss to defective autophagy, reduced RhoA, myelin pathology, and altered immune signature, broadening its physiological footprint.\",\n      \"evidence\": \"CRISPR knockout with autophagy reporters and RAB26QL rescue in U251 cells; Plekhg5 knockout mouse with nerve EM, T-cell profiling, and RNAseq\",\n      \"pmids\": [\"33318498\", \"32733205\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting RhoA and Rab26 outputs to autophagy not unified\", \"Cell-autonomous vs non-autonomous immune effects undefined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Biophysical and computational characterization of purified PLEKHG5 supported PH-domain allosteric regulation of DH-domain GEF activity toward membrane-embedded RhoA.\",\n      \"evidence\": \"Recombinant expression, circular dichroism, size-exclusion co-elution with RhoA, homology modeling, and molecular dynamics on a realistic membrane\",\n      \"pmids\": [\"35639414\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Allosteric site not validated by mutagenesis\", \"No experimental structure of the full protein\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"PH-domain self-association required for GEF activity and apical localization, with a disease mutation abolishing it, mechanistically connected a structural feature to function and pathology.\",\n      \"evidence\": \"Xenopus deletion/point mutants, self-association Co-IP, in trans rescue, and apical constriction readouts\",\n      \"pmids\": [\"39196644\", \"39366938\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Oligomeric stoichiometry not defined\", \"Whether self-association is regulated by signaling unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"An RND3–PLEKHG5 axis was reported to modulate autophagy and oxidative stress in endometriosis, extending Rnd3 regulation to a new disease context.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, autophagy and oxidative stress markers, and a mouse EMS model\",\n      \"pmids\": [\"41137701\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single Co-IP without reciprocal structural mapping in this context\", \"Reported transcriptional upregulation of PLEKHG5 by RND3 mechanism unexplained\", \"Direct vs indirect effects on autophagy not separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PLEKHG5 selects between its RhoA and Rab26 substrates, and how its scaffolds, phosphorylation, and PH-domain self-association are integrated to choose between junction control, migration, apical constriction, and synaptic-vesicle autophagy in a given cell, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model of RhoA vs Rab26 substrate switching\", \"No full-length experimental structure\", \"Context-specific regulatory inputs incompletely mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005085\", \"supporting_discovery_ids\": [0, 2, 5, 10, 12, 16, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 6, 8, 12, 20]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [8, 11, 19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 6, 9]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [10, 14, 21]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 12, 19]}\n    ],\n    \"complexes\": [\n      \"Amot–Patj/MUPP1 ternary complex\",\n      \"NRP1–GIPC1–Syx complex\"\n    ],\n    \"partners\": [\n      \"RHOA\",\n      \"RAB26\",\n      \"AMOT\",\n      \"MPDZ\",\n      \"GIPC1\",\n      \"NRP1\",\n      \"RND3\",\n      \"YWHAB\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}