{"gene":"PREX1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2002,"finding":"P-Rex1 is a 185 kDa guanine-nucleotide exchange factor (GEF) for Rac, purified from neutrophil cytosol, whose Rac-GEF activity is directly, substantially, and synergistically activated by PtdIns(3,4,5)P3 and Gβγ subunits both in vitro and in vivo. Antisense knockdown of P-Rex1 reduced C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line.","method":"Biochemical purification from neutrophil cytosol, in vitro GEF activity assay, antisense knockdown with ROS readout","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of GEF activity with purified components, replicated in vivo, foundational paper with multiple orthogonal methods","pmids":["11955434"],"is_preprint":false},{"year":2005,"finding":"P-Rex1 preferentially activates Rac2 over Rac1 in mouse neutrophils; P-Rex1-deficient neutrophils show impaired Rac2 (but not Rac1) activation in response to fMLP, reduced F-actin formation, superoxide production, and chemotactic migration rate. P-Rex1 showed higher affinity for dominant-negative Rac2(S17N) than Rac1(S17N) by co-immunoprecipitation.","method":"P-Rex1 knockout mouse, PBD pulldown GTPase activation assay, co-immunoprecipitation with dominant-negative Rac isoforms, superoxide assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined cellular phenotype, replicated by two independent KO studies in the same journal issue","pmids":["16243036","16243035"],"is_preprint":false},{"year":2005,"finding":"In P-Rex1-deficient mice, GPCR-dependent Rac2 activation is impaired; LPS-primed P-Rex1−/− neutrophils lack GPCR-dependent ROS formation; recruitment of P-Rex1−/− neutrophils to inflammatory sites is impaired. Chemotaxis of isolated neutrophils is only mildly reduced, with normal polarization and directionality.","method":"P-Rex1 knockout mouse, in vivo peritonitis model, ROS assay, chemotaxis assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with multiple defined cellular phenotypes, replicated independently","pmids":["16243035"],"is_preprint":false},{"year":2005,"finding":"PKA phosphorylates P-Rex1 in vitro and in cells, making Gβγ 47-fold less potent at activating phosphorylated P-Rex1 compared to dephosphorylated P-Rex1. Gs-coupled receptor activation (isoproterenol) increases P-Rex1 phosphorylation and reduces GTP-bound Rac in cells.","method":"In vitro kinase assay with purified PKA, 32P metabolic labeling in HEK293T cells, GTP-Rac pulldown assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins combined with cell-based phosphorylation and Rac activation assays, single lab","pmids":["16301320"],"is_preprint":false},{"year":2005,"finding":"Among Gβγ dimer isoforms, Gβ1–4/γ2 dimers activate P-Rex1 GEF activity (EC50 10–20 nM) while Gβ5γ2 cannot. Gβ1 paired with different γ subunits shows variable potency; Gβ1γ11 (abundant in hematopoietic cells) and Gβ1γ12 are less effective activators. Gα subunits (Gs, Gi, Gq, G12, G13) do not activate P-Rex1.","method":"In vitro GEF activity assay with purified recombinant Gβγ dimers reconstituted into synthetic lipid vesicles","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified recombinant proteins, systematic isoform survey, single lab","pmids":["16301321"],"is_preprint":false},{"year":2005,"finding":"P-Rex1 is expressed in developing mouse brain neurons and localizes to the leading process of migrating neurons. In PC12 cells, P-Rex1 is activated by NGF to increase GTP-bound Rac1 and cell motility. Dominant-negative P-Rex1 (lacking DH domain) or siRNA knockdown impairs neurotrophin/EGF-induced cell migration of PC12 cells and primary cortical neurons. In utero electroporation of the dominant-negative form perturbs radial neuronal migration.","method":"In situ hybridization, immunofluorescence localization, Rac-GTP pulldown, siRNA knockdown, dominant-negative construct, in utero electroporation","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function with multiple approaches (siRNA, dominant-negative, in vivo electroporation) with defined migration phenotype","pmids":["15858067"],"is_preprint":false},{"year":2007,"finding":"P-Rex1 membrane translocation requires synergistic action of Gβγ and PI3K (PIP3); neither alone causes significant translocation. The DH/PH domain tandem is sufficient for membrane localization; GEF activity is not required. The DEP, PDZ, and IP4P domains promote cytosolic retention in basal cells. Membrane-derived P-Rex1 has higher basal Rac2-GEF activity than cytosol-derived P-Rex1.","method":"Subcellular fractionation of Sf9 cells co-expressing P-Rex1 with Gβγ and/or PI3K; P-Rex1 domain mutants; in vitro Rac2-GEF activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — fractionation combined with in vitro GEF assay and domain mutagenesis, single lab","pmids":["17698854"],"is_preprint":false},{"year":2007,"finding":"P-Rex1 interacts with mTOR through its tandem DEP domains and is associated with both mTORC1 and mTORC2 complexes. P-Rex1 dominant-negative constructs and shRNA knockdown decrease mTOR-dependent leucine-induced Rac activation and cell migration. P-Rex1 is only active when in the mTORC2 complex (rapamycin does not inhibit Rac activity or migration induced by leucine).","method":"Co-immunoprecipitation of P-Rex1 with mTOR complexes, dominant-negative constructs, shRNA knockdown, Rac activation assay, cell migration assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP and functional knockdown, single lab, mTORC2 specificity inferred from rapamycin insensitivity","pmids":["17565979"],"is_preprint":false},{"year":2007,"finding":"Endogenous P-Rex1 translocates from cytoplasm to the leading edge of polarized human neutrophils upon chemoattractant stimulation, colocalizing with F-actin and Rac2, in a Gβγ- and PIP3-dependent manner. This translocation requires tyrosine kinase activity, is modulated by cell adhesion, and is inhibited by PKA activation.","method":"Immunofluorescence microscopy of endogenous P-Rex1 in activated human neutrophils, pharmacological inhibitors","journal":"Journal of leukocyte biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct localization of endogenous protein with functional manipulation, single lab","pmids":["17227822"],"is_preprint":false},{"year":2008,"finding":"The second DEP and first PDZ domains of P-Rex1 associate with the IP4P domain (intramolecular interaction). Mutations in the PDZ protein-binding pocket or C-terminal truncation of IP4P abolish this interaction. Gβγ can activate a complex of P-Rex1 lacking IP4P together with isolated IP4P domain, as well as full-length P-Rex1. PKA phosphorylation prevents domain-domain interaction and Gβγ binding.","method":"Co-immunoprecipitation of P-Rex1 domain mutants, in vitro GEF assay, PAK1/2 phosphorylation assay, actin reorganization assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal domain interaction assays with functional GEF readout, single lab","pmids":["18514484"],"is_preprint":false},{"year":2008,"finding":"P-Rex1 localizes to distal tips of developing neurites and growth cones of hippocampal neurons. P-Rex1 expression inhibits NGF-stimulated neurite differentiation in PC12 cells via its Rac-GEF activity; low-dose cytochalasin D rescues this. P-Rex1 activates Rac3 GTPase in PC12 cells. siRNA knockdown of P-Rex1 reduces Rac1 and Rac3 activity and promotes spontaneous neurite formation and NGF-induced hyper-elongation.","method":"Immunofluorescence localization, siRNA knockdown, GTPase activation assays (Rac1 and Rac3), GEF-dead mutant, cytochalasin D treatment","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — localization tied to functional GEF-dead rescue experiments, Rac3-GEF activity shown, single lab","pmids":["18697831"],"is_preprint":false},{"year":2009,"finding":"P-Rex1 PDZ domains interact directly with the carboxyl-terminal tail of S1P receptor S1P1 (including full-length receptor monomers and dimers). Co-expression of P-Rex1 reduces S1P1 trafficking to intracellular compartments, and expression of P-Rex1 PDZ domains increases endothelial cell migration to S1P.","method":"Co-immunoprecipitation, cell surface trafficking assay, migration assay with PDZ domain expression","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct binding shown by pulldown/CoIP, functional consequence in migration, single lab","pmids":["20036214"],"is_preprint":false},{"year":2010,"finding":"P-Rex1 is required for ErbB receptor-driven Rac1 activation, motility, cell growth, and tumorigenesis in breast cancer cells. Activation of P-Rex1 in breast cancer cells requires convergent inputs from ErbB receptors and a Gβγ/PI3Kγ-dependent pathway. The GPCR CXCR4 is identified as a mediator of P-Rex1/Rac1 activation in response to ErbB ligands.","method":"siRNA knockdown, Rac1 activation assay (GTP pulldown), cell motility assay, xenograft tumor model, epistasis using PI3Kγ and CXCR4 manipulation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal loss-of-function approaches, in vivo validation, epistasis analysis, replicated across studies","pmids":["21172654"],"is_preprint":false},{"year":2010,"finding":"ErbB/HER receptor activation triggers a phosphorylation/dephosphorylation cycle of P-Rex1. Dephosphorylation of inhibitory residues (Ser313, Ser319) and phosphorylation of activating residues (Ser605, Ser1169) together promote Rac activation. P-Rex1 knockdown impairs breast cancer cell migration/invasion and in vivo tumorigenic potential.","method":"Phospho-specific antibody analysis, siRNA knockdown, Rac activation assay, in vivo tumor assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-site specific readouts combined with functional knockdown, single lab","pmids":["21042280"],"is_preprint":false},{"year":2010,"finding":"P-Rex1 and Vav1 cooperate synergistically in controlling fMLF-stimulated Rac1 and Rac2 activation, ROS formation, adhesion, and chemotaxis in neutrophils. Double-deficient P-Rex1/Vav1 neutrophils show more severe impairment than single knockouts or other combinations, with reduced Mac-1 surface expression.","method":"Double-knockout mouse genetics, Rac1/Rac2 activation assays, ROS assay, adhesion/chemotaxis assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with double-KO, multiple orthogonal phenotypic readouts, single lab","pmids":["21178006"],"is_preprint":false},{"year":2011,"finding":"P-Rex1 deficiency causes a melanoblast migration defect during development (white belly phenotype) and P-Rex1−/− mice are resistant to melanoma metastasis in a murine melanoma model. P-Rex1 drives invasion in a Rac-dependent manner.","method":"P-Rex1 knockout mouse crossed to melanoma model, developmental phenotype scoring, invasion assay with Rac pathway readout","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic model with developmental and metastasis phenotypes, mechanistic link to Rac established","pmids":["22109529"],"is_preprint":false},{"year":2011,"finding":"P-Rex1 promotes GLUT4 trafficking to the plasma membrane in adipocytes at submaximal insulin concentrations in a PI3K- and Rac1-dependent manner. This requires a functional actin network and membrane ruffle formation; Cdc42 or Rho expression did not affect P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or dominant-negative mutant reduced glucose uptake.","method":"siRNA knockdown, dominant-negative P-Rex1, GLUT4 trafficking assay, actin disruption (cytochalasin D), glucose uptake assay in 3T3-L1 adipocytes","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with multiple approaches and specific Rac1-dependence shown, single lab","pmids":["22002247"],"is_preprint":false},{"year":2011,"finding":"P-Rex1 is expressed in platelets and associates with Rac1 (identified by mass spectrometry from Rac1 pulldown of platelet lysates). However, platelets from P-Rex1−/− mice respond normally to platelet agonists and activating surfaces, indicating P-Rex1 is not required for Rac1-mediated platelet activation.","method":"Mass spectrometry of Rac1-associated proteins, western blot, P-Rex1 knockout platelet function assays (spreading, aggregation)","journal":"Journal of molecular signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein interaction identified by MS/pulldown; negative functional result in KO platelets is well-controlled","pmids":["21884615"],"is_preprint":false},{"year":2012,"finding":"PP1α binds P-Rex1 through an RVxF-type docking motif and directly activates P-Rex1 GEF activity in vitro, additively to PIP3 and Gβγ. PP1α also activates P-Rex1 in vivo. Mass spectrometry identified three PP1α dephosphorylation sites on P-Rex1: Ser834, Ser1001, and Ser1165. Mutagenesis of Ser1165 to alanine activated P-Rex1 similarly to PP1α, confirming it as a key inhibitory phosphorylation site.","method":"In vitro GEF activity assay with purified PP1α and P-Rex1, RVxF docking motif analysis, mass spectrometry phosphosite identification, site-directed mutagenesis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins, MS phosphosite mapping, mutagenesis validation, single lab","pmids":["22242915"],"is_preprint":false},{"year":2012,"finding":"In zebrafish gastrulation, the Rac-specific GEF Prex1 is a Nodal signaling target and mediates Nodal-dependent random motility of endodermal cells. Reducing Rac1 activity in endoderm cells caused them to bypass random migration and aberrantly contribute to mesodermal tissues.","method":"Zebrafish transgenic line for actin visualization, morpholino knockdown of prex1, Rac1 inhibition, cell fate analysis","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function in zebrafish embryo with actin dynamics and cell fate readouts, single lab","pmids":["22945937"],"is_preprint":false},{"year":2013,"finding":"P-Rex1 is the PIP3-dependent GEF for Rac1 responsible for regulation of the Rac1/C-RAF/MEK/ERK pathway (not involving RAS) in PIK3CA-mutant and HER2-amplified breast cancers. PI3K inhibition suppresses this Rac1/PAK/C-RAF/MEK/ERK axis, leading to BIM upregulation and apoptosis. Constitutively active Rac1 expression blocked PI3Ki-induced ERK suppression and apoptosis.","method":"siRNA knockdown of P-Rex1, constitutively active Rac1 rescue, PI3K inhibitor treatment, phospho-ERK/MEK/BIM assays, in vivo xenograft","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — pathway placement by epistasis (CA-Rac1 rescue), multiple orthogonal assays, in vivo validation, single lab","pmids":["24327733"],"is_preprint":false},{"year":2013,"finding":"P-Rex1 and PDGFRβ are components of the same macromolecular complex (co-immunoprecipitation). P-Rex1 expression drives invasion in fibroblasts in a PDGFRβ-dependent manner; siRNA of either P-Rex1 or PDGFRβ opposes invasiveness in melanoma cells.","method":"Co-immunoprecipitation, siRNA knockdown, 3D invasion assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single co-IP showing complex formation, functional loss-of-function supporting interaction, single lab","pmids":["23382862"],"is_preprint":false},{"year":2013,"finding":"Phosphorylation of P-Rex1 at Ser1169 is induced by IGF-1R and FGFR activation and is required for IGF-1-induced Rac activation and cell proliferation, as well as IGF-1-induced adhesion in MCF7 breast cancer cells.","method":"Phospho-specific antibody analysis, siRNA knockdown, Rac activation assay, proliferation and adhesion assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-site linked to functional outcome by knockdown, single lab","pmids":["23899556"],"is_preprint":false},{"year":2014,"finding":"P-Rex1 acts as a GEF for RhoG both in vitro and in GPCR-stimulated primary mouse neutrophils, in addition to its known Rac-GEF activity. Loss of either P-Rex1 or RhoG caused equivalent reductions in GPCR-driven Rac activation and NADPH oxidase activity. Loss of RhoG impaired GPCR-driven recruitment of the Rac-GEF DOCK2 and F-actin to the leading edge, establishing a signaling hierarchy: P-Rex1→RhoG→DOCK2→Rac.","method":"In vitro GEF activity assay for RhoG, primary neutrophils from P-Rex1 and RhoG knockout mice, NADPH oxidase assay, F-actin localization, DOCK2 recruitment assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro GEF assay plus genetic epistasis in primary cells with multiple readouts, single lab","pmids":["24659802"],"is_preprint":false},{"year":2014,"finding":"P-REX1 overexpression activates Rac1 and increases PI3K/AKT, MEK/ERK signaling and IGF-1R activation in a PTEN-independent manner. Loss of P-REX1 suppresses PI3K/AKT and MEK/ERK. P-REX1 provides positive feedback to activators upstream of PI3K.","method":"P-Rex1 overexpression/knockdown, phosphoproteomic analysis, Rac1 activation assay, IGF-1R activation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple signaling readouts with gain and loss of function, single lab","pmids":["25284585"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of the P-Rex1 DH-PH tandem domain in complex with Rac1 at 1.95 Å resolution. Interface mutagenesis revealed a critical role for the P-Rex1·Rac1 complex in signaling downstream of RTKs and GPCRs. Structural analysis indicated PIP3/Gβγ binding sites are on the opposite surface from the Rac1 interface, supporting a model whereby PIP3/Gβγ binding releases inhibitory C-terminal domains to expose the Rac1 binding site.","method":"X-ray crystallography (1.95 Å), interface mutagenesis with functional signaling readouts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with mutagenesis validation, single lab","pmids":["26112412"],"is_preprint":false},{"year":2015,"finding":"Genetic deletion or knockdown of P-Rex1 in the CA1 hippocampus causes autism-like social behavior linked to defective LTD via alteration of AMPA receptor endocytosis mediated by a postsynaptic PP1α-P-Rex1-Rac1 signaling pathway.","method":"Conditional knockout, hippocampal siRNA knockdown, LTD electrophysiology, AMPA receptor endocytosis assay, behavioral testing","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with defined synaptic and behavioral phenotype, pathway placement via PP1α-P-Rex1-Rac1 axis, single lab","pmids":["26621702"],"is_preprint":false},{"year":2016,"finding":"Norbin (Neurochondrin, NCDN) is a direct binding partner of P-Rex1, interacting through the PH domain of P-Rex1. Direct interaction with Norbin increases the basal, PIP3-, and Gβγ-stimulated Rac-GEF activity of P-Rex1. Co-expression of P-Rex1 and Norbin induces translocation of both proteins from cytosol to plasma membrane and promotes cell spreading and lamellipodia formation.","method":"Pulldown from brain fractions, co-immunoprecipitation, in vitro binding with purified recombinant proteins, in vitro Rac-GEF assay, PAK-CRIB pulldown, immunofluorescence and subcellular fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct interaction shown with purified proteins, in vitro GEF stimulation, membrane translocation demonstrated, single lab","pmids":["26792863"],"is_preprint":false},{"year":2016,"finding":"Type I PKA regulatory subunit RIα interacts with P-Rex1 PDZ domains via the CNB-B domain of RIα. P-Rex1 activation localizes PKA to the cell periphery. PKA phosphorylates the P-Rex1 DEP1 domain at Ser436, which inhibits the DH-PH catalytic cassette by direct interaction. A P-Rex1 S436A mutant shows increased RacGEF activity and prevents the inhibitory effect of PKA on cell migration.","method":"Co-immunoprecipitation of endogenous proteins, PKA activity assays, phospho-site mutagenesis (S436A), Rac-GEF activity assay, endothelial cell migration assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — co-IP of endogenous complex, phospho-site mutagenesis with functional rescue, single lab","pmids":["26797121"],"is_preprint":false},{"year":2016,"finding":"PAK kinases phosphorylate PREX1 downstream of insulin, neuregulin, and IGF-1 receptor stimulation. PAK-mediated phosphorylation reduces PREX1 binding to PIP3 and negatively regulates PREX1 GEF activity. PREX1 phosphorylation onset is delayed compared to AKT, supporting a negative feedback model. GPCR-stimulated PREX1 phosphorylation is partially PAK-dependent and also involves PKA.","method":"PAK inhibitors, phospho-mass spectrometry, PIP3 binding assay, in vitro GEF activity assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-site identification by MS, PIP3 binding assay, GEF activity measurement, single lab","pmids":["27481946"],"is_preprint":false},{"year":2016,"finding":"PKC isoform PKCδ directly phosphorylates P-Rex1 at Ser313. PKC activation causes phosphorylation of Ser313, Ser319, and Ser1169. Phosphorylation at Ser313 negatively regulates P-Rex1 exchange activity. Growth factor receptor-induced Ser1169 phosphorylation occurs through a mechanism independent of PKC, indicating different kinases control different regulatory serines.","method":"In vitro kinase assay with purified PKCδ, phospho-specific antibody analysis, P-Rex1 mutant expression (S313A), GEF activity assay","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay plus mutant functional analysis, single lab","pmids":["27788493"],"is_preprint":false},{"year":2016,"finding":"PREX1 GEF activity drives ERK1/2 MAPK activation downstream of EGF/IGF-1 stimulation, promoting cyclin D1 and p21(WAF1) induction and anchorage-independent cell growth. GEF-dead PREX1 fails to increase ERK1/2 phosphorylation, anchorage-independent growth, or xenograft tumor growth. MEK1/2/ERK1/2 inhibition suppresses PREX1-mediated effects.","method":"Wild-type vs GEF-dead PREX1 expression, shRNA knockdown, phospho-ERK1/2 assay, anchorage-independent growth assay, xenograft tumor model","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — GEF-dead mutant as mechanistic control, in vivo validation, multiple orthogonal assays, single lab","pmids":["27358402"],"is_preprint":false},{"year":2016,"finding":"ERK/MAPK signaling drives PREX1 overexpression in BRAF- and NRAS-mutant melanoma by both increasing PREX1 gene transcription and promoting PREX1 protein stability. Pharmacologic ERK inhibition reduces PREX1 transcription and protein levels. PREX1-dependent invasion in melanoma is attributable to RAC1 but not CDC42 activation.","method":"ERK pathway inhibitors, siRNA knockdown, RAC1/CDC42 activation assays, invasion assay","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological manipulation combined with functional readouts, single lab","pmids":["27418645"],"is_preprint":false},{"year":2018,"finding":"Gαq and Gα13 directly inhibit Gβγ signaling to P-REX1. GTPase-deficient GαqQL and Gα13QL form stable complexes with Gβγ, preventing its interaction with P-REX1. Gβγ and AKT kinase associate with active P-REX1 during SDF-1/CXCL12 stimulation. GαqQL and Gα13QL also prevent CXCR4-dependent cell migration.","method":"Pulldown assays with chimeric Gα constructs, DREADDs chemogenetic control, co-immunoprecipitation of active P-REX1 complexes, cell migration assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — pulldown and co-IP with chimeric Gα constructs plus functional migration readout, single lab","pmids":["30446620"],"is_preprint":false},{"year":2018,"finding":"GRK2 mediates TCR-induced phosphorylation of CXCR4 at Ser339 and TCR-CXCR4 complex formation. This complex signals via PI3Kγ to recruit PREX1 to the membrane, which activates a Rac1-dependent pathway stabilizing cytokine mRNAs and promoting robust cytokine secretion by T cells.","method":"siRNA depletion of GRK2, PI3Kγ, and PREX1; phospho-CXCR4 assay; membrane recruitment of PREX1 by fractionation; cytokine ELISA; mRNA stability assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple siRNA knockdowns with pathway placement, single lab","pmids":["30018141"],"is_preprint":false},{"year":2018,"finding":"PKA regulatory subunit RIα directly activates P-REX1 in vitro and promotes P-REX1-mediated Rac activation and cell migration via Gs-coupled EP2 receptors in a cAMP-dependent manner. RIα interacts with P-REX1 PDZ domains via its CNB-B domain. Active P-REX1 fraction is not phosphorylated, while inactive P-REX1 is phosphorylated, indicating co-existence of stimulatory (RIα) and inhibitory (catalytic subunit Cα) PKA effects.","method":"In vitro activation assay with purified RIα and P-REX1, cAMP pulldown, siRNA knockdown of RIα, Rac activation assay, endothelial cell migration assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro reconstitution with purified proteins plus cell-based validation, single lab","pmids":["30530493"],"is_preprint":false},{"year":2019,"finding":"Cryo-EM structure of the P-Rex1-Gβγ complex at 3.2 Å reveals that the C-terminal half of P-Rex1 adopts a fold similar to Legionella phosphoinositide phosphatases, forming an extensive docking site for Gβγ together with a DEP domain and two PDZ domains. Hydrogen-deuterium exchange MS suggests Gβγ binding induces allosteric changes in P-Rex1. Functional assays indicate membrane localization is also required for full activation.","method":"Cryo-EM structure determination (3.2 Å), hydrogen-deuterium exchange mass spectrometry, functional activation assays","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with HDX-MS allosteric mapping and functional validation, single lab","pmids":["31663027"],"is_preprint":false},{"year":2020,"finding":"The DEP1 domain of P-Rex1 autoinhibits the DH/PH catalytic module through direct interaction. Crystal structure of DEP1 at 3.1 Å shows a domain-swap involving an exposed basic loop containing the primary PKA phosphorylation site (Ser436). PKA phosphorylation of DEP1 does not affect DH/PH-DEP1 fragment activity in solution but inhibits DEP1 domain binding to phosphatidic acid-containing liposomes, suggesting PKA inhibits P-Rex1 membrane binding rather than its catalytic activity directly.","method":"Crystal structure (3.1 Å), in vitro GEF activity assay with DH/PH-DEP1 fragments, liposome binding assay, PKA phosphorylation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with in vitro biochemical assays and liposome binding, single lab","pmids":["32661198"],"is_preprint":false},{"year":2020,"finding":"Small molecules targeting the P-Rex1 PH domain block PIP3 binding and inhibit fMLP-induced spreading and Rac2 activation in human neutrophils. One compound reduces neutrophil velocity and inhibits neutrophil recruitment to inflammation in a zebrafish model.","method":"Differential scanning fluorimetry screen, PIP3 competition binding assay, human neutrophil spreading/Rac2 activation assay, zebrafish in vivo inflammation assay","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — biochemical binding assays combined with cell-based and in vivo functional validation, single lab","pmids":["31900312"],"is_preprint":false},{"year":2020,"finding":"P-Rex1 is expressed in pancreatic β-cells (INS-1 832/13 cells, rat and human islets). siRNA-mediated knockdown of P-Rex1 attenuates glucose-induced Rac1 activation, membrane association, and insulin secretion. RhoG knockdown did not affect glucose-stimulated insulin secretion.","method":"siRNA knockdown, Rac1 activation pulldown assay, membrane fractionation, ELISA for insulin secretion","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with defined functional readout and specificity controls (RhoG negative), single lab","pmids":["33347743"],"is_preprint":false},{"year":2021,"finding":"Gβγ recruits and activates P-Rex1 via two independent binding interfaces: Gβγ binds both the P-Rex1 DH/PH domains and the PDZ-PDZ tandem. The DEP-DEP tandem and PDZ-PDZ interact intramolecularly and dissociate upon Gβγ binding. The PDZ-PDZ interface mediates P-Rex1 recruitment to the plasma membrane; the DH/PH interface is required for activation.","method":"Pulldown assays with domain fragments, synthetic chimeric RhoGEF (Q-Rhox) to dissect recruitment from activation, co-immunoprecipitation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — domain dissection with chimeric GEF to parse binding interfaces, single lab","pmids":["33412417"],"is_preprint":false},{"year":2023,"finding":"NRBP1 (a pseudokinase) binds P-Rex1 and acts as a scaffold to enhance GTP-bound Rac1 and Cdc42 levels in a P-Rex1-dependent manner. NRBP1 overexpression-driven cell migration and invasion are P-Rex1-dependent. ROS generation via a NRBP1/P-Rex1 pathway contributes to oncogenic roles in triple-negative breast cancer.","method":"BioID/MS proximity proteomics, co-immunoprecipitation, Rac1/Cdc42 activation assays, siRNA knockdown epistasis, invasion assay, ROS assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — BioID identifies interaction, co-IP confirms, epistasis in functional assays, single lab","pmids":["36693952"],"is_preprint":false},{"year":2023,"finding":"P-Rex1 is a novel substrate of the E3 ubiquitin ligase Malin (EPM2B); Malin ubiquitinates P-Rex1 and this is associated with altered glucose uptake relevant to Lafora disease pathology.","method":"Unbiased E3 ligase substrate identification approach, ubiquitination assay, glucose uptake functional assay","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — novel ubiquitination substrate identification with functional consequence, single lab","pmids":["36638890"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structure of P-Rex1·IP4 complex reveals an autoinhibited conformation where the PH domain occludes the DH domain active site, stabilized by DH-DEP1 and PH-4HB interdomain contacts. IP4 inhibits P-Rex1 GEF activity and reduces backbone dynamics broadly. Disruption of DH-DEP1 or PH-4HB interfaces increases activity and confers a more extended conformation. Mutations constraining the occluded conformation reduce GEF activity. PIP3-containing liposomes disrupt these interfaces and increase dynamics. This autoinhibited structure is confirmed in living cells by gain-of-function variants showing enhanced activity during chemokine-induced migration.","method":"Cryo-EM structure, HDX-MS, in vitro GEF activity assays with interface mutants, liposome binding assay, cell migration assay with interface mutant variants","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure with HDX-MS, mutagenesis, biochemical reconstitution, and cellular validation, multiple orthogonal methods in a single study","pmids":["39082940"],"is_preprint":false},{"year":2024,"finding":"PREX1 promotes nuclear translocation of phosphorylated STAT5 in naive CD4+ T cells, supporting homeostatic proliferation in response to IL-7, and biases differentiation toward effector T cells.","method":"PREX1 expression analysis in aged naive CD4+ T cells, nuclear fractionation for phospho-STAT5, functional proliferation assays with PREX1 manipulation","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — nuclear translocation of pSTAT5 linked to PREX1 expression and IL-7 responsiveness, single lab","pmids":["38329813"],"is_preprint":false},{"year":2025,"finding":"P-Rex1 limits GPCR (S1PR1, CXCR4, PAR4, GLP1R) internalization independently of its Rac-GEF activity, through its PDZ, DEP, and IP4P domains. P-Rex1 blocks phosphorylation required for GPCR internalization. P-Rex1 binds GRK2 both in vitro and in cells, but does not regulate GRK2 activity.","method":"Catalytically inactive Prex1GD knock-in mice, GPCR internalization assays, phosphorylation assays, in vitro binding of P-Rex1 with GRK2, domain deletion analysis","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — catalytically inactive knock-in mouse, in vitro binding, multiple GPCR substrates tested, mechanistic separation of GEF and adaptor functions, single lab","pmids":["41100251"],"is_preprint":false},{"year":2025,"finding":"P-Rex1 limits hepatocyte glucose uptake and mitochondrial ATP production independently of its Rac-GEF catalytic activity through the orphan GPCR Gpr21. P-Rex1 controls Gpr21 trafficking (retaining it at plasma membrane) and controls Glut2 surface levels, mitochondrial morphology, membrane potential, and ATP production in hepatocytes. P-Rex1 GEF activity is required for maintaining fasting blood glucose and insulin sensitivity.","method":"Prex1−/− and catalytically inactive Prex1GD mice, high-fat diet diabetes model, hepatocyte glucose uptake assay, mitochondrial membrane potential/ATP assays, Gpr21 trafficking by cell fractionation, Glut2 surface assay, pharmacological Gpr21 inverse agonist (GRA2)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — genetic separation of GEF-dependent and GEF-independent functions using knock-in mice, multiple orthogonal metabolic and cell biology readouts, single lab","pmids":["41046518"],"is_preprint":false},{"year":2025,"finding":"P-Rex1 mediates phagocytosis of IgG-opsonized particles and bactericidal activity in neutrophils independently of its Rac-GEF catalytic activity, through mechanisms not related to its GPCR trafficking or glucose uptake adaptor functions. P-Rex1 mediates both integrin-dependent and Fc receptor-dependent phagocytosis and the Fc receptor-dependent activation of Rac and Syk. P-Rex1 GEF activity is required for migration, ROS, and NET formation.","method":"Prex1−/− and catalytically inactive Prex1GD mice, phagocytosis assays (IgG-opsonized zymosan), in vivo septic peritonitis bacteria clearance, Syk activation assay, Rac activation assay","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic separation using KO and catalytically inactive knock-in, multiple phagocytic and bactericidal readouts, single lab","pmids":["41098722"],"is_preprint":false}],"current_model":"P-Rex1 is a large, multidomain Rac-GEF (Rac1, Rac2, Rac3, and RhoG substrates) that functions as a coincidence detector, synergistically activated at the plasma membrane by PIP3 (via its PH domain) and Gβγ subunits (via DEP-PDZ and DH/PH interfaces); in its resting state it adopts an autoinhibited conformation in which the PH domain occludes the DH active site through DH-DEP1 and PH-4HB interdomain contacts. Activation is further regulated by dephosphorylation via PP1α (at Ser1165, Ser1001, Ser834), activating phosphorylation at Ser605 and Ser1169 by RTK-linked kinases, and inhibitory phosphorylation at Ser313/Ser319 by PKCδ, Ser436 by PKA catalytic subunit (which also impairs membrane binding), and multiple sites by PAKs. RIα regulatory subunits of type I PKA directly bind the P-Rex1 PDZ domains and can activate P-Rex1 in a cAMP-dependent manner, while Gαq and Gα13 sequester Gβγ to suppress P-Rex1 signaling. Beyond Rac-GEF activity, P-Rex1 serves as an adaptor that limits agonist-induced GPCR internalization by binding GRK2 and controls hepatocyte Gpr21 surface trafficking and mitochondrial glucose metabolism; these adaptor functions are independent of catalytic activity."},"narrative":{"mechanistic_narrative":"P-Rex1 is a large multidomain guanine-nucleotide exchange factor that activates the Rho-family GTPases Rac1, Rac2, Rac3 and RhoG to drive actin remodeling, cell migration, ROS production and invasion across neutrophils, neurons, and cancer cells [PMID:11955434, PMID:16243036, PMID:16243035, PMID:18697831, PMID:24659802]. Its defining property is coincidence detection at the plasma membrane: Rac-GEF activity is synergistically and directly stimulated by PtdIns(3,4,5)P3 (via the PH domain) and Gβγ subunits, neither input alone being sufficient for full membrane translocation and activation [PMID:11955434, PMID:17698854]. Structural work resolves the underlying logic — in the resting state the PH domain occludes the DH active site, stabilized by DH-DEP1 and PH-4HB interdomain contacts and by IP4 binding, while PIP3 and Gβγ disrupt these autoinhibitory interfaces to expose the Rac-binding surface [PMID:26112412, PMID:31663027, PMID:39082940]. Gβγ engages two independent interfaces (DH/PH for activation and the PDZ-PDZ tandem for membrane recruitment), and Gαq/Gα13 suppress signaling by sequestering Gβγ [PMID:30446620, PMID:33412417]. Activity is further tuned by a phosphorylation cycle: PP1α docks via an RVxF motif and dephosphorylates inhibitory sites (Ser1165, Ser1001, Ser834) to activate the enzyme [PMID:22242915], RTK-linked inputs drive activating phosphorylation at Ser605/Ser1169 [PMID:21042280, PMID:23899556], while PKCδ (Ser313/Ser319), PKA (Ser436, which impairs membrane binding), and PAKs phosphorylate inhibitory sites, the last forming a delayed negative-feedback loop [PMID:26797121, PMID:27788493, PMID:32661198, PMID:27481946]; type I PKA RIα subunits bind the PDZ domains and conversely activate P-Rex1 in a cAMP-dependent manner [PMID:26797121, PMID:30530493]. Through ErbB/CXCR4-, IGF-1R- and PI3K-coupled inputs P-Rex1 activates Rac1 to drive a C-RAF/MEK/ERK growth axis and tumorigenesis, and its overexpression is reinforced by ERK-driven transcription and protein stabilization in melanoma [PMID:21172654, PMID:24327733, PMID:27358402, PMID:27418645]. P-Rex1 deficiency impairs neutrophil recruitment, melanoblast migration and melanoma metastasis, and contributes to hippocampal LTD and social behavior via a postsynaptic PP1α-P-Rex1-Rac1 pathway controlling AMPA-receptor endocytosis [PMID:16243035, PMID:22109529, PMID:26621702]. Beyond catalysis, P-Rex1 acts as a Rac-GEF-independent adaptor that limits agonist-induced GPCR internalization by binding GRK2, controls hepatocyte Gpr21 surface trafficking and mitochondrial glucose metabolism, and supports Fc-receptor-dependent phagocytosis [PMID:41100251, PMID:41046518, PMID:41098722].","teleology":[{"year":2002,"claim":"Established the existence and core biochemistry of P-Rex1, answering whether a PIP3- and Gβγ-responsive Rac-GEF links PI3K and GPCR inputs in neutrophils.","evidence":"Biochemical purification from neutrophil cytosol with in vitro reconstituted GEF assays and antisense knockdown with ROS readout","pmids":["11955434"],"confidence":"High","gaps":["Substrate specificity among Rac isoforms not resolved","Structural basis of synergistic activation unknown"]},{"year":2005,"claim":"Genetic loss-of-function defined P-Rex1 as a physiologically required, Rac2-preferring GEF in neutrophil GPCR signaling, linking it to ROS, F-actin and inflammatory recruitment.","evidence":"P-Rex1 knockout mice with PBD pulldown GTPase assays, superoxide assays, and in vivo peritonitis model; replicated independently","pmids":["16243036","16243035"],"confidence":"High","gaps":["Mechanism of Rac2-over-Rac1 selectivity unclear","Only mildly reduced chemotaxis left activation regulation open"]},{"year":2005,"claim":"Identified the upstream activators and inhibitors that gate P-Rex1, showing Gβγ isoform selectivity and PKA-driven phosphorylation as an off-switch.","evidence":"In vitro GEF assays with purified Gβγ dimers in lipid vesicles, in vitro PKA kinase assays, and cell-based Rac activation assays","pmids":["16301320","16301321"],"confidence":"High","gaps":["PKA phosphorylation sites not mapped","Mechanism connecting phosphorylation to Gβγ responsiveness unknown"]},{"year":2005,"claim":"Extended P-Rex1 function beyond immunity by showing it drives neurotrophin-induced Rac1 activation and neuronal migration during brain development.","evidence":"In situ hybridization, immunofluorescence, siRNA, dominant-negative constructs and in utero electroporation","pmids":["15858067"],"confidence":"High","gaps":["Receptor inputs upstream in neurons not defined","Rac isoform usage in neurons unresolved"]},{"year":2007,"claim":"Dissected how P-Rex1 reaches the membrane, establishing that Gβγ and PIP3 act synergistically for translocation while inhibitory domains retain it in cytosol.","evidence":"Subcellular fractionation in Sf9 cells with domain mutants and in vitro GEF assays; Co-IP placing P-Rex1 with mTORC2","pmids":["17698854","17565979"],"confidence":"High","gaps":["Structural basis of intramolecular autoinhibition not yet defined","mTORC2 association rests on Co-IP in a single lab"]},{"year":2008,"claim":"Mapped intramolecular autoinhibitory contacts and tied PKA phosphorylation to disruption of domain-domain interaction and Gβγ binding.","evidence":"Co-IP of domain mutants, in vitro GEF assays, and neuronal localization with GEF-dead rescue experiments","pmids":["18514484","18697831"],"confidence":"Medium","gaps":["Atomic structure of autoinhibited state still lacking","Single-lab domain-interaction assays"]},{"year":2010,"claim":"Placed P-Rex1 at the convergence of RTK and GPCR inputs in cancer, defining a phospho-regulatory cycle and a requirement for tumorigenesis.","evidence":"siRNA knockdown, Rac1 activation assays, phospho-specific antibodies, xenografts, and CXCR4/PI3Kγ epistasis in breast cancer cells","pmids":["21172654","21042280"],"confidence":"High","gaps":["Identity of kinases for individual activating sites not fully resolved","Direct versus indirect ErbB-P-Rex1 coupling unclear"]},{"year":2011,"claim":"Demonstrated developmental and metastatic roles in melanocyte lineage and broadened substrate/cellular contexts to adipocyte GLUT4 trafficking and platelets.","evidence":"P-Rex1 knockout melanoma model, GLUT4/glucose uptake assays in adipocytes, and Rac1-pulldown mass spectrometry in platelets","pmids":["22109529","22002247","21884615"],"confidence":"High","gaps":["Why P-Rex1 is dispensable in platelets despite Rac1 binding unexplained","Tissue-specific upstream activators not defined"]},{"year":2012,"claim":"Identified PP1α as a direct activating phosphatase and mapped its dephosphorylation sites, establishing reversible phospho-control of GEF activity.","evidence":"In vitro GEF assays with purified PP1α, RVxF motif analysis, MS phosphosite mapping, and S1165A mutagenesis; zebrafish Nodal/Prex1 morpholino study","pmids":["22242915","22945937"],"confidence":"High","gaps":["Coordination between PP1α and antagonizing kinases in vivo unclear","Spatial control of dephosphorylation unknown"]},{"year":2013,"claim":"Defined the Rac1/C-RAF/MEK/ERK signaling axis downstream of P-Rex1 in PI3K-pathway-mutant cancers and identified PDGFRβ as a complex partner driving invasion.","evidence":"siRNA, constitutively active Rac1 rescue, PI3K-inhibitor treatment, phospho-ERK/BIM assays, xenografts, and Co-IP in fibroblasts","pmids":["24327733","23382862","23899556"],"confidence":"High","gaps":["Whether ERK activation is fully RAS-independent in all contexts unresolved","PDGFRβ complex stoichiometry undefined"]},{"year":2014,"claim":"Showed P-Rex1 is also a RhoG-GEF defining a P-Rex1→RhoG→DOCK2→Rac hierarchy, and acts in a positive feedback loop upstream of PI3K in cancer.","evidence":"In vitro RhoG GEF assays, P-Rex1 and RhoG knockout neutrophils with DOCK2 recruitment readouts, and phosphoproteomics with gain/loss of function","pmids":["24659802","25284585"],"confidence":"High","gaps":["Direct mechanism of PI3K-upstream feedback unclear","Relative contribution of Rac- versus RhoG-GEF activity per tissue unresolved"]},{"year":2015,"claim":"Provided the first atomic view of the DH-PH·Rac1 catalytic complex and connected a PP1α-P-Rex1-Rac1 pathway to synaptic plasticity and autism-like behavior.","evidence":"X-ray crystallography at 1.95 Å with interface mutagenesis; conditional KO with LTD electrophysiology, AMPAR endocytosis, and behavioral assays","pmids":["26112412","26621702"],"confidence":"High","gaps":["Full-length autoinhibited architecture not yet solved","How PIP3/Gβγ release inhibitory domains shown only by inference"]},{"year":2016,"claim":"Expanded the regulatory network with direct activators (Norbin, RIα) and inhibitory kinases (PKCδ, PAKs), and confirmed GEF activity drives ERK-dependent proliferation and a melanoma feedforward loop.","evidence":"Purified-protein binding and GEF assays, phospho-site mutagenesis (S436A, S313A), PAK inhibitors with MS, GEF-dead controls, xenografts, and ERK-inhibitor manipulation","pmids":["26792863","26797121","27481946","27788493","27358402","27418645"],"confidence":"High","gaps":["Integration of competing positive and negative regulators in time/space not modeled","Distinct kinases for each serine only partially assigned"]},{"year":2018,"claim":"Resolved how Gα subunits restrain P-Rex1 by Gβγ sequestration and detailed GRK2/CXCR4-PI3Kγ recruitment driving T-cell cytokine output.","evidence":"Pulldown with chimeric Gα constructs, DREADD chemogenetics, Co-IP of active complexes, siRNA epistasis, and cytokine/mRNA stability assays","pmids":["30446620","30018141"],"confidence":"Medium","gaps":["Stoichiometry of Gβγ sequestration in cells unclear","GRK2 binding role versus catalytic recruitment not separated here"]},{"year":2019,"claim":"Cryo-EM of the P-Rex1·Gβγ complex revealed a Legionella-phosphatase-like C-terminal fold forming an extensive Gβγ docking site and allosteric activation.","evidence":"Cryo-EM at 3.2 Å, HDX-MS, and functional activation assays","pmids":["31663027"],"confidence":"High","gaps":["Membrane-bound active conformation not captured","Coupling of Gβγ docking to active-site exposure incompletely defined"]},{"year":2020,"claim":"Solved the DEP1 autoinhibition mechanism, showed PKA acts on membrane binding rather than catalysis directly, and demonstrated PH-domain small-molecule inhibition.","evidence":"Crystal structure of DEP1 at 3.1 Å, liposome binding, in vitro GEF assays with fragments, DSF screen with neutrophil and zebrafish validation; β-cell siRNA insulin secretion study","pmids":["32661198","31900312","33347743"],"confidence":"High","gaps":["Full-length autoinhibited structure still pending","Selectivity and pharmacology of PH-domain inhibitors not optimized"]},{"year":2021,"claim":"Separated Gβγ-mediated recruitment from activation, assigning the PDZ-PDZ tandem to membrane targeting and the DH/PH interface to catalytic activation.","evidence":"Domain-fragment pulldowns and a synthetic chimeric RhoGEF (Q-Rhox) with Co-IP","pmids":["33412417"],"confidence":"Medium","gaps":["Quantitative contribution of each interface in vivo unresolved","Dissection relies on chimeric constructs in a single lab"]},{"year":2024,"claim":"Defined the complete autoinhibited architecture and its release mechanism, showing the PH domain occludes the DH active site and PIP3 disrupts DH-DEP1/PH-4HB interfaces to activate the enzyme.","evidence":"Cryo-EM of P-Rex1·IP4, HDX-MS, interface-mutant GEF and liposome assays, and cellular gain-of-function migration validation; pSTAT5/IL-7 T-cell study","pmids":["39082940","38329813"],"confidence":"High","gaps":["Order of PIP3 and Gβγ engagement during activation not fully kinetically resolved","How phospho-regulation maps onto the autoinhibited structure unclear"]},{"year":2025,"claim":"Genetic separation of catalytic from adaptor functions revealed Rac-GEF-independent roles in limiting GPCR internalization, controlling hepatocyte Gpr21/glucose metabolism, and supporting Fc-receptor phagocytosis.","evidence":"Catalytically inactive Prex1GD knock-in mice with GPCR internalization, in vitro GRK2 binding, hepatocyte metabolic assays, Gpr21 trafficking, and phagocytosis/bactericidal assays","pmids":["41100251","41046518","41098722"],"confidence":"High","gaps":["Molecular mechanism by which P-Rex1 blocks GPCR phosphorylation undefined","How a single scaffold mediates distinct GEF-independent outputs unresolved"]},{"year":null,"claim":"How the full regulatory layers — autoinhibition, phospho-cycling, multi-interface Gβγ engagement, and Rac-GEF-independent adaptor functions — are integrated spatiotemporally within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified kinetic model linking activation inputs to output selectivity","Structural basis of adaptor (GEF-independent) functions uncharacterized","Direct Mendelian disease causation not established in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,23,25]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[6,37,38,43]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[45,46]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[18,28,35]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,8,27,40]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,6,8,27]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,12,20,33]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,2,14,47]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[12,15,20,31]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[16,39,46]}],"complexes":["mTORC2","P-Rex1-Gβγ complex"],"partners":["GΒΓ","PP1Α","GRK2","NORBIN (NCDN)","PDGFRΒ","NRBP1","RIΑ (PRKAR1A)","RAC1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TCU6","full_name":"Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein","aliases":[],"length_aa":1659,"mass_kda":186.2,"function":"Functions as a RAC guanine nucleotide exchange factor (GEF), which activates the Rac proteins by exchanging bound GDP for free GTP. Its activity is synergistically activated by phosphatidylinositol 3,4,5-trisphosphate and the beta gamma subunits of heterotrimeric G protein. May function downstream of heterotrimeric G proteins in neutrophils","subcellular_location":"Cytoplasm, cytosol; Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q8TCU6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PREX1","classification":"Not Classified","n_dependent_lines":23,"n_total_lines":1208,"dependency_fraction":0.01903973509933775},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PREX1","total_profiled":1310},"omim":[{"mim_id":"612139","title":"PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE-DEPENDENT RAC EXCHANGER 2; PREX2","url":"https://www.omim.org/entry/612139"},{"mim_id":"606905","title":"PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE-DEPENDENT RAC EXCHANGER 1; PREX1","url":"https://www.omim.org/entry/606905"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":49.6}],"url":"https://www.proteinatlas.org/search/PREX1"},"hgnc":{"alias_symbol":["KIAA1415","P-REX1"],"prev_symbol":[]},"alphafold":{"accession":"Q8TCU6","domains":[{"cath_id":"1.20.900.10","chopping":"39-250","consensus_level":"high","plddt":91.7606,"start":39,"end":250},{"cath_id":"2.30.29.30","chopping":"260-306_324-397","consensus_level":"high","plddt":89.1245,"start":260,"end":397},{"cath_id":"1.10.10.10","chopping":"423-495","consensus_level":"high","plddt":83.2385,"start":423,"end":495},{"cath_id":"1.10.10.10","chopping":"540-554_561-612","consensus_level":"medium","plddt":84.0321,"start":540,"end":612},{"cath_id":"2.30.42.10","chopping":"624-707","consensus_level":"medium","plddt":91.8014,"start":624,"end":707},{"cath_id":"2.30.42.10","chopping":"709-802_840-848","consensus_level":"medium","plddt":87.9031,"start":709,"end":848}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCU6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCU6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TCU6-F1-predicted_aligned_error_v6.png","plddt_mean":78.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PREX1","jax_strain_url":"https://www.jax.org/strain/search?query=PREX1"},"sequence":{"accession":"Q8TCU6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TCU6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TCU6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TCU6"}},"corpus_meta":[{"pmid":"11955434","id":"PMC_11955434","title":"P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac.","date":"2002","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/11955434","citation_count":448,"is_preprint":false},{"pmid":"24327733","id":"PMC_24327733","title":"PI3K regulates MEK/ERK signaling in breast cancer via the Rac-GEF, P-Rex1.","date":"2013","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/24327733","citation_count":185,"is_preprint":false},{"pmid":"21172654","id":"PMC_21172654","title":"Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer.","date":"2010","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/21172654","citation_count":173,"is_preprint":false},{"pmid":"22109529","id":"PMC_22109529","title":"P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.","date":"2011","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/22109529","citation_count":154,"is_preprint":false},{"pmid":"16243035","id":"PMC_16243035","title":"P-Rex1 regulates neutrophil function.","date":"2005","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/16243035","citation_count":142,"is_preprint":false},{"pmid":"17565979","id":"PMC_17565979","title":"P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17565979","citation_count":130,"is_preprint":false},{"pmid":"19305425","id":"PMC_19305425","title":"Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis.","date":"2009","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/19305425","citation_count":129,"is_preprint":false},{"pmid":"16243036","id":"PMC_16243036","title":"P-Rex1 is a primary Rac2 guanine nucleotide exchange factor in mouse neutrophils.","date":"2005","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/16243036","citation_count":110,"is_preprint":false},{"pmid":"15858067","id":"PMC_15858067","title":"Involvement of a Rac activator,P-Rex1, in neurotrophin-derived signaling and neuronal migration.","date":"2005","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/15858067","citation_count":98,"is_preprint":false},{"pmid":"21042280","id":"PMC_21042280","title":"P-Rex1 participates in Neuregulin-ErbB signal transduction and its expression correlates with patient outcome in breast cancer.","date":"2010","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/21042280","citation_count":81,"is_preprint":false},{"pmid":"25284585","id":"PMC_25284585","title":"P-REX1 creates a positive feedback loop to activate growth factor receptor, PI3K/AKT and MEK/ERK signaling in breast 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Carcinoma","date":"2025-09-21","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.19.25336108","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":51452,"output_tokens":12765,"usd":0.172915,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":24512,"output_tokens":6541,"usd":0.143042,"stage2_stop_reason":"end_turn"},"total_usd":0.315957,"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\": 2002,\n      \"finding\": \"P-Rex1 is a 185 kDa guanine-nucleotide exchange factor (GEF) for Rac, purified from neutrophil cytosol, whose Rac-GEF activity is directly, substantially, and synergistically activated by PtdIns(3,4,5)P3 and Gβγ subunits both in vitro and in vivo. Antisense knockdown of P-Rex1 reduced C5a-stimulated reactive oxygen species formation in a neutrophil-like cell line.\",\n      \"method\": \"Biochemical purification from neutrophil cytosol, in vitro GEF activity assay, antisense knockdown with ROS readout\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of GEF activity with purified components, replicated in vivo, foundational paper with multiple orthogonal methods\",\n      \"pmids\": [\"11955434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"P-Rex1 preferentially activates Rac2 over Rac1 in mouse neutrophils; P-Rex1-deficient neutrophils show impaired Rac2 (but not Rac1) activation in response to fMLP, reduced F-actin formation, superoxide production, and chemotactic migration rate. P-Rex1 showed higher affinity for dominant-negative Rac2(S17N) than Rac1(S17N) by co-immunoprecipitation.\",\n      \"method\": \"P-Rex1 knockout mouse, PBD pulldown GTPase activation assay, co-immunoprecipitation with dominant-negative Rac isoforms, superoxide assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined cellular phenotype, replicated by two independent KO studies in the same journal issue\",\n      \"pmids\": [\"16243036\", \"16243035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In P-Rex1-deficient mice, GPCR-dependent Rac2 activation is impaired; LPS-primed P-Rex1−/− neutrophils lack GPCR-dependent ROS formation; recruitment of P-Rex1−/− neutrophils to inflammatory sites is impaired. Chemotaxis of isolated neutrophils is only mildly reduced, with normal polarization and directionality.\",\n      \"method\": \"P-Rex1 knockout mouse, in vivo peritonitis model, ROS assay, chemotaxis assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with multiple defined cellular phenotypes, replicated independently\",\n      \"pmids\": [\"16243035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PKA phosphorylates P-Rex1 in vitro and in cells, making Gβγ 47-fold less potent at activating phosphorylated P-Rex1 compared to dephosphorylated P-Rex1. Gs-coupled receptor activation (isoproterenol) increases P-Rex1 phosphorylation and reduces GTP-bound Rac in cells.\",\n      \"method\": \"In vitro kinase assay with purified PKA, 32P metabolic labeling in HEK293T cells, GTP-Rac pulldown assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins combined with cell-based phosphorylation and Rac activation assays, single lab\",\n      \"pmids\": [\"16301320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Among Gβγ dimer isoforms, Gβ1–4/γ2 dimers activate P-Rex1 GEF activity (EC50 10–20 nM) while Gβ5γ2 cannot. Gβ1 paired with different γ subunits shows variable potency; Gβ1γ11 (abundant in hematopoietic cells) and Gβ1γ12 are less effective activators. Gα subunits (Gs, Gi, Gq, G12, G13) do not activate P-Rex1.\",\n      \"method\": \"In vitro GEF activity assay with purified recombinant Gβγ dimers reconstituted into synthetic lipid vesicles\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified recombinant proteins, systematic isoform survey, single lab\",\n      \"pmids\": [\"16301321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"P-Rex1 is expressed in developing mouse brain neurons and localizes to the leading process of migrating neurons. In PC12 cells, P-Rex1 is activated by NGF to increase GTP-bound Rac1 and cell motility. Dominant-negative P-Rex1 (lacking DH domain) or siRNA knockdown impairs neurotrophin/EGF-induced cell migration of PC12 cells and primary cortical neurons. In utero electroporation of the dominant-negative form perturbs radial neuronal migration.\",\n      \"method\": \"In situ hybridization, immunofluorescence localization, Rac-GTP pulldown, siRNA knockdown, dominant-negative construct, in utero electroporation\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with multiple approaches (siRNA, dominant-negative, in vivo electroporation) with defined migration phenotype\",\n      \"pmids\": [\"15858067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"P-Rex1 membrane translocation requires synergistic action of Gβγ and PI3K (PIP3); neither alone causes significant translocation. The DH/PH domain tandem is sufficient for membrane localization; GEF activity is not required. The DEP, PDZ, and IP4P domains promote cytosolic retention in basal cells. Membrane-derived P-Rex1 has higher basal Rac2-GEF activity than cytosol-derived P-Rex1.\",\n      \"method\": \"Subcellular fractionation of Sf9 cells co-expressing P-Rex1 with Gβγ and/or PI3K; P-Rex1 domain mutants; in vitro Rac2-GEF activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — fractionation combined with in vitro GEF assay and domain mutagenesis, single lab\",\n      \"pmids\": [\"17698854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"P-Rex1 interacts with mTOR through its tandem DEP domains and is associated with both mTORC1 and mTORC2 complexes. P-Rex1 dominant-negative constructs and shRNA knockdown decrease mTOR-dependent leucine-induced Rac activation and cell migration. P-Rex1 is only active when in the mTORC2 complex (rapamycin does not inhibit Rac activity or migration induced by leucine).\",\n      \"method\": \"Co-immunoprecipitation of P-Rex1 with mTOR complexes, dominant-negative constructs, shRNA knockdown, Rac activation assay, cell migration assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP and functional knockdown, single lab, mTORC2 specificity inferred from rapamycin insensitivity\",\n      \"pmids\": [\"17565979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Endogenous P-Rex1 translocates from cytoplasm to the leading edge of polarized human neutrophils upon chemoattractant stimulation, colocalizing with F-actin and Rac2, in a Gβγ- and PIP3-dependent manner. This translocation requires tyrosine kinase activity, is modulated by cell adhesion, and is inhibited by PKA activation.\",\n      \"method\": \"Immunofluorescence microscopy of endogenous P-Rex1 in activated human neutrophils, pharmacological inhibitors\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct localization of endogenous protein with functional manipulation, single lab\",\n      \"pmids\": [\"17227822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The second DEP and first PDZ domains of P-Rex1 associate with the IP4P domain (intramolecular interaction). Mutations in the PDZ protein-binding pocket or C-terminal truncation of IP4P abolish this interaction. Gβγ can activate a complex of P-Rex1 lacking IP4P together with isolated IP4P domain, as well as full-length P-Rex1. PKA phosphorylation prevents domain-domain interaction and Gβγ binding.\",\n      \"method\": \"Co-immunoprecipitation of P-Rex1 domain mutants, in vitro GEF assay, PAK1/2 phosphorylation assay, actin reorganization assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal domain interaction assays with functional GEF readout, single lab\",\n      \"pmids\": [\"18514484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"P-Rex1 localizes to distal tips of developing neurites and growth cones of hippocampal neurons. P-Rex1 expression inhibits NGF-stimulated neurite differentiation in PC12 cells via its Rac-GEF activity; low-dose cytochalasin D rescues this. P-Rex1 activates Rac3 GTPase in PC12 cells. siRNA knockdown of P-Rex1 reduces Rac1 and Rac3 activity and promotes spontaneous neurite formation and NGF-induced hyper-elongation.\",\n      \"method\": \"Immunofluorescence localization, siRNA knockdown, GTPase activation assays (Rac1 and Rac3), GEF-dead mutant, cytochalasin D treatment\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — localization tied to functional GEF-dead rescue experiments, Rac3-GEF activity shown, single lab\",\n      \"pmids\": [\"18697831\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"P-Rex1 PDZ domains interact directly with the carboxyl-terminal tail of S1P receptor S1P1 (including full-length receptor monomers and dimers). Co-expression of P-Rex1 reduces S1P1 trafficking to intracellular compartments, and expression of P-Rex1 PDZ domains increases endothelial cell migration to S1P.\",\n      \"method\": \"Co-immunoprecipitation, cell surface trafficking assay, migration assay with PDZ domain expression\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct binding shown by pulldown/CoIP, functional consequence in migration, single lab\",\n      \"pmids\": [\"20036214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"P-Rex1 is required for ErbB receptor-driven Rac1 activation, motility, cell growth, and tumorigenesis in breast cancer cells. Activation of P-Rex1 in breast cancer cells requires convergent inputs from ErbB receptors and a Gβγ/PI3Kγ-dependent pathway. The GPCR CXCR4 is identified as a mediator of P-Rex1/Rac1 activation in response to ErbB ligands.\",\n      \"method\": \"siRNA knockdown, Rac1 activation assay (GTP pulldown), cell motility assay, xenograft tumor model, epistasis using PI3Kγ and CXCR4 manipulation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal loss-of-function approaches, in vivo validation, epistasis analysis, replicated across studies\",\n      \"pmids\": [\"21172654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ErbB/HER receptor activation triggers a phosphorylation/dephosphorylation cycle of P-Rex1. Dephosphorylation of inhibitory residues (Ser313, Ser319) and phosphorylation of activating residues (Ser605, Ser1169) together promote Rac activation. P-Rex1 knockdown impairs breast cancer cell migration/invasion and in vivo tumorigenic potential.\",\n      \"method\": \"Phospho-specific antibody analysis, siRNA knockdown, Rac activation assay, in vivo tumor assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-site specific readouts combined with functional knockdown, single lab\",\n      \"pmids\": [\"21042280\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"P-Rex1 and Vav1 cooperate synergistically in controlling fMLF-stimulated Rac1 and Rac2 activation, ROS formation, adhesion, and chemotaxis in neutrophils. Double-deficient P-Rex1/Vav1 neutrophils show more severe impairment than single knockouts or other combinations, with reduced Mac-1 surface expression.\",\n      \"method\": \"Double-knockout mouse genetics, Rac1/Rac2 activation assays, ROS assay, adhesion/chemotaxis assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with double-KO, multiple orthogonal phenotypic readouts, single lab\",\n      \"pmids\": [\"21178006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"P-Rex1 deficiency causes a melanoblast migration defect during development (white belly phenotype) and P-Rex1−/− mice are resistant to melanoma metastasis in a murine melanoma model. P-Rex1 drives invasion in a Rac-dependent manner.\",\n      \"method\": \"P-Rex1 knockout mouse crossed to melanoma model, developmental phenotype scoring, invasion assay with Rac pathway readout\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic model with developmental and metastasis phenotypes, mechanistic link to Rac established\",\n      \"pmids\": [\"22109529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"P-Rex1 promotes GLUT4 trafficking to the plasma membrane in adipocytes at submaximal insulin concentrations in a PI3K- and Rac1-dependent manner. This requires a functional actin network and membrane ruffle formation; Cdc42 or Rho expression did not affect P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or dominant-negative mutant reduced glucose uptake.\",\n      \"method\": \"siRNA knockdown, dominant-negative P-Rex1, GLUT4 trafficking assay, actin disruption (cytochalasin D), glucose uptake assay in 3T3-L1 adipocytes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with multiple approaches and specific Rac1-dependence shown, single lab\",\n      \"pmids\": [\"22002247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"P-Rex1 is expressed in platelets and associates with Rac1 (identified by mass spectrometry from Rac1 pulldown of platelet lysates). However, platelets from P-Rex1−/− mice respond normally to platelet agonists and activating surfaces, indicating P-Rex1 is not required for Rac1-mediated platelet activation.\",\n      \"method\": \"Mass spectrometry of Rac1-associated proteins, western blot, P-Rex1 knockout platelet function assays (spreading, aggregation)\",\n      \"journal\": \"Journal of molecular signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein interaction identified by MS/pulldown; negative functional result in KO platelets is well-controlled\",\n      \"pmids\": [\"21884615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PP1α binds P-Rex1 through an RVxF-type docking motif and directly activates P-Rex1 GEF activity in vitro, additively to PIP3 and Gβγ. PP1α also activates P-Rex1 in vivo. Mass spectrometry identified three PP1α dephosphorylation sites on P-Rex1: Ser834, Ser1001, and Ser1165. Mutagenesis of Ser1165 to alanine activated P-Rex1 similarly to PP1α, confirming it as a key inhibitory phosphorylation site.\",\n      \"method\": \"In vitro GEF activity assay with purified PP1α and P-Rex1, RVxF docking motif analysis, mass spectrometry phosphosite identification, site-directed mutagenesis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins, MS phosphosite mapping, mutagenesis validation, single lab\",\n      \"pmids\": [\"22242915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In zebrafish gastrulation, the Rac-specific GEF Prex1 is a Nodal signaling target and mediates Nodal-dependent random motility of endodermal cells. Reducing Rac1 activity in endoderm cells caused them to bypass random migration and aberrantly contribute to mesodermal tissues.\",\n      \"method\": \"Zebrafish transgenic line for actin visualization, morpholino knockdown of prex1, Rac1 inhibition, cell fate analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in zebrafish embryo with actin dynamics and cell fate readouts, single lab\",\n      \"pmids\": [\"22945937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"P-Rex1 is the PIP3-dependent GEF for Rac1 responsible for regulation of the Rac1/C-RAF/MEK/ERK pathway (not involving RAS) in PIK3CA-mutant and HER2-amplified breast cancers. PI3K inhibition suppresses this Rac1/PAK/C-RAF/MEK/ERK axis, leading to BIM upregulation and apoptosis. Constitutively active Rac1 expression blocked PI3Ki-induced ERK suppression and apoptosis.\",\n      \"method\": \"siRNA knockdown of P-Rex1, constitutively active Rac1 rescue, PI3K inhibitor treatment, phospho-ERK/MEK/BIM assays, in vivo xenograft\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway placement by epistasis (CA-Rac1 rescue), multiple orthogonal assays, in vivo validation, single lab\",\n      \"pmids\": [\"24327733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"P-Rex1 and PDGFRβ are components of the same macromolecular complex (co-immunoprecipitation). P-Rex1 expression drives invasion in fibroblasts in a PDGFRβ-dependent manner; siRNA of either P-Rex1 or PDGFRβ opposes invasiveness in melanoma cells.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, 3D invasion assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single co-IP showing complex formation, functional loss-of-function supporting interaction, single lab\",\n      \"pmids\": [\"23382862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Phosphorylation of P-Rex1 at Ser1169 is induced by IGF-1R and FGFR activation and is required for IGF-1-induced Rac activation and cell proliferation, as well as IGF-1-induced adhesion in MCF7 breast cancer cells.\",\n      \"method\": \"Phospho-specific antibody analysis, siRNA knockdown, Rac activation assay, proliferation and adhesion assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-site linked to functional outcome by knockdown, single lab\",\n      \"pmids\": [\"23899556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"P-Rex1 acts as a GEF for RhoG both in vitro and in GPCR-stimulated primary mouse neutrophils, in addition to its known Rac-GEF activity. Loss of either P-Rex1 or RhoG caused equivalent reductions in GPCR-driven Rac activation and NADPH oxidase activity. Loss of RhoG impaired GPCR-driven recruitment of the Rac-GEF DOCK2 and F-actin to the leading edge, establishing a signaling hierarchy: P-Rex1→RhoG→DOCK2→Rac.\",\n      \"method\": \"In vitro GEF activity assay for RhoG, primary neutrophils from P-Rex1 and RhoG knockout mice, NADPH oxidase assay, F-actin localization, DOCK2 recruitment assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro GEF assay plus genetic epistasis in primary cells with multiple readouts, single lab\",\n      \"pmids\": [\"24659802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"P-REX1 overexpression activates Rac1 and increases PI3K/AKT, MEK/ERK signaling and IGF-1R activation in a PTEN-independent manner. Loss of P-REX1 suppresses PI3K/AKT and MEK/ERK. P-REX1 provides positive feedback to activators upstream of PI3K.\",\n      \"method\": \"P-Rex1 overexpression/knockdown, phosphoproteomic analysis, Rac1 activation assay, IGF-1R activation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple signaling readouts with gain and loss of function, single lab\",\n      \"pmids\": [\"25284585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of the P-Rex1 DH-PH tandem domain in complex with Rac1 at 1.95 Å resolution. Interface mutagenesis revealed a critical role for the P-Rex1·Rac1 complex in signaling downstream of RTKs and GPCRs. Structural analysis indicated PIP3/Gβγ binding sites are on the opposite surface from the Rac1 interface, supporting a model whereby PIP3/Gβγ binding releases inhibitory C-terminal domains to expose the Rac1 binding site.\",\n      \"method\": \"X-ray crystallography (1.95 Å), interface mutagenesis with functional signaling readouts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with mutagenesis validation, single lab\",\n      \"pmids\": [\"26112412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Genetic deletion or knockdown of P-Rex1 in the CA1 hippocampus causes autism-like social behavior linked to defective LTD via alteration of AMPA receptor endocytosis mediated by a postsynaptic PP1α-P-Rex1-Rac1 signaling pathway.\",\n      \"method\": \"Conditional knockout, hippocampal siRNA knockdown, LTD electrophysiology, AMPA receptor endocytosis assay, behavioral testing\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with defined synaptic and behavioral phenotype, pathway placement via PP1α-P-Rex1-Rac1 axis, single lab\",\n      \"pmids\": [\"26621702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Norbin (Neurochondrin, NCDN) is a direct binding partner of P-Rex1, interacting through the PH domain of P-Rex1. Direct interaction with Norbin increases the basal, PIP3-, and Gβγ-stimulated Rac-GEF activity of P-Rex1. Co-expression of P-Rex1 and Norbin induces translocation of both proteins from cytosol to plasma membrane and promotes cell spreading and lamellipodia formation.\",\n      \"method\": \"Pulldown from brain fractions, co-immunoprecipitation, in vitro binding with purified recombinant proteins, in vitro Rac-GEF assay, PAK-CRIB pulldown, immunofluorescence and subcellular fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct interaction shown with purified proteins, in vitro GEF stimulation, membrane translocation demonstrated, single lab\",\n      \"pmids\": [\"26792863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Type I PKA regulatory subunit RIα interacts with P-Rex1 PDZ domains via the CNB-B domain of RIα. P-Rex1 activation localizes PKA to the cell periphery. PKA phosphorylates the P-Rex1 DEP1 domain at Ser436, which inhibits the DH-PH catalytic cassette by direct interaction. A P-Rex1 S436A mutant shows increased RacGEF activity and prevents the inhibitory effect of PKA on cell migration.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, PKA activity assays, phospho-site mutagenesis (S436A), Rac-GEF activity assay, endothelial cell migration assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — co-IP of endogenous complex, phospho-site mutagenesis with functional rescue, single lab\",\n      \"pmids\": [\"26797121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PAK kinases phosphorylate PREX1 downstream of insulin, neuregulin, and IGF-1 receptor stimulation. PAK-mediated phosphorylation reduces PREX1 binding to PIP3 and negatively regulates PREX1 GEF activity. PREX1 phosphorylation onset is delayed compared to AKT, supporting a negative feedback model. GPCR-stimulated PREX1 phosphorylation is partially PAK-dependent and also involves PKA.\",\n      \"method\": \"PAK inhibitors, phospho-mass spectrometry, PIP3 binding assay, in vitro GEF activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-site identification by MS, PIP3 binding assay, GEF activity measurement, single lab\",\n      \"pmids\": [\"27481946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PKC isoform PKCδ directly phosphorylates P-Rex1 at Ser313. PKC activation causes phosphorylation of Ser313, Ser319, and Ser1169. Phosphorylation at Ser313 negatively regulates P-Rex1 exchange activity. Growth factor receptor-induced Ser1169 phosphorylation occurs through a mechanism independent of PKC, indicating different kinases control different regulatory serines.\",\n      \"method\": \"In vitro kinase assay with purified PKCδ, phospho-specific antibody analysis, P-Rex1 mutant expression (S313A), GEF activity assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay plus mutant functional analysis, single lab\",\n      \"pmids\": [\"27788493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PREX1 GEF activity drives ERK1/2 MAPK activation downstream of EGF/IGF-1 stimulation, promoting cyclin D1 and p21(WAF1) induction and anchorage-independent cell growth. GEF-dead PREX1 fails to increase ERK1/2 phosphorylation, anchorage-independent growth, or xenograft tumor growth. MEK1/2/ERK1/2 inhibition suppresses PREX1-mediated effects.\",\n      \"method\": \"Wild-type vs GEF-dead PREX1 expression, shRNA knockdown, phospho-ERK1/2 assay, anchorage-independent growth assay, xenograft tumor model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GEF-dead mutant as mechanistic control, in vivo validation, multiple orthogonal assays, single lab\",\n      \"pmids\": [\"27358402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ERK/MAPK signaling drives PREX1 overexpression in BRAF- and NRAS-mutant melanoma by both increasing PREX1 gene transcription and promoting PREX1 protein stability. Pharmacologic ERK inhibition reduces PREX1 transcription and protein levels. PREX1-dependent invasion in melanoma is attributable to RAC1 but not CDC42 activation.\",\n      \"method\": \"ERK pathway inhibitors, siRNA knockdown, RAC1/CDC42 activation assays, invasion assay\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological manipulation combined with functional readouts, single lab\",\n      \"pmids\": [\"27418645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Gαq and Gα13 directly inhibit Gβγ signaling to P-REX1. GTPase-deficient GαqQL and Gα13QL form stable complexes with Gβγ, preventing its interaction with P-REX1. Gβγ and AKT kinase associate with active P-REX1 during SDF-1/CXCL12 stimulation. GαqQL and Gα13QL also prevent CXCR4-dependent cell migration.\",\n      \"method\": \"Pulldown assays with chimeric Gα constructs, DREADDs chemogenetic control, co-immunoprecipitation of active P-REX1 complexes, cell migration assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — pulldown and co-IP with chimeric Gα constructs plus functional migration readout, single lab\",\n      \"pmids\": [\"30446620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GRK2 mediates TCR-induced phosphorylation of CXCR4 at Ser339 and TCR-CXCR4 complex formation. This complex signals via PI3Kγ to recruit PREX1 to the membrane, which activates a Rac1-dependent pathway stabilizing cytokine mRNAs and promoting robust cytokine secretion by T cells.\",\n      \"method\": \"siRNA depletion of GRK2, PI3Kγ, and PREX1; phospho-CXCR4 assay; membrane recruitment of PREX1 by fractionation; cytokine ELISA; mRNA stability assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple siRNA knockdowns with pathway placement, single lab\",\n      \"pmids\": [\"30018141\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PKA regulatory subunit RIα directly activates P-REX1 in vitro and promotes P-REX1-mediated Rac activation and cell migration via Gs-coupled EP2 receptors in a cAMP-dependent manner. RIα interacts with P-REX1 PDZ domains via its CNB-B domain. Active P-REX1 fraction is not phosphorylated, while inactive P-REX1 is phosphorylated, indicating co-existence of stimulatory (RIα) and inhibitory (catalytic subunit Cα) PKA effects.\",\n      \"method\": \"In vitro activation assay with purified RIα and P-REX1, cAMP pulldown, siRNA knockdown of RIα, Rac activation assay, endothelial cell migration assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro reconstitution with purified proteins plus cell-based validation, single lab\",\n      \"pmids\": [\"30530493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cryo-EM structure of the P-Rex1-Gβγ complex at 3.2 Å reveals that the C-terminal half of P-Rex1 adopts a fold similar to Legionella phosphoinositide phosphatases, forming an extensive docking site for Gβγ together with a DEP domain and two PDZ domains. Hydrogen-deuterium exchange MS suggests Gβγ binding induces allosteric changes in P-Rex1. Functional assays indicate membrane localization is also required for full activation.\",\n      \"method\": \"Cryo-EM structure determination (3.2 Å), hydrogen-deuterium exchange mass spectrometry, functional activation assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with HDX-MS allosteric mapping and functional validation, single lab\",\n      \"pmids\": [\"31663027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The DEP1 domain of P-Rex1 autoinhibits the DH/PH catalytic module through direct interaction. Crystal structure of DEP1 at 3.1 Å shows a domain-swap involving an exposed basic loop containing the primary PKA phosphorylation site (Ser436). PKA phosphorylation of DEP1 does not affect DH/PH-DEP1 fragment activity in solution but inhibits DEP1 domain binding to phosphatidic acid-containing liposomes, suggesting PKA inhibits P-Rex1 membrane binding rather than its catalytic activity directly.\",\n      \"method\": \"Crystal structure (3.1 Å), in vitro GEF activity assay with DH/PH-DEP1 fragments, liposome binding assay, PKA phosphorylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with in vitro biochemical assays and liposome binding, single lab\",\n      \"pmids\": [\"32661198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Small molecules targeting the P-Rex1 PH domain block PIP3 binding and inhibit fMLP-induced spreading and Rac2 activation in human neutrophils. One compound reduces neutrophil velocity and inhibits neutrophil recruitment to inflammation in a zebrafish model.\",\n      \"method\": \"Differential scanning fluorimetry screen, PIP3 competition binding assay, human neutrophil spreading/Rac2 activation assay, zebrafish in vivo inflammation assay\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — biochemical binding assays combined with cell-based and in vivo functional validation, single lab\",\n      \"pmids\": [\"31900312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"P-Rex1 is expressed in pancreatic β-cells (INS-1 832/13 cells, rat and human islets). siRNA-mediated knockdown of P-Rex1 attenuates glucose-induced Rac1 activation, membrane association, and insulin secretion. RhoG knockdown did not affect glucose-stimulated insulin secretion.\",\n      \"method\": \"siRNA knockdown, Rac1 activation pulldown assay, membrane fractionation, ELISA for insulin secretion\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with defined functional readout and specificity controls (RhoG negative), single lab\",\n      \"pmids\": [\"33347743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Gβγ recruits and activates P-Rex1 via two independent binding interfaces: Gβγ binds both the P-Rex1 DH/PH domains and the PDZ-PDZ tandem. The DEP-DEP tandem and PDZ-PDZ interact intramolecularly and dissociate upon Gβγ binding. The PDZ-PDZ interface mediates P-Rex1 recruitment to the plasma membrane; the DH/PH interface is required for activation.\",\n      \"method\": \"Pulldown assays with domain fragments, synthetic chimeric RhoGEF (Q-Rhox) to dissect recruitment from activation, co-immunoprecipitation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — domain dissection with chimeric GEF to parse binding interfaces, single lab\",\n      \"pmids\": [\"33412417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NRBP1 (a pseudokinase) binds P-Rex1 and acts as a scaffold to enhance GTP-bound Rac1 and Cdc42 levels in a P-Rex1-dependent manner. NRBP1 overexpression-driven cell migration and invasion are P-Rex1-dependent. ROS generation via a NRBP1/P-Rex1 pathway contributes to oncogenic roles in triple-negative breast cancer.\",\n      \"method\": \"BioID/MS proximity proteomics, co-immunoprecipitation, Rac1/Cdc42 activation assays, siRNA knockdown epistasis, invasion assay, ROS assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — BioID identifies interaction, co-IP confirms, epistasis in functional assays, single lab\",\n      \"pmids\": [\"36693952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"P-Rex1 is a novel substrate of the E3 ubiquitin ligase Malin (EPM2B); Malin ubiquitinates P-Rex1 and this is associated with altered glucose uptake relevant to Lafora disease pathology.\",\n      \"method\": \"Unbiased E3 ligase substrate identification approach, ubiquitination assay, glucose uptake functional assay\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — novel ubiquitination substrate identification with functional consequence, single lab\",\n      \"pmids\": [\"36638890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structure of P-Rex1·IP4 complex reveals an autoinhibited conformation where the PH domain occludes the DH domain active site, stabilized by DH-DEP1 and PH-4HB interdomain contacts. IP4 inhibits P-Rex1 GEF activity and reduces backbone dynamics broadly. Disruption of DH-DEP1 or PH-4HB interfaces increases activity and confers a more extended conformation. Mutations constraining the occluded conformation reduce GEF activity. PIP3-containing liposomes disrupt these interfaces and increase dynamics. This autoinhibited structure is confirmed in living cells by gain-of-function variants showing enhanced activity during chemokine-induced migration.\",\n      \"method\": \"Cryo-EM structure, HDX-MS, in vitro GEF activity assays with interface mutants, liposome binding assay, cell migration assay with interface mutant variants\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure with HDX-MS, mutagenesis, biochemical reconstitution, and cellular validation, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"39082940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PREX1 promotes nuclear translocation of phosphorylated STAT5 in naive CD4+ T cells, supporting homeostatic proliferation in response to IL-7, and biases differentiation toward effector T cells.\",\n      \"method\": \"PREX1 expression analysis in aged naive CD4+ T cells, nuclear fractionation for phospho-STAT5, functional proliferation assays with PREX1 manipulation\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — nuclear translocation of pSTAT5 linked to PREX1 expression and IL-7 responsiveness, single lab\",\n      \"pmids\": [\"38329813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"P-Rex1 limits GPCR (S1PR1, CXCR4, PAR4, GLP1R) internalization independently of its Rac-GEF activity, through its PDZ, DEP, and IP4P domains. P-Rex1 blocks phosphorylation required for GPCR internalization. P-Rex1 binds GRK2 both in vitro and in cells, but does not regulate GRK2 activity.\",\n      \"method\": \"Catalytically inactive Prex1GD knock-in mice, GPCR internalization assays, phosphorylation assays, in vitro binding of P-Rex1 with GRK2, domain deletion analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — catalytically inactive knock-in mouse, in vitro binding, multiple GPCR substrates tested, mechanistic separation of GEF and adaptor functions, single lab\",\n      \"pmids\": [\"41100251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"P-Rex1 limits hepatocyte glucose uptake and mitochondrial ATP production independently of its Rac-GEF catalytic activity through the orphan GPCR Gpr21. P-Rex1 controls Gpr21 trafficking (retaining it at plasma membrane) and controls Glut2 surface levels, mitochondrial morphology, membrane potential, and ATP production in hepatocytes. P-Rex1 GEF activity is required for maintaining fasting blood glucose and insulin sensitivity.\",\n      \"method\": \"Prex1−/− and catalytically inactive Prex1GD mice, high-fat diet diabetes model, hepatocyte glucose uptake assay, mitochondrial membrane potential/ATP assays, Gpr21 trafficking by cell fractionation, Glut2 surface assay, pharmacological Gpr21 inverse agonist (GRA2)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — genetic separation of GEF-dependent and GEF-independent functions using knock-in mice, multiple orthogonal metabolic and cell biology readouts, single lab\",\n      \"pmids\": [\"41046518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"P-Rex1 mediates phagocytosis of IgG-opsonized particles and bactericidal activity in neutrophils independently of its Rac-GEF catalytic activity, through mechanisms not related to its GPCR trafficking or glucose uptake adaptor functions. P-Rex1 mediates both integrin-dependent and Fc receptor-dependent phagocytosis and the Fc receptor-dependent activation of Rac and Syk. P-Rex1 GEF activity is required for migration, ROS, and NET formation.\",\n      \"method\": \"Prex1−/− and catalytically inactive Prex1GD mice, phagocytosis assays (IgG-opsonized zymosan), in vivo septic peritonitis bacteria clearance, Syk activation assay, Rac activation assay\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic separation using KO and catalytically inactive knock-in, multiple phagocytic and bactericidal readouts, single lab\",\n      \"pmids\": [\"41098722\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"P-Rex1 is a large, multidomain Rac-GEF (Rac1, Rac2, Rac3, and RhoG substrates) that functions as a coincidence detector, synergistically activated at the plasma membrane by PIP3 (via its PH domain) and Gβγ subunits (via DEP-PDZ and DH/PH interfaces); in its resting state it adopts an autoinhibited conformation in which the PH domain occludes the DH active site through DH-DEP1 and PH-4HB interdomain contacts. Activation is further regulated by dephosphorylation via PP1α (at Ser1165, Ser1001, Ser834), activating phosphorylation at Ser605 and Ser1169 by RTK-linked kinases, and inhibitory phosphorylation at Ser313/Ser319 by PKCδ, Ser436 by PKA catalytic subunit (which also impairs membrane binding), and multiple sites by PAKs. RIα regulatory subunits of type I PKA directly bind the P-Rex1 PDZ domains and can activate P-Rex1 in a cAMP-dependent manner, while Gαq and Gα13 sequester Gβγ to suppress P-Rex1 signaling. Beyond Rac-GEF activity, P-Rex1 serves as an adaptor that limits agonist-induced GPCR internalization by binding GRK2 and controls hepatocyte Gpr21 surface trafficking and mitochondrial glucose metabolism; these adaptor functions are independent of catalytic activity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"P-Rex1 is a large multidomain guanine-nucleotide exchange factor that activates the Rho-family GTPases Rac1, Rac2, Rac3 and RhoG to drive actin remodeling, cell migration, ROS production and invasion across neutrophils, neurons, and cancer cells [#0, #1, #10, #23]. Its defining property is coincidence detection at the plasma membrane: Rac-GEF activity is synergistically and directly stimulated by PtdIns(3,4,5)P3 (via the PH domain) and Gβγ subunits, neither input alone being sufficient for full membrane translocation and activation [#0, #6]. Structural work resolves the underlying logic — in the resting state the PH domain occludes the DH active site, stabilized by DH-DEP1 and PH-4HB interdomain contacts and by IP4 binding, while PIP3 and Gβγ disrupt these autoinhibitory interfaces to expose the Rac-binding surface [#25, #36, #43]. Gβγ engages two independent interfaces (DH/PH for activation and the PDZ-PDZ tandem for membrane recruitment), and Gαq/Gα13 suppress signaling by sequestering Gβγ [#33, #40]. Activity is further tuned by a phosphorylation cycle: PP1α docks via an RVxF motif and dephosphorylates inhibitory sites (Ser1165, Ser1001, Ser834) to activate the enzyme [#18], RTK-linked inputs drive activating phosphorylation at Ser605/Ser1169 [#13, #22], while PKCδ (Ser313/Ser319), PKA (Ser436, which impairs membrane binding), and PAKs phosphorylate inhibitory sites, the last forming a delayed negative-feedback loop [#28, #30, #37, #29]; type I PKA RIα subunits bind the PDZ domains and conversely activate P-Rex1 in a cAMP-dependent manner [#28, #35]. Through ErbB/CXCR4-, IGF-1R- and PI3K-coupled inputs P-Rex1 activates Rac1 to drive a C-RAF/MEK/ERK growth axis and tumorigenesis, and its overexpression is reinforced by ERK-driven transcription and protein stabilization in melanoma [#12, #20, #31, #32]. P-Rex1 deficiency impairs neutrophil recruitment, melanoblast migration and melanoma metastasis, and contributes to hippocampal LTD and social behavior via a postsynaptic PP1α-P-Rex1-Rac1 pathway controlling AMPA-receptor endocytosis [#2, #15, #26]. Beyond catalysis, P-Rex1 acts as a Rac-GEF-independent adaptor that limits agonist-induced GPCR internalization by binding GRK2, controls hepatocyte Gpr21 surface trafficking and mitochondrial glucose metabolism, and supports Fc-receptor-dependent phagocytosis [#45, #46, #47].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established the existence and core biochemistry of P-Rex1, answering whether a PIP3- and Gβγ-responsive Rac-GEF links PI3K and GPCR inputs in neutrophils.\",\n      \"evidence\": \"Biochemical purification from neutrophil cytosol with in vitro reconstituted GEF assays and antisense knockdown with ROS readout\",\n      \"pmids\": [\"11955434\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrate specificity among Rac isoforms not resolved\", \"Structural basis of synergistic activation unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Genetic loss-of-function defined P-Rex1 as a physiologically required, Rac2-preferring GEF in neutrophil GPCR signaling, linking it to ROS, F-actin and inflammatory recruitment.\",\n      \"evidence\": \"P-Rex1 knockout mice with PBD pulldown GTPase assays, superoxide assays, and in vivo peritonitis model; replicated independently\",\n      \"pmids\": [\"16243036\", \"16243035\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of Rac2-over-Rac1 selectivity unclear\", \"Only mildly reduced chemotaxis left activation regulation open\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified the upstream activators and inhibitors that gate P-Rex1, showing Gβγ isoform selectivity and PKA-driven phosphorylation as an off-switch.\",\n      \"evidence\": \"In vitro GEF assays with purified Gβγ dimers in lipid vesicles, in vitro PKA kinase assays, and cell-based Rac activation assays\",\n      \"pmids\": [\"16301320\", \"16301321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PKA phosphorylation sites not mapped\", \"Mechanism connecting phosphorylation to Gβγ responsiveness unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Extended P-Rex1 function beyond immunity by showing it drives neurotrophin-induced Rac1 activation and neuronal migration during brain development.\",\n      \"evidence\": \"In situ hybridization, immunofluorescence, siRNA, dominant-negative constructs and in utero electroporation\",\n      \"pmids\": [\"15858067\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor inputs upstream in neurons not defined\", \"Rac isoform usage in neurons unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Dissected how P-Rex1 reaches the membrane, establishing that Gβγ and PIP3 act synergistically for translocation while inhibitory domains retain it in cytosol.\",\n      \"evidence\": \"Subcellular fractionation in Sf9 cells with domain mutants and in vitro GEF assays; Co-IP placing P-Rex1 with mTORC2\",\n      \"pmids\": [\"17698854\", \"17565979\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of intramolecular autoinhibition not yet defined\", \"mTORC2 association rests on Co-IP in a single lab\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Mapped intramolecular autoinhibitory contacts and tied PKA phosphorylation to disruption of domain-domain interaction and Gβγ binding.\",\n      \"evidence\": \"Co-IP of domain mutants, in vitro GEF assays, and neuronal localization with GEF-dead rescue experiments\",\n      \"pmids\": [\"18514484\", \"18697831\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Atomic structure of autoinhibited state still lacking\", \"Single-lab domain-interaction assays\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Placed P-Rex1 at the convergence of RTK and GPCR inputs in cancer, defining a phospho-regulatory cycle and a requirement for tumorigenesis.\",\n      \"evidence\": \"siRNA knockdown, Rac1 activation assays, phospho-specific antibodies, xenografts, and CXCR4/PI3Kγ epistasis in breast cancer cells\",\n      \"pmids\": [\"21172654\", \"21042280\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of kinases for individual activating sites not fully resolved\", \"Direct versus indirect ErbB-P-Rex1 coupling unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated developmental and metastatic roles in melanocyte lineage and broadened substrate/cellular contexts to adipocyte GLUT4 trafficking and platelets.\",\n      \"evidence\": \"P-Rex1 knockout melanoma model, GLUT4/glucose uptake assays in adipocytes, and Rac1-pulldown mass spectrometry in platelets\",\n      \"pmids\": [\"22109529\", \"22002247\", \"21884615\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why P-Rex1 is dispensable in platelets despite Rac1 binding unexplained\", \"Tissue-specific upstream activators not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified PP1α as a direct activating phosphatase and mapped its dephosphorylation sites, establishing reversible phospho-control of GEF activity.\",\n      \"evidence\": \"In vitro GEF assays with purified PP1α, RVxF motif analysis, MS phosphosite mapping, and S1165A mutagenesis; zebrafish Nodal/Prex1 morpholino study\",\n      \"pmids\": [\"22242915\", \"22945937\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coordination between PP1α and antagonizing kinases in vivo unclear\", \"Spatial control of dephosphorylation unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the Rac1/C-RAF/MEK/ERK signaling axis downstream of P-Rex1 in PI3K-pathway-mutant cancers and identified PDGFRβ as a complex partner driving invasion.\",\n      \"evidence\": \"siRNA, constitutively active Rac1 rescue, PI3K-inhibitor treatment, phospho-ERK/BIM assays, xenografts, and Co-IP in fibroblasts\",\n      \"pmids\": [\"24327733\", \"23382862\", \"23899556\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ERK activation is fully RAS-independent in all contexts unresolved\", \"PDGFRβ complex stoichiometry undefined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed P-Rex1 is also a RhoG-GEF defining a P-Rex1→RhoG→DOCK2→Rac hierarchy, and acts in a positive feedback loop upstream of PI3K in cancer.\",\n      \"evidence\": \"In vitro RhoG GEF assays, P-Rex1 and RhoG knockout neutrophils with DOCK2 recruitment readouts, and phosphoproteomics with gain/loss of function\",\n      \"pmids\": [\"24659802\", \"25284585\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mechanism of PI3K-upstream feedback unclear\", \"Relative contribution of Rac- versus RhoG-GEF activity per tissue unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Provided the first atomic view of the DH-PH·Rac1 catalytic complex and connected a PP1α-P-Rex1-Rac1 pathway to synaptic plasticity and autism-like behavior.\",\n      \"evidence\": \"X-ray crystallography at 1.95 Å with interface mutagenesis; conditional KO with LTD electrophysiology, AMPAR endocytosis, and behavioral assays\",\n      \"pmids\": [\"26112412\", \"26621702\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length autoinhibited architecture not yet solved\", \"How PIP3/Gβγ release inhibitory domains shown only by inference\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Expanded the regulatory network with direct activators (Norbin, RIα) and inhibitory kinases (PKCδ, PAKs), and confirmed GEF activity drives ERK-dependent proliferation and a melanoma feedforward loop.\",\n      \"evidence\": \"Purified-protein binding and GEF assays, phospho-site mutagenesis (S436A, S313A), PAK inhibitors with MS, GEF-dead controls, xenografts, and ERK-inhibitor manipulation\",\n      \"pmids\": [\"26792863\", \"26797121\", \"27481946\", \"27788493\", \"27358402\", \"27418645\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of competing positive and negative regulators in time/space not modeled\", \"Distinct kinases for each serine only partially assigned\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved how Gα subunits restrain P-Rex1 by Gβγ sequestration and detailed GRK2/CXCR4-PI3Kγ recruitment driving T-cell cytokine output.\",\n      \"evidence\": \"Pulldown with chimeric Gα constructs, DREADD chemogenetics, Co-IP of active complexes, siRNA epistasis, and cytokine/mRNA stability assays\",\n      \"pmids\": [\"30446620\", \"30018141\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry of Gβγ sequestration in cells unclear\", \"GRK2 binding role versus catalytic recruitment not separated here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Cryo-EM of the P-Rex1·Gβγ complex revealed a Legionella-phosphatase-like C-terminal fold forming an extensive Gβγ docking site and allosteric activation.\",\n      \"evidence\": \"Cryo-EM at 3.2 Å, HDX-MS, and functional activation assays\",\n      \"pmids\": [\"31663027\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Membrane-bound active conformation not captured\", \"Coupling of Gβγ docking to active-site exposure incompletely defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Solved the DEP1 autoinhibition mechanism, showed PKA acts on membrane binding rather than catalysis directly, and demonstrated PH-domain small-molecule inhibition.\",\n      \"evidence\": \"Crystal structure of DEP1 at 3.1 Å, liposome binding, in vitro GEF assays with fragments, DSF screen with neutrophil and zebrafish validation; β-cell siRNA insulin secretion study\",\n      \"pmids\": [\"32661198\", \"31900312\", \"33347743\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length autoinhibited structure still pending\", \"Selectivity and pharmacology of PH-domain inhibitors not optimized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Separated Gβγ-mediated recruitment from activation, assigning the PDZ-PDZ tandem to membrane targeting and the DH/PH interface to catalytic activation.\",\n      \"evidence\": \"Domain-fragment pulldowns and a synthetic chimeric RhoGEF (Q-Rhox) with Co-IP\",\n      \"pmids\": [\"33412417\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative contribution of each interface in vivo unresolved\", \"Dissection relies on chimeric constructs in a single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the complete autoinhibited architecture and its release mechanism, showing the PH domain occludes the DH active site and PIP3 disrupts DH-DEP1/PH-4HB interfaces to activate the enzyme.\",\n      \"evidence\": \"Cryo-EM of P-Rex1·IP4, HDX-MS, interface-mutant GEF and liposome assays, and cellular gain-of-function migration validation; pSTAT5/IL-7 T-cell study\",\n      \"pmids\": [\"39082940\", \"38329813\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order of PIP3 and Gβγ engagement during activation not fully kinetically resolved\", \"How phospho-regulation maps onto the autoinhibited structure unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Genetic separation of catalytic from adaptor functions revealed Rac-GEF-independent roles in limiting GPCR internalization, controlling hepatocyte Gpr21/glucose metabolism, and supporting Fc-receptor phagocytosis.\",\n      \"evidence\": \"Catalytically inactive Prex1GD knock-in mice with GPCR internalization, in vitro GRK2 binding, hepatocyte metabolic assays, Gpr21 trafficking, and phagocytosis/bactericidal assays\",\n      \"pmids\": [\"41100251\", \"41046518\", \"41098722\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which P-Rex1 blocks GPCR phosphorylation undefined\", \"How a single scaffold mediates distinct GEF-independent outputs unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the full regulatory layers — autoinhibition, phospho-cycling, multi-interface Gβγ engagement, and Rac-GEF-independent adaptor functions — are integrated spatiotemporally within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified kinetic model linking activation inputs to output selectivity\", \"Structural basis of adaptor (GEF-independent) functions uncharacterized\", \"Direct Mendelian disease causation not established in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005085\", \"supporting_discovery_ids\": [0, 1, 23]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 23, 25]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [6, 37, 38, 43]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [45, 46]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [18, 28, 35]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 8, 27, 40]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 6, 8, 27]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 12, 20, 33]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 2, 14, 47]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [12, 15, 20, 31]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [16, 39, 46]}\n    ],\n    \"complexes\": [\n      \"mTORC2\",\n      \"P-Rex1-Gβγ complex\"\n    ],\n    \"partners\": [\n      \"Gβγ\",\n      \"PP1α\",\n      \"GRK2\",\n      \"Norbin (NCDN)\",\n      \"PDGFRβ\",\n      \"NRBP1\",\n      \"RIα (PRKAR1A)\",\n      \"Rac1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}