{"gene":"RALGDS","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":1994,"finding":"RALGDS interacts directly with the GTP-bound (active) form of Ras p21 through the effector loop of Ras, but not with the GDP-bound form or an effector-loop mutant of Ras. In insect cells, RalGDS formed a complex with v-Ras but not with dominant-negative Ras. RalGDS inhibited both NF1 GAP activity toward Ras and the interaction of Raf with Ras in vitro, indicating it competes with Raf and NF1 for the Ras effector loop.","method":"Yeast two-hybrid, co-immunoprecipitation in insect cells, in vitro binding/competition assays with GTP/GDP-loaded Ras","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (yeast two-hybrid, insect-cell co-IP, in vitro binding), replicated across assay systems in one study","pmids":["7935463"],"is_preprint":false},{"year":1996,"finding":"EGF induces the interaction of c-Ras and RalGDS in mammalian COS cells in an effector-loop-dependent manner. Cyclic AMP-dependent protein kinase (PKA) phosphorylates RalGDS but does not alter its affinity for Ras or its GDP/GTP exchange activity toward Ral p24. PKA phosphorylation of (1-149)Raf reduces its affinity for Ras, thereby shifting Ras binding selectively toward RalGDS under cAMP-elevated conditions.","method":"Co-immunoprecipitation in COS cells, in vitro kinase assay (PKA phosphorylation), GEF activity assay, forskolin treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP plus in vitro biochemical assays, multiple orthogonal methods in one study","pmids":["8550624"],"is_preprint":false},{"year":1997,"finding":"RalGDS functions as an effector of Ras in cAMP-mediated growth stimulation in thyroid cells. A Ras effector-domain mutant (RasV12G37) defective in Raf-1 binding still bound RalGDS by yeast two-hybrid and co-immunoprecipitation. Microinjection of dominant-negative RalA (which sequesters RalGDS family members) reduced Ras-stimulated and cAMP-stimulated DNA synthesis.","method":"Yeast two-hybrid, co-immunoprecipitation in thyroid cell extracts, microinjection of dominant-negative RalA, DNA synthesis assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (yeast two-hybrid, co-IP, microinjection phenotypic assay) in one study","pmids":["9038168"],"is_preprint":false},{"year":1997,"finding":"The Ras-interacting domain (RID) of RalGDS adopts an ubiquitin-like fold. Mutational analysis identified three residues in the RID critical for interaction with Ras.","method":"NMR structure determination, site-directed mutagenesis, binding assays","journal":"Nature structural biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure plus mutagenesis validation, single lab","pmids":["9253406"],"is_preprint":false},{"year":1998,"finding":"Crystal structure (2.1 Å) of the Ras–RalGDS RID complex reveals that the beta-sheet of the RID joins the switch I region of Ras to form an extended beta-sheet, analogous to but geometrically distinct from the Rap-Raf complex. A second RID molecule also contacts the switch II region of Ras, providing a structural basis for cross-talk between the Ras and Ral pathways.","method":"X-ray crystallography at 2.1 Å resolution","journal":"Nature structural biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure, independently consistent with other structural and biochemical studies","pmids":["9628477"],"is_preprint":false},{"year":1998,"finding":"RalGDS mediates Ras-dependent inhibition of skeletal myogenesis. An H-Ras effector mutant (G12V,E37G) that binds RalGDS but not Raf inhibits muscle-specific reporter gene activity. Membrane-targeted RalGDS (RalGDS-CAAX) also inhibits alpha-actin-Luc and SRF-dependent transcription, while a RalGDS mutant defective in Ras interaction has no effect. RalGDS and activated RalA additionally inhibit the SRF-dependent component of H-Ras G12V,E37G-induced transcription.","method":"Transient transfection of Ras effector-loop mutants and RalGDS constructs, luciferase reporter assays, dominant-negative and constitutively active Ral constructs","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean loss-of-function (Ras-interaction-defective RalGDS as control) plus gain-of-function with membrane-targeted construct, single lab","pmids":["9651367"],"is_preprint":false},{"year":1999,"finding":"X-ray crystal structure of the Ras–RalGDS RBD (RGS-RBD) complex shows an inter-protein beta-sheet between Ras switch I and the RGS-RBD beta-strand, analogous to Rap-RafRBD but with markedly different interface side-chain interactions. Gel filtration confirms the complex is a 1:1 monomer. Mutational studies define interface residues contributing to binding affinity.","method":"X-ray crystallography, size-exclusion chromatography, site-directed mutagenesis with binding affinity measurements","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis and biochemical validation, consistent with independent structure from Huang et al. 1998","pmids":["10371160"],"is_preprint":false},{"year":1999,"finding":"RalGEFs (specifically Rlf/RGL) are required for Ras-induced primitive endoderm differentiation of F9 embryonal carcinoma cells. Constitutively active Rlf-CAAX is sufficient to induce differentiation; dominant-negative RalA completely abolishes Rlf-CAAX– and RasV12G37-induced differentiation. RalGEF-induced differentiation requires basal MEK/ERK activity (MEK inhibition blocks it), but Rlf-CAAX itself does not increase ERK activity.","method":"Ras effector-domain mutants, constitutively active RalGEF (Rlf-CAAX), dominant-negative RalA, MEK inhibitor (PD98059), morphological differentiation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis with multiple genetic tools, single lab","pmids":["10442634"],"is_preprint":false},{"year":2001,"finding":"RalGDS activation by Ras involves membrane translocation (RBD-dependent) but additionally requires a Ras switch II interaction: Ras Y64W is impaired in RalGDS activation but not in RBD binding. An artificially membrane-targeted RalGDS lacking its RBD can still be activated by Ras, indicating the RBD is important but not sufficient. Rap1 antagonizes Ras-mediated RalGDS activation only when the RBD is intact, by forming a long-lived complex (KD ~0.6 μM) that sequesters RalGDS, whereas the Ras×RalGDS complex is short-lived (~0.1 s).","method":"RalA-GTP loading assay, Ras/RalGDS mutant constructs, kinetic binding measurements (surface plasmon resonance/stopped-flow), membrane-targeted RalGDS constructs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro biochemical assays with GTP measurement, kinetic studies, and multiple mutant constructs converging on same conclusion","pmids":["11748241"],"is_preprint":false},{"year":2002,"finding":"Beta-arrestins bind RalGDS (identified by yeast two-hybrid and co-immunoprecipitation from human PMNs). Under basal conditions, RalGDS is held inactive in a cytosolic complex with beta-arrestin. Upon fMLP receptor stimulation, the complex dissociates, RalGDS translocates to the plasma membrane, and Ral is activated independently of Ras, driving cytoskeletal reorganization. Re-association of the complex correlates with Ral signal termination.","method":"Yeast two-hybrid screen, co-immunoprecipitation from PMNs, subcellular fractionation/translocation assay, dominant-negative Ral constructs, cytoskeletal phenotype readout","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP plus translocation imaging plus functional (cytoskeletal) readout, multiple orthogonal methods","pmids":["12105416"],"is_preprint":false},{"year":2002,"finding":"PDK1 enhances RalGDS catalytic (GEF) activity by a kinase-independent mechanism. The non-catalytic N-terminus of PDK1 forms an EGF-induced complex with the N-terminus of RalGDS, relieving its auto-inhibitory effect on the catalytic domain. This represents cooperation between the PI3K-PDK1 Ras effector pathway and the RalGEF pathway to promote Ral activation.","method":"Co-immunoprecipitation, kinase-dead PDK1 constructs, Ral-GTP loading assay, N-terminal deletion mutants of RalGDS","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple mutant constructs, kinase-dead controls, GTP-loading readout, co-IP; multiple orthogonal approaches in one study","pmids":["11889038"],"is_preprint":false},{"year":2002,"finding":"Ras regulates choline kinase activity through its direct effectors RalGDS and PI3K, but not through Raf-1. Ras effector-domain mutants specifically activating RalGDS (RasV12G37) or PI3K (RasV12C40), but not Raf (RasV12S35), activated choline kinase, establishing RalGDS as a required intermediary.","method":"Ras effector-loop mutants (RasV12G37, V12C40, V12S35), choline kinase activity assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic dissection with effector-domain mutants, single lab, single primary readout","pmids":["11840339"],"is_preprint":false},{"year":2002,"finding":"Growth factors activate ATF2 Thr69 phosphorylation via a Ral-RalGDS-Src-p38 pathway, distinct from the Raf-MEK-ERK pathway that phosphorylates Thr71. Cooperation between ERK and p38 is required for full ATF2 dual-phosphorylation by growth factors such as insulin and EGF.","method":"Phosphorylation-site-specific ATF2 antibodies, dominant-negative and constitutively active pathway constructs, kinase inhibitors, Ras effector-domain mutants","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal approaches (specific phospho-antibodies, pathway mutants, inhibitors), two-step mechanism validated","pmids":["12110590"],"is_preprint":false},{"year":2004,"finding":"The Ras/RalGEF/p38 pathway dictates host cell permissiveness to reovirus infection. Ras effector-domain mutant RasV12G37 (retaining RalGEF signaling but not Raf or PI3K) renders NIH 3T3 cells permissive. Activated RalGEF (Rlf) alone confers permissiveness; dominant-negative RalA renders H-Ras cells resistant. p38 inhibition blocks reovirus replication downstream of RalGEF.","method":"Ras effector-domain mutants, activated RalGEF (Rlf) overexpression, dominant-negative RalA, p38/JNK inhibitors, viral replication assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple tools converging on RalGEF→p38 as the required pathway, functional viral replication readout","pmids":["15263068"],"is_preprint":false},{"year":2005,"finding":"RalGDS is required for Ras-induced oncogenesis in vivo. RalGDS-knockout mice develop normally but show reduced tumor incidence, size, and malignant progression in multistage skin carcinogenesis. RalGDS does not regulate cell proliferation but controls survival of transformed cells, apparently through activation of the JNK/SAPK pathway.","method":"Ralgds-/- knockout mice, DMBA/TPA skin carcinogenesis model, tissue culture transformation assays, apoptosis/survival assays, JNK activity measurements in tumor-derived cells","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic knockout with multiple phenotypic readouts and mechanistic follow-up (JNK activity); independently discussed by Rodriguez-Viciana & McCormick 2005","pmids":["15766660"],"is_preprint":false},{"year":2007,"finding":"Activation of the RalGEF/Ral pathway promotes prostate cancer bone metastasis. In DU145 cells, activation of the RalGEF pathway (RasV12G37) but not Raf/ERK or PI3K promotes bone metastasis. Loss of RalA in metastatic PC3 cells inhibits bone metastasis but not subcutaneous tumor growth, specifically suppressing expansive growth in bone.","method":"Ras effector-domain mutants, RalA RNAi knockdown, in vivo metastasis assay (intracardiac injection), subcutaneous tumor growth assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic dissection with effector mutants plus RNAi knockdown in vivo, specific organ-metastasis readout","pmids":["17709381"],"is_preprint":false},{"year":2008,"finding":"RalGDS mediates thrombin- and epinephrine-induced exocytosis of Weibel-Palade bodies (WPBs) from endothelial cells. RNAi knockdown of RalGDS inhibits WPB exocytosis; overexpression promotes it; a dominant-negative exchange-domain-deleted RalGDS blocks WPB release. RalGDS binds calmodulin (CaM) via an N-terminal CaM-binding domain, and a cell-permeable peptide comprising this domain inhibits Ral activation and WPB exocytosis.","method":"RNAi knockdown, overexpression, dominant-negative mutant, calmodulin pulldown, cell-permeable peptide inhibitor, WPB exocytosis assay (VWF secretion), Ral-GTP loading assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal loss- and gain-of-function approaches with defined molecular mechanism (CaM binding domain), single lab","pmids":["18417737"],"is_preprint":false},{"year":2008,"finding":"RalGDS has a second function beyond Ral GEF activity: it promotes Akt phosphorylation by PDK1 by acting as a scaffold that brings PDK1 and Akt together. The N-terminus of RalGDS complexes PDK1 while the central region associates with Akt via JIP1. An N-terminally deleted RalGDS retains Ral activation but cannot promote Akt phosphorylation and fails to support cell proliferation.","method":"RNAi suppression of RalGDS, co-immunoprecipitation (PDK1/RalGDS, Akt/RalGDS via JIP1), deletion mutant analysis, phospho-Akt immunoblot, cell proliferation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNAi knockdown, reciprocal co-IP, domain-deletion mapping, functional proliferation readout; multiple orthogonal methods","pmids":["18285454"],"is_preprint":false},{"year":2013,"finding":"RalGDS-mediated cardiomyocyte autophagy is required for load-induced cardiac hypertrophy. RalGDS-null hearts show blunted hypertrophic growth and suppressed autophagy in response to pressure-overload (TAC), while ventricular function is preserved and fetal gene program activation is unaffected. In NRCMs, RalB (a downstream target of RalGDS) specifically mediates mTOR-dependent autophagy.","method":"Ralgds-/- knockout mice, transverse aortic constriction (TAC) model, RalA/RalB RNAi in neonatal rat cardiomyocytes, autophagy markers (LC3, p62), echocardiography, fetal gene expression","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knockout with pressure-overload model plus in vitro RNAi epistasis, multiple phenotypic readouts","pmids":["23473774"],"is_preprint":false},{"year":2015,"finding":"RILP (Rab7-interacting lysosomal protein) interacts with RalGDS via its N-terminal region binding to the GEF domain of RalGDS. RILP overexpression recruits RalGDS to late endosomal compartments and inhibits RalA activity and breast cancer cell invasion; RILP knockdown promotes invasion. The interaction is confirmed by co-immunoprecipitation and truncation analysis.","method":"Co-immunoprecipitation, truncation/deletion mapping, immunofluorescence microscopy, RalA-GTP loading assay, cell invasion assay, RNAi knockdown","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP with domain mapping, subcellular localization imaging, functional GEF-activity readout; single lab","pmids":["26469971"],"is_preprint":false},{"year":2001,"finding":"JAK/STAT3 activation in M1 myeloid leukemia cells induces RalGDS expression, leading to RalA-GTP activation. Dominant-negative STAT3 and the JAK inhibitor JAB/SOCS1 suppress both RalGDS expression and RalA activation. Full RalA activation also requires Ras activity, revealing cross-talk between the JAK/STAT3 and Ras/RalGDS/Ral pathways.","method":"Representational difference analysis (RalGDS cloning), dominant-negative STAT3, JAK inhibitor, Ras inhibitor, Ral-GTP loading assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RalGDS identified as STAT3 target, functional Ral-GTP readout with pathway inhibitors; single lab","pmids":["11432872"],"is_preprint":false},{"year":2017,"finding":"Single-molecule fluorescence imaging shows that EGF-induced Ras activation increases the membrane density of RalGDS via its RBD (not the REMCDC domain). The RBD increases the RalGDS association rate to the membrane, while the REMCDC domain decreases the dissociation rate after Ras activation or Ral overexpression. Ras Y64 residue and RalGDS cluster formation on the membrane are involved in stabilizing RalGDS–Ral interaction upon Ras activation.","method":"Single-molecule fluorescence imaging, GFP-tagged RalGDS domain constructs in living HeLa cells, EGF stimulation, membrane density quantitation, rate constant measurements","journal":"Biophysics and physicobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct live-cell single-molecule imaging of RalGDS translocation with domain dissection; single lab, single technique","pmids":["28744424"],"is_preprint":false},{"year":2019,"finding":"In C. elegans, the RalGEF ortholog RGL-1 plays two genetically separable roles in vulval cell fate patterning: (1) a canonical GEF-dependent promotion of 2° fate via RAL-1 activation, and (2) a non-canonical GEF-independent promotion of 1° fate that acts through the AGE-1/PI3K-PDK-1-AKT-1 cascade. Loss of RGL-1 increases VPC patterning error rates 15-fold, indicating RGL-1 orchestrates opposing modulatory cascades to reduce developmental stochasticity.","method":"C. elegans genetic epistasis, RGL-1 GEF-dead mutants, double-mutant analysis with PI3K/PDK pathway components, VPC fate scoring","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — rigorous C. elegans genetic epistasis with GEF-dead separation-of-function mutants; ortholog study, single lab","pmids":["31086367"],"is_preprint":false}],"current_model":"RALGDS is a guanine nucleotide exchange factor (GEF) that is recruited to the plasma membrane by binding its Ras-binding domain (RBD) to GTP-Ras (via the Ras switch I effector loop), whereupon it activates Ral GTPases (RalA/RalB); its catalytic activity can be further enhanced by a kinase-independent PDK1 interaction that relieves autoinhibition; additional regulation is provided by beta-arrestin sequestration in the cytosol, calmodulin binding required for Ca²⁺-stimulated Ral activation and Weibel-Palade body exocytosis, and RILP-mediated recruitment to late endosomes to suppress RalA activity; beyond Ral activation, RalGDS scaffolds PDK1 and Akt (via JIP1) to promote Akt phosphorylation and cell survival, and mediates Ras-driven oncogenesis, bone metastasis, cardiomyocyte autophagy/hypertrophy, and ATF2 Thr69 phosphorylation through a Ral-Src-p38 axis."},"narrative":{"mechanistic_narrative":"RALGDS is a Ras effector and guanine-nucleotide exchange factor that couples activated Ras to the Ral GTPase branch of Ras signaling, thereby controlling cell survival, oncogenic transformation, and regulated exocytosis [PMID:7935463, PMID:15766660]. Its Ras-interacting domain adopts a ubiquitin-like fold and engages GTP-bound Ras through an inter-protein beta-sheet between the RalGDS beta-strand and the Ras switch I effector loop, binding only the active conformation and competing with Raf and NF1 for the same surface [PMID:7935463, PMID:9253406, PMID:9628477, PMID:10371160]. Ras engagement recruits RalGDS to the plasma membrane—the RBD raising the membrane association rate while a second, switch-II-dependent contact (Ras Y64) and RalGDS clustering stabilize the productive complex—whereupon RalGDS catalyzes nucleotide exchange on RalA/RalB [PMID:11748241, PMID:28744424]. Membrane recruitment and catalysis are gated by additional inputs: PDK1 binds the RalGDS N-terminus by a kinase-independent mechanism that relieves N-terminal autoinhibition of the catalytic domain, beta-arrestin sequesters RalGDS in an inactive cytosolic complex until receptor stimulation releases it, calmodulin binding through an N-terminal CaM-binding domain is required for Ca2+-stimulated Ral activation, and RILP recruits RalGDS to late endosomes to suppress RalA activity [PMID:11889038, PMID:12105416, PMID:18417737, PMID:26469971]. Beyond exchange activity, RalGDS acts as a scaffold that bridges PDK1 and Akt (the latter via JIP1) to promote Akt phosphorylation and proliferation, a function genetically separable from Ral activation [PMID:18285454]. Downstream, RalGDS-driven Ral signaling phosphorylates ATF2 Thr69 through a Ral-Src-p38 axis and promotes JNK/SAPK-dependent survival of transformed cells [PMID:12110590, PMID:15766660]. Physiologically and pathologically, RalGDS is required for Ras-induced skin carcinogenesis, prostate cancer bone metastasis, endothelial Weibel-Palade body exocytosis, and load-induced cardiomyocyte autophagy and hypertrophy [PMID:15766660, PMID:17709381, PMID:18417737, PMID:23473774].","teleology":[{"year":1994,"claim":"Established that RalGDS is a bona fide Ras effector by showing it binds selectively to GTP-Ras through the effector loop and competes with the canonical effectors Raf and NF1.","evidence":"Yeast two-hybrid, insect-cell co-IP, and in vitro binding/competition assays with GTP/GDP-loaded Ras","pmids":["7935463"],"confidence":"High","gaps":["Did not identify the downstream GTPase target","Did not establish whether binding leads to catalytic output"]},{"year":1996,"claim":"Showed that receptor (EGF) signaling drives the Ras-RalGDS interaction and that cAMP/PKA biases Ras toward RalGDS by lowering Raf affinity, defining a context-dependent effector switch.","evidence":"Co-IP in COS cells, in vitro PKA kinase assay, GEF activity assay with forskolin","pmids":["8550624"],"confidence":"High","gaps":["PKA phosphorylation of RalGDS itself had no functional consequence identified","Physiological setting of the cAMP switch not defined"]},{"year":1997,"claim":"Provided structural and genetic evidence that RalGDS uses a ubiquitin-like Ras-interacting domain and functions as an effector in cAMP/Ras-stimulated growth, distinguishing the Ral arm from the Raf arm.","evidence":"NMR structure with mutagenesis; effector-domain Ras mutant binding plus dominant-negative RalA microinjection and DNA synthesis assay","pmids":["9253406","9038168"],"confidence":"High","gaps":["Atomic interface geometry not yet resolved","Direct demonstration of Ral exchange in the growth response absent"]},{"year":1998,"claim":"Crystal structures defined the Ras-RalGDS interface as an inter-protein beta-sheet at switch I plus a second contact at switch II, giving a structural basis for Ras-Ral cross-talk and effector discrimination.","evidence":"X-ray crystallography of the Ras-RalGDS RID complex at 2.1 A","pmids":["9628477"],"confidence":"High","gaps":["Functional weight of the switch-II contact not yet tested in cells","Did not address membrane recruitment dynamics"]},{"year":1998,"claim":"Demonstrated a biological output for the Ral arm by showing RalGDS/RalA mediates Ras-dependent inhibition of myogenic SRF-driven transcription, separable from Raf.","evidence":"Ras effector-loop mutants, membrane-targeted and Ras-binding-defective RalGDS, luciferase reporter assays","pmids":["9651367"],"confidence":"Medium","gaps":["Single-lab reporter readout","Endogenous RalGDS contribution not tested by loss-of-function"]},{"year":1999,"claim":"Refined the structural model with a second Ras-RalGDS RBD complex and extended Ral-pathway function to Ras-induced endoderm differentiation, linking RalGEF activity to a developmental program requiring basal MEK/ERK.","evidence":"X-ray crystallography, gel filtration, mutagenesis; Rlf-CAAX and dominant-negative RalA in F9 differentiation assay with MEK inhibitor","pmids":["10371160","10442634"],"confidence":"High","gaps":["Mechanism of MEK/ERK cooperation with the Ral arm unresolved","Differentiation study used the RGL/Rlf GEF rather than RalGDS directly"]},{"year":2001,"claim":"Dissected the activation mechanism, showing membrane translocation via the RBD is necessary but not sufficient and that a switch-II (Ras Y64) interaction is additionally required, while Rap1 antagonizes activation by sequestering RalGDS in a long-lived complex.","evidence":"RalA-GTP loading assays, Ras/RalGDS mutant constructs, kinetic binding measurements, membrane-targeted RalGDS","pmids":["11748241"],"confidence":"High","gaps":["Conformational basis of switch-II-dependent activation not resolved","In vivo relevance of Rap1 antagonism not tested"]},{"year":2001,"claim":"Identified transcriptional regulation of RalGDS, showing JAK/STAT3 signaling induces RalGDS expression to drive RalA activation, revealing cross-talk between cytokine and Ras/Ral pathways.","evidence":"RDA cloning, dominant-negative STAT3, JAK and Ras inhibitors, Ral-GTP loading assay in M1 leukemia cells","pmids":["11432872"],"confidence":"Medium","gaps":["Single-lab study","Direct STAT3 occupancy of the RALGDS promoter not shown"]},{"year":2002,"claim":"Uncovered allosteric regulation by PDK1, which relieves RalGDS N-terminal autoinhibition through a kinase-independent interaction, integrating the PI3K-PDK1 and RalGEF effector pathways.","evidence":"Co-IP, kinase-dead PDK1, RalGDS N-terminal deletions, Ral-GTP loading assay","pmids":["11889038"],"confidence":"High","gaps":["Structural basis of autoinhibition release not defined","Stoichiometry and cellular dynamics of the PDK1-RalGDS complex unresolved"]},{"year":2002,"claim":"Mapped specific RalGDS-dependent downstream outputs—choline kinase activation and ATF2 Thr69 phosphorylation via a Ral-Src-p38 axis—distinguishing them from Raf-MEK-ERK signaling.","evidence":"Ras effector-domain mutants, phospho-site-specific ATF2 antibodies, pathway mutants and inhibitors","pmids":["11840339","12110590"],"confidence":"High","gaps":["Direct enzymatic link between Ral and Src/choline kinase not established","Endogenous RalGDS requirement not tested by knockout"]},{"year":2002,"claim":"Defined beta-arrestin as a negative regulator that holds RalGDS inactive in the cytosol and releases it for membrane translocation and Ras-independent Ral activation upon receptor stimulation.","evidence":"Yeast two-hybrid, co-IP from PMNs, translocation/fractionation assay, dominant-negative Ral, cytoskeletal readout","pmids":["12105416"],"confidence":"High","gaps":["Structural basis of beta-arrestin sequestration unknown","Generality beyond fMLP/GPCR receptors not tested"]},{"year":2004,"claim":"Extended the RalGEF/p38 pathway to host-pathogen biology, showing it dictates cellular permissiveness to reovirus infection downstream of Ras.","evidence":"Ras effector-domain mutants, activated Rlf, dominant-negative RalA, p38 inhibitor, viral replication assay","pmids":["15263068"],"confidence":"High","gaps":["Molecular target of p38 enabling viral replication unknown","Used the Rlf GEF as a tool rather than endogenous RalGDS"]},{"year":2005,"claim":"Provided in vivo proof that RalGDS is required for Ras-induced tumorigenesis, controlling survival rather than proliferation of transformed cells through JNK/SAPK activation.","evidence":"Ralgds-/- mice in DMBA/TPA skin carcinogenesis, transformation and survival assays, JNK activity measurement","pmids":["15766660"],"confidence":"High","gaps":["Mechanism linking RalGDS to JNK not fully defined","Whether the survival role is Ral-dependent not isolated"]},{"year":2007,"claim":"Showed the RalGEF/Ral arm specifically drives prostate cancer bone metastasis, distinct from primary tumor growth and from Raf/PI3K signaling.","evidence":"Ras effector-domain mutants, RalA RNAi, intracardiac metastasis and subcutaneous growth assays","pmids":["17709381"],"confidence":"High","gaps":["Effector(s) of RalA mediating bone-specific growth unknown","Direct requirement for RalGDS (vs other RalGEFs) not isolated"]},{"year":2008,"claim":"Revealed two regulatory and functional dimensions of RalGDS: a Ca2+/calmodulin requirement for Ral activation driving Weibel-Palade body exocytosis, and a scaffolding function bridging PDK1 and Akt (via JIP1) to promote Akt phosphorylation and proliferation independent of Ral exchange.","evidence":"RNAi, overexpression, dominant-negative and deletion mutants, calmodulin pulldown and CaM-peptide inhibitor, WPB exocytosis and Ral-GTP assays; reciprocal co-IP and domain mapping with phospho-Akt and proliferation readouts","pmids":["18417737","18285454"],"confidence":"High","gaps":["How CaM binding couples to catalytic activation not structurally resolved","Whether scaffolding and GEF functions are spatially partitioned unclear"]},{"year":2013,"claim":"Established a physiological role for RalGDS in the heart, where it is required for load-induced cardiomyocyte autophagy and hypertrophy via RalB-mediated mTOR-dependent autophagy.","evidence":"Ralgds-/- mice with transverse aortic constriction, RalA/RalB RNAi in cardiomyocytes, autophagy markers and echocardiography","pmids":["23473774"],"confidence":"High","gaps":["Upstream signal activating RalGDS under pressure overload unknown","Link between RalB and mTOR mechanistically undefined"]},{"year":2015,"claim":"Identified RILP as a spatial regulator that recruits RalGDS to late endosomes through its GEF domain to suppress RalA activity and tumor cell invasion.","evidence":"Co-IP, truncation mapping, immunofluorescence, RalA-GTP and invasion assays, RNAi","pmids":["26469971"],"confidence":"Medium","gaps":["Single-lab study","How endosomal localization mechanistically dampens GEF activity unresolved"]},{"year":2017,"claim":"Resolved the membrane recruitment kinetics at single-molecule resolution, showing the RBD accelerates RalGDS membrane association while the REMCDC domain slows dissociation and Ras Y64 plus RalGDS clustering stabilize the Ral-engaged state.","evidence":"Single-molecule fluorescence imaging of GFP-tagged RalGDS domains in living HeLa cells with EGF stimulation","pmids":["28744424"],"confidence":"Medium","gaps":["Single technique, single lab","Functional consequence of clustering for Ral exchange rate not quantified"]},{"year":2019,"claim":"Demonstrated in vivo that the RalGEF ortholog orchestrates two genetically separable outputs—GEF-dependent Ral activation and GEF-independent PI3K-PDK-AKT signaling—to reduce developmental stochasticity, mirroring the dual Ral/scaffold roles seen in mammals.","evidence":"C. elegans genetic epistasis with GEF-dead RGL-1 mutants and PI3K/PDK pathway double mutants, VPC fate scoring","pmids":["31086367"],"confidence":"Medium","gaps":["Ortholog study; mammalian RalGDS not directly tested here","Molecular basis of the GEF-independent activity in this context not defined"]},{"year":null,"claim":"How the multiple regulatory inputs (PDK1 autoinhibition relief, CaM, beta-arrestin, RILP, clustering) are integrated to set RalGDS catalytic output in a given cellular context, and the structural basis of switch-II-dependent activation, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural model of autoinhibited vs activated RalGDS","Spatial/temporal coordination of GEF versus Akt-scaffold functions not resolved","Crosstalk hierarchy among regulators in vivo unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,8,10,16]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,8]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[17]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8,9,21]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[9]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[19]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,8,12]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[14,15]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[18]}],"complexes":[],"partners":["HRAS","RALA","RALB","PDPK1","AKT1","CALM1","RILP","ARRB2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q12967","full_name":"Ral guanine nucleotide dissociation stimulator","aliases":["Ral guanine nucleotide exchange factor","RalGEF"],"length_aa":914,"mass_kda":100.6,"function":"Functions as a guanine nucleotide exchange factor (GEF) activating either RalA or RalB GTPases and plays an important role in intracellular transport. Interacts and acts as an effector molecule for R-Ras, H-Ras, K-Ras, and Rap (By similarity). During bacterial clearance, recognizes 'Lys-33'-linked polyubiquitinated TRAF3 and subsequently mediates assembly of the exocyst complex (PubMed:27438768)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q12967/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RALGDS","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RALGDS","total_profiled":1310},"omim":[{"mim_id":"616743","title":"RAL GUANINE NUCLEOTIDE DISSOCIATION STIMULATOR-LIKE 3; RGL3","url":"https://www.omim.org/entry/616743"},{"mim_id":"612214","title":"RAL GUANINE NUCLEOTIDE DISSOCIATION STIMULATOR-LIKE 4; RGL4","url":"https://www.omim.org/entry/612214"},{"mim_id":"609592","title":"RIC-LIKE PROTEIN WITHOUT CAAX MOTIF 2; RIT2","url":"https://www.omim.org/entry/609592"},{"mim_id":"609591","title":"RIC-LIKE PROTEIN WITHOUT CAAX MOTIF 1; RIT1","url":"https://www.omim.org/entry/609591"},{"mim_id":"605801","title":"RALA-BINDING PROTEIN 1; RALBP1","url":"https://www.omim.org/entry/605801"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nucleoplasm","reliability":"Uncertain"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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RalGDS inhibited both NF1 GAP activity toward Ras and the interaction of Raf with Ras in vitro, indicating it competes with Raf and NF1 for the Ras effector loop.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation in insect cells, in vitro binding/competition assays with GTP/GDP-loaded Ras\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (yeast two-hybrid, insect-cell co-IP, in vitro binding), replicated across assay systems in one study\",\n      \"pmids\": [\"7935463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"EGF induces the interaction of c-Ras and RalGDS in mammalian COS cells in an effector-loop-dependent manner. Cyclic AMP-dependent protein kinase (PKA) phosphorylates RalGDS but does not alter its affinity for Ras or its GDP/GTP exchange activity toward Ral p24. PKA phosphorylation of (1-149)Raf reduces its affinity for Ras, thereby shifting Ras binding selectively toward RalGDS under cAMP-elevated conditions.\",\n      \"method\": \"Co-immunoprecipitation in COS cells, in vitro kinase assay (PKA phosphorylation), GEF activity assay, forskolin treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP plus in vitro biochemical assays, multiple orthogonal methods in one study\",\n      \"pmids\": [\"8550624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"RalGDS functions as an effector of Ras in cAMP-mediated growth stimulation in thyroid cells. A Ras effector-domain mutant (RasV12G37) defective in Raf-1 binding still bound RalGDS by yeast two-hybrid and co-immunoprecipitation. Microinjection of dominant-negative RalA (which sequesters RalGDS family members) reduced Ras-stimulated and cAMP-stimulated DNA synthesis.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation in thyroid cell extracts, microinjection of dominant-negative RalA, DNA synthesis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (yeast two-hybrid, co-IP, microinjection phenotypic assay) in one study\",\n      \"pmids\": [\"9038168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The Ras-interacting domain (RID) of RalGDS adopts an ubiquitin-like fold. Mutational analysis identified three residues in the RID critical for interaction with Ras.\",\n      \"method\": \"NMR structure determination, site-directed mutagenesis, binding assays\",\n      \"journal\": \"Nature structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure plus mutagenesis validation, single lab\",\n      \"pmids\": [\"9253406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Crystal structure (2.1 Å) of the Ras–RalGDS RID complex reveals that the beta-sheet of the RID joins the switch I region of Ras to form an extended beta-sheet, analogous to but geometrically distinct from the Rap-Raf complex. A second RID molecule also contacts the switch II region of Ras, providing a structural basis for cross-talk between the Ras and Ral pathways.\",\n      \"method\": \"X-ray crystallography at 2.1 Å resolution\",\n      \"journal\": \"Nature structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure, independently consistent with other structural and biochemical studies\",\n      \"pmids\": [\"9628477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"RalGDS mediates Ras-dependent inhibition of skeletal myogenesis. An H-Ras effector mutant (G12V,E37G) that binds RalGDS but not Raf inhibits muscle-specific reporter gene activity. Membrane-targeted RalGDS (RalGDS-CAAX) also inhibits alpha-actin-Luc and SRF-dependent transcription, while a RalGDS mutant defective in Ras interaction has no effect. RalGDS and activated RalA additionally inhibit the SRF-dependent component of H-Ras G12V,E37G-induced transcription.\",\n      \"method\": \"Transient transfection of Ras effector-loop mutants and RalGDS constructs, luciferase reporter assays, dominant-negative and constitutively active Ral constructs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean loss-of-function (Ras-interaction-defective RalGDS as control) plus gain-of-function with membrane-targeted construct, single lab\",\n      \"pmids\": [\"9651367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"X-ray crystal structure of the Ras–RalGDS RBD (RGS-RBD) complex shows an inter-protein beta-sheet between Ras switch I and the RGS-RBD beta-strand, analogous to Rap-RafRBD but with markedly different interface side-chain interactions. Gel filtration confirms the complex is a 1:1 monomer. Mutational studies define interface residues contributing to binding affinity.\",\n      \"method\": \"X-ray crystallography, size-exclusion chromatography, site-directed mutagenesis with binding affinity measurements\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis and biochemical validation, consistent with independent structure from Huang et al. 1998\",\n      \"pmids\": [\"10371160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"RalGEFs (specifically Rlf/RGL) are required for Ras-induced primitive endoderm differentiation of F9 embryonal carcinoma cells. Constitutively active Rlf-CAAX is sufficient to induce differentiation; dominant-negative RalA completely abolishes Rlf-CAAX– and RasV12G37-induced differentiation. RalGEF-induced differentiation requires basal MEK/ERK activity (MEK inhibition blocks it), but Rlf-CAAX itself does not increase ERK activity.\",\n      \"method\": \"Ras effector-domain mutants, constitutively active RalGEF (Rlf-CAAX), dominant-negative RalA, MEK inhibitor (PD98059), morphological differentiation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis with multiple genetic tools, single lab\",\n      \"pmids\": [\"10442634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"RalGDS activation by Ras involves membrane translocation (RBD-dependent) but additionally requires a Ras switch II interaction: Ras Y64W is impaired in RalGDS activation but not in RBD binding. An artificially membrane-targeted RalGDS lacking its RBD can still be activated by Ras, indicating the RBD is important but not sufficient. Rap1 antagonizes Ras-mediated RalGDS activation only when the RBD is intact, by forming a long-lived complex (KD ~0.6 μM) that sequesters RalGDS, whereas the Ras×RalGDS complex is short-lived (~0.1 s).\",\n      \"method\": \"RalA-GTP loading assay, Ras/RalGDS mutant constructs, kinetic binding measurements (surface plasmon resonance/stopped-flow), membrane-targeted RalGDS constructs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro biochemical assays with GTP measurement, kinetic studies, and multiple mutant constructs converging on same conclusion\",\n      \"pmids\": [\"11748241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Beta-arrestins bind RalGDS (identified by yeast two-hybrid and co-immunoprecipitation from human PMNs). Under basal conditions, RalGDS is held inactive in a cytosolic complex with beta-arrestin. Upon fMLP receptor stimulation, the complex dissociates, RalGDS translocates to the plasma membrane, and Ral is activated independently of Ras, driving cytoskeletal reorganization. Re-association of the complex correlates with Ral signal termination.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation from PMNs, subcellular fractionation/translocation assay, dominant-negative Ral constructs, cytoskeletal phenotype readout\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP plus translocation imaging plus functional (cytoskeletal) readout, multiple orthogonal methods\",\n      \"pmids\": [\"12105416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"PDK1 enhances RalGDS catalytic (GEF) activity by a kinase-independent mechanism. The non-catalytic N-terminus of PDK1 forms an EGF-induced complex with the N-terminus of RalGDS, relieving its auto-inhibitory effect on the catalytic domain. This represents cooperation between the PI3K-PDK1 Ras effector pathway and the RalGEF pathway to promote Ral activation.\",\n      \"method\": \"Co-immunoprecipitation, kinase-dead PDK1 constructs, Ral-GTP loading assay, N-terminal deletion mutants of RalGDS\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple mutant constructs, kinase-dead controls, GTP-loading readout, co-IP; multiple orthogonal approaches in one study\",\n      \"pmids\": [\"11889038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Ras regulates choline kinase activity through its direct effectors RalGDS and PI3K, but not through Raf-1. Ras effector-domain mutants specifically activating RalGDS (RasV12G37) or PI3K (RasV12C40), but not Raf (RasV12S35), activated choline kinase, establishing RalGDS as a required intermediary.\",\n      \"method\": \"Ras effector-loop mutants (RasV12G37, V12C40, V12S35), choline kinase activity assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic dissection with effector-domain mutants, single lab, single primary readout\",\n      \"pmids\": [\"11840339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Growth factors activate ATF2 Thr69 phosphorylation via a Ral-RalGDS-Src-p38 pathway, distinct from the Raf-MEK-ERK pathway that phosphorylates Thr71. Cooperation between ERK and p38 is required for full ATF2 dual-phosphorylation by growth factors such as insulin and EGF.\",\n      \"method\": \"Phosphorylation-site-specific ATF2 antibodies, dominant-negative and constitutively active pathway constructs, kinase inhibitors, Ras effector-domain mutants\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal approaches (specific phospho-antibodies, pathway mutants, inhibitors), two-step mechanism validated\",\n      \"pmids\": [\"12110590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The Ras/RalGEF/p38 pathway dictates host cell permissiveness to reovirus infection. Ras effector-domain mutant RasV12G37 (retaining RalGEF signaling but not Raf or PI3K) renders NIH 3T3 cells permissive. Activated RalGEF (Rlf) alone confers permissiveness; dominant-negative RalA renders H-Ras cells resistant. p38 inhibition blocks reovirus replication downstream of RalGEF.\",\n      \"method\": \"Ras effector-domain mutants, activated RalGEF (Rlf) overexpression, dominant-negative RalA, p38/JNK inhibitors, viral replication assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple tools converging on RalGEF→p38 as the required pathway, functional viral replication readout\",\n      \"pmids\": [\"15263068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RalGDS is required for Ras-induced oncogenesis in vivo. RalGDS-knockout mice develop normally but show reduced tumor incidence, size, and malignant progression in multistage skin carcinogenesis. RalGDS does not regulate cell proliferation but controls survival of transformed cells, apparently through activation of the JNK/SAPK pathway.\",\n      \"method\": \"Ralgds-/- knockout mice, DMBA/TPA skin carcinogenesis model, tissue culture transformation assays, apoptosis/survival assays, JNK activity measurements in tumor-derived cells\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic knockout with multiple phenotypic readouts and mechanistic follow-up (JNK activity); independently discussed by Rodriguez-Viciana & McCormick 2005\",\n      \"pmids\": [\"15766660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Activation of the RalGEF/Ral pathway promotes prostate cancer bone metastasis. In DU145 cells, activation of the RalGEF pathway (RasV12G37) but not Raf/ERK or PI3K promotes bone metastasis. Loss of RalA in metastatic PC3 cells inhibits bone metastasis but not subcutaneous tumor growth, specifically suppressing expansive growth in bone.\",\n      \"method\": \"Ras effector-domain mutants, RalA RNAi knockdown, in vivo metastasis assay (intracardiac injection), subcutaneous tumor growth assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic dissection with effector mutants plus RNAi knockdown in vivo, specific organ-metastasis readout\",\n      \"pmids\": [\"17709381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RalGDS mediates thrombin- and epinephrine-induced exocytosis of Weibel-Palade bodies (WPBs) from endothelial cells. RNAi knockdown of RalGDS inhibits WPB exocytosis; overexpression promotes it; a dominant-negative exchange-domain-deleted RalGDS blocks WPB release. RalGDS binds calmodulin (CaM) via an N-terminal CaM-binding domain, and a cell-permeable peptide comprising this domain inhibits Ral activation and WPB exocytosis.\",\n      \"method\": \"RNAi knockdown, overexpression, dominant-negative mutant, calmodulin pulldown, cell-permeable peptide inhibitor, WPB exocytosis assay (VWF secretion), Ral-GTP loading assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal loss- and gain-of-function approaches with defined molecular mechanism (CaM binding domain), single lab\",\n      \"pmids\": [\"18417737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RalGDS has a second function beyond Ral GEF activity: it promotes Akt phosphorylation by PDK1 by acting as a scaffold that brings PDK1 and Akt together. The N-terminus of RalGDS complexes PDK1 while the central region associates with Akt via JIP1. An N-terminally deleted RalGDS retains Ral activation but cannot promote Akt phosphorylation and fails to support cell proliferation.\",\n      \"method\": \"RNAi suppression of RalGDS, co-immunoprecipitation (PDK1/RalGDS, Akt/RalGDS via JIP1), deletion mutant analysis, phospho-Akt immunoblot, cell proliferation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNAi knockdown, reciprocal co-IP, domain-deletion mapping, functional proliferation readout; multiple orthogonal methods\",\n      \"pmids\": [\"18285454\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RalGDS-mediated cardiomyocyte autophagy is required for load-induced cardiac hypertrophy. RalGDS-null hearts show blunted hypertrophic growth and suppressed autophagy in response to pressure-overload (TAC), while ventricular function is preserved and fetal gene program activation is unaffected. In NRCMs, RalB (a downstream target of RalGDS) specifically mediates mTOR-dependent autophagy.\",\n      \"method\": \"Ralgds-/- knockout mice, transverse aortic constriction (TAC) model, RalA/RalB RNAi in neonatal rat cardiomyocytes, autophagy markers (LC3, p62), echocardiography, fetal gene expression\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knockout with pressure-overload model plus in vitro RNAi epistasis, multiple phenotypic readouts\",\n      \"pmids\": [\"23473774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RILP (Rab7-interacting lysosomal protein) interacts with RalGDS via its N-terminal region binding to the GEF domain of RalGDS. RILP overexpression recruits RalGDS to late endosomal compartments and inhibits RalA activity and breast cancer cell invasion; RILP knockdown promotes invasion. The interaction is confirmed by co-immunoprecipitation and truncation analysis.\",\n      \"method\": \"Co-immunoprecipitation, truncation/deletion mapping, immunofluorescence microscopy, RalA-GTP loading assay, cell invasion assay, RNAi knockdown\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP with domain mapping, subcellular localization imaging, functional GEF-activity readout; single lab\",\n      \"pmids\": [\"26469971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"JAK/STAT3 activation in M1 myeloid leukemia cells induces RalGDS expression, leading to RalA-GTP activation. Dominant-negative STAT3 and the JAK inhibitor JAB/SOCS1 suppress both RalGDS expression and RalA activation. Full RalA activation also requires Ras activity, revealing cross-talk between the JAK/STAT3 and Ras/RalGDS/Ral pathways.\",\n      \"method\": \"Representational difference analysis (RalGDS cloning), dominant-negative STAT3, JAK inhibitor, Ras inhibitor, Ral-GTP loading assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RalGDS identified as STAT3 target, functional Ral-GTP readout with pathway inhibitors; single lab\",\n      \"pmids\": [\"11432872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Single-molecule fluorescence imaging shows that EGF-induced Ras activation increases the membrane density of RalGDS via its RBD (not the REMCDC domain). The RBD increases the RalGDS association rate to the membrane, while the REMCDC domain decreases the dissociation rate after Ras activation or Ral overexpression. Ras Y64 residue and RalGDS cluster formation on the membrane are involved in stabilizing RalGDS–Ral interaction upon Ras activation.\",\n      \"method\": \"Single-molecule fluorescence imaging, GFP-tagged RalGDS domain constructs in living HeLa cells, EGF stimulation, membrane density quantitation, rate constant measurements\",\n      \"journal\": \"Biophysics and physicobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct live-cell single-molecule imaging of RalGDS translocation with domain dissection; single lab, single technique\",\n      \"pmids\": [\"28744424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In C. elegans, the RalGEF ortholog RGL-1 plays two genetically separable roles in vulval cell fate patterning: (1) a canonical GEF-dependent promotion of 2° fate via RAL-1 activation, and (2) a non-canonical GEF-independent promotion of 1° fate that acts through the AGE-1/PI3K-PDK-1-AKT-1 cascade. Loss of RGL-1 increases VPC patterning error rates 15-fold, indicating RGL-1 orchestrates opposing modulatory cascades to reduce developmental stochasticity.\",\n      \"method\": \"C. elegans genetic epistasis, RGL-1 GEF-dead mutants, double-mutant analysis with PI3K/PDK pathway components, VPC fate scoring\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — rigorous C. elegans genetic epistasis with GEF-dead separation-of-function mutants; ortholog study, single lab\",\n      \"pmids\": [\"31086367\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RALGDS is a guanine nucleotide exchange factor (GEF) that is recruited to the plasma membrane by binding its Ras-binding domain (RBD) to GTP-Ras (via the Ras switch I effector loop), whereupon it activates Ral GTPases (RalA/RalB); its catalytic activity can be further enhanced by a kinase-independent PDK1 interaction that relieves autoinhibition; additional regulation is provided by beta-arrestin sequestration in the cytosol, calmodulin binding required for Ca²⁺-stimulated Ral activation and Weibel-Palade body exocytosis, and RILP-mediated recruitment to late endosomes to suppress RalA activity; beyond Ral activation, RalGDS scaffolds PDK1 and Akt (via JIP1) to promote Akt phosphorylation and cell survival, and mediates Ras-driven oncogenesis, bone metastasis, cardiomyocyte autophagy/hypertrophy, and ATF2 Thr69 phosphorylation through a Ral-Src-p38 axis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RALGDS is a Ras effector and guanine-nucleotide exchange factor that couples activated Ras to the Ral GTPase branch of Ras signaling, thereby controlling cell survival, oncogenic transformation, and regulated exocytosis [#0, #14]. Its Ras-interacting domain adopts a ubiquitin-like fold and engages GTP-bound Ras through an inter-protein beta-sheet between the RalGDS beta-strand and the Ras switch I effector loop, binding only the active conformation and competing with Raf and NF1 for the same surface [#0, #3, #4, #6]. Ras engagement recruits RalGDS to the plasma membrane—the RBD raising the membrane association rate while a second, switch-II-dependent contact (Ras Y64) and RalGDS clustering stabilize the productive complex—whereupon RalGDS catalyzes nucleotide exchange on RalA/RalB [#8, #21]. Membrane recruitment and catalysis are gated by additional inputs: PDK1 binds the RalGDS N-terminus by a kinase-independent mechanism that relieves N-terminal autoinhibition of the catalytic domain, beta-arrestin sequesters RalGDS in an inactive cytosolic complex until receptor stimulation releases it, calmodulin binding through an N-terminal CaM-binding domain is required for Ca2+-stimulated Ral activation, and RILP recruits RalGDS to late endosomes to suppress RalA activity [#10, #9, #16, #19]. Beyond exchange activity, RalGDS acts as a scaffold that bridges PDK1 and Akt (the latter via JIP1) to promote Akt phosphorylation and proliferation, a function genetically separable from Ral activation [#17]. Downstream, RalGDS-driven Ral signaling phosphorylates ATF2 Thr69 through a Ral-Src-p38 axis and promotes JNK/SAPK-dependent survival of transformed cells [#12, #14]. Physiologically and pathologically, RalGDS is required for Ras-induced skin carcinogenesis, prostate cancer bone metastasis, endothelial Weibel-Palade body exocytosis, and load-induced cardiomyocyte autophagy and hypertrophy [#14, #15, #16, #18].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established that RalGDS is a bona fide Ras effector by showing it binds selectively to GTP-Ras through the effector loop and competes with the canonical effectors Raf and NF1.\",\n      \"evidence\": \"Yeast two-hybrid, insect-cell co-IP, and in vitro binding/competition assays with GTP/GDP-loaded Ras\",\n      \"pmids\": [\"7935463\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the downstream GTPase target\", \"Did not establish whether binding leads to catalytic output\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Showed that receptor (EGF) signaling drives the Ras-RalGDS interaction and that cAMP/PKA biases Ras toward RalGDS by lowering Raf affinity, defining a context-dependent effector switch.\",\n      \"evidence\": \"Co-IP in COS cells, in vitro PKA kinase assay, GEF activity assay with forskolin\",\n      \"pmids\": [\"8550624\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PKA phosphorylation of RalGDS itself had no functional consequence identified\", \"Physiological setting of the cAMP switch not defined\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Provided structural and genetic evidence that RalGDS uses a ubiquitin-like Ras-interacting domain and functions as an effector in cAMP/Ras-stimulated growth, distinguishing the Ral arm from the Raf arm.\",\n      \"evidence\": \"NMR structure with mutagenesis; effector-domain Ras mutant binding plus dominant-negative RalA microinjection and DNA synthesis assay\",\n      \"pmids\": [\"9253406\", \"9038168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic interface geometry not yet resolved\", \"Direct demonstration of Ral exchange in the growth response absent\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Crystal structures defined the Ras-RalGDS interface as an inter-protein beta-sheet at switch I plus a second contact at switch II, giving a structural basis for Ras-Ral cross-talk and effector discrimination.\",\n      \"evidence\": \"X-ray crystallography of the Ras-RalGDS RID complex at 2.1 A\",\n      \"pmids\": [\"9628477\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional weight of the switch-II contact not yet tested in cells\", \"Did not address membrane recruitment dynamics\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstrated a biological output for the Ral arm by showing RalGDS/RalA mediates Ras-dependent inhibition of myogenic SRF-driven transcription, separable from Raf.\",\n      \"evidence\": \"Ras effector-loop mutants, membrane-targeted and Ras-binding-defective RalGDS, luciferase reporter assays\",\n      \"pmids\": [\"9651367\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab reporter readout\", \"Endogenous RalGDS contribution not tested by loss-of-function\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Refined the structural model with a second Ras-RalGDS RBD complex and extended Ral-pathway function to Ras-induced endoderm differentiation, linking RalGEF activity to a developmental program requiring basal MEK/ERK.\",\n      \"evidence\": \"X-ray crystallography, gel filtration, mutagenesis; Rlf-CAAX and dominant-negative RalA in F9 differentiation assay with MEK inhibitor\",\n      \"pmids\": [\"10371160\", \"10442634\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of MEK/ERK cooperation with the Ral arm unresolved\", \"Differentiation study used the RGL/Rlf GEF rather than RalGDS directly\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Dissected the activation mechanism, showing membrane translocation via the RBD is necessary but not sufficient and that a switch-II (Ras Y64) interaction is additionally required, while Rap1 antagonizes activation by sequestering RalGDS in a long-lived complex.\",\n      \"evidence\": \"RalA-GTP loading assays, Ras/RalGDS mutant constructs, kinetic binding measurements, membrane-targeted RalGDS\",\n      \"pmids\": [\"11748241\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational basis of switch-II-dependent activation not resolved\", \"In vivo relevance of Rap1 antagonism not tested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified transcriptional regulation of RalGDS, showing JAK/STAT3 signaling induces RalGDS expression to drive RalA activation, revealing cross-talk between cytokine and Ras/Ral pathways.\",\n      \"evidence\": \"RDA cloning, dominant-negative STAT3, JAK and Ras inhibitors, Ral-GTP loading assay in M1 leukemia cells\",\n      \"pmids\": [\"11432872\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"Direct STAT3 occupancy of the RALGDS promoter not shown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Uncovered allosteric regulation by PDK1, which relieves RalGDS N-terminal autoinhibition through a kinase-independent interaction, integrating the PI3K-PDK1 and RalGEF effector pathways.\",\n      \"evidence\": \"Co-IP, kinase-dead PDK1, RalGDS N-terminal deletions, Ral-GTP loading assay\",\n      \"pmids\": [\"11889038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of autoinhibition release not defined\", \"Stoichiometry and cellular dynamics of the PDK1-RalGDS complex unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapped specific RalGDS-dependent downstream outputs—choline kinase activation and ATF2 Thr69 phosphorylation via a Ral-Src-p38 axis—distinguishing them from Raf-MEK-ERK signaling.\",\n      \"evidence\": \"Ras effector-domain mutants, phospho-site-specific ATF2 antibodies, pathway mutants and inhibitors\",\n      \"pmids\": [\"11840339\", \"12110590\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymatic link between Ral and Src/choline kinase not established\", \"Endogenous RalGDS requirement not tested by knockout\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined beta-arrestin as a negative regulator that holds RalGDS inactive in the cytosol and releases it for membrane translocation and Ras-independent Ral activation upon receptor stimulation.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP from PMNs, translocation/fractionation assay, dominant-negative Ral, cytoskeletal readout\",\n      \"pmids\": [\"12105416\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of beta-arrestin sequestration unknown\", \"Generality beyond fMLP/GPCR receptors not tested\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Extended the RalGEF/p38 pathway to host-pathogen biology, showing it dictates cellular permissiveness to reovirus infection downstream of Ras.\",\n      \"evidence\": \"Ras effector-domain mutants, activated Rlf, dominant-negative RalA, p38 inhibitor, viral replication assay\",\n      \"pmids\": [\"15263068\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular target of p38 enabling viral replication unknown\", \"Used the Rlf GEF as a tool rather than endogenous RalGDS\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Provided in vivo proof that RalGDS is required for Ras-induced tumorigenesis, controlling survival rather than proliferation of transformed cells through JNK/SAPK activation.\",\n      \"evidence\": \"Ralgds-/- mice in DMBA/TPA skin carcinogenesis, transformation and survival assays, JNK activity measurement\",\n      \"pmids\": [\"15766660\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking RalGDS to JNK not fully defined\", \"Whether the survival role is Ral-dependent not isolated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed the RalGEF/Ral arm specifically drives prostate cancer bone metastasis, distinct from primary tumor growth and from Raf/PI3K signaling.\",\n      \"evidence\": \"Ras effector-domain mutants, RalA RNAi, intracardiac metastasis and subcutaneous growth assays\",\n      \"pmids\": [\"17709381\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effector(s) of RalA mediating bone-specific growth unknown\", \"Direct requirement for RalGDS (vs other RalGEFs) not isolated\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed two regulatory and functional dimensions of RalGDS: a Ca2+/calmodulin requirement for Ral activation driving Weibel-Palade body exocytosis, and a scaffolding function bridging PDK1 and Akt (via JIP1) to promote Akt phosphorylation and proliferation independent of Ral exchange.\",\n      \"evidence\": \"RNAi, overexpression, dominant-negative and deletion mutants, calmodulin pulldown and CaM-peptide inhibitor, WPB exocytosis and Ral-GTP assays; reciprocal co-IP and domain mapping with phospho-Akt and proliferation readouts\",\n      \"pmids\": [\"18417737\", \"18285454\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CaM binding couples to catalytic activation not structurally resolved\", \"Whether scaffolding and GEF functions are spatially partitioned unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established a physiological role for RalGDS in the heart, where it is required for load-induced cardiomyocyte autophagy and hypertrophy via RalB-mediated mTOR-dependent autophagy.\",\n      \"evidence\": \"Ralgds-/- mice with transverse aortic constriction, RalA/RalB RNAi in cardiomyocytes, autophagy markers and echocardiography\",\n      \"pmids\": [\"23473774\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signal activating RalGDS under pressure overload unknown\", \"Link between RalB and mTOR mechanistically undefined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified RILP as a spatial regulator that recruits RalGDS to late endosomes through its GEF domain to suppress RalA activity and tumor cell invasion.\",\n      \"evidence\": \"Co-IP, truncation mapping, immunofluorescence, RalA-GTP and invasion assays, RNAi\",\n      \"pmids\": [\"26469971\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"How endosomal localization mechanistically dampens GEF activity unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved the membrane recruitment kinetics at single-molecule resolution, showing the RBD accelerates RalGDS membrane association while the REMCDC domain slows dissociation and Ras Y64 plus RalGDS clustering stabilize the Ral-engaged state.\",\n      \"evidence\": \"Single-molecule fluorescence imaging of GFP-tagged RalGDS domains in living HeLa cells with EGF stimulation\",\n      \"pmids\": [\"28744424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single technique, single lab\", \"Functional consequence of clustering for Ral exchange rate not quantified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated in vivo that the RalGEF ortholog orchestrates two genetically separable outputs—GEF-dependent Ral activation and GEF-independent PI3K-PDK-AKT signaling—to reduce developmental stochasticity, mirroring the dual Ral/scaffold roles seen in mammals.\",\n      \"evidence\": \"C. elegans genetic epistasis with GEF-dead RGL-1 mutants and PI3K/PDK pathway double mutants, VPC fate scoring\",\n      \"pmids\": [\"31086367\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ortholog study; mammalian RalGDS not directly tested here\", \"Molecular basis of the GEF-independent activity in this context not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple regulatory inputs (PDK1 autoinhibition relief, CaM, beta-arrestin, RILP, clustering) are integrated to set RalGDS catalytic output in a given cellular context, and the structural basis of switch-II-dependent activation, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural model of autoinhibited vs activated RalGDS\", \"Spatial/temporal coordination of GEF versus Akt-scaffold functions not resolved\", \"Crosstalk hierarchy among regulators in vivo unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 8, 10, 16]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8, 9, 21]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 8, 12]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [14, 15]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"HRAS\", \"RALA\", \"RALB\", \"PDPK1\", \"AKT1\", \"CALM1\", \"RILP\", \"ARRB2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}