{"gene":"RRAGD","run_date":"2026-04-28T20:42:06","timeline":{"discoveries":[{"year":2021,"finding":"Heterozygous missense variants in RRAGD cause constitutive activation of mTORC1 signaling in vitro, establishing that RagD GTPase mediates amino acid signaling to mTORC1, and that its dysregulation leads to kidney tubulopathy (hypomagnesemia, hypokalemia, nephrocalcinosis) and dilated cardiomyopathy (ADKH-RRAGD syndrome). RagD expression was confirmed along the mammalian nephron including the thick ascending limb and distal convoluted tubule.","method":"Whole-exome/genome sequencing of patient cohort, in vitro functional analyses of RRAGD variants measuring mTOR signaling activation, immunolocalization along nephron segments","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods (patient genetics, in vitro mTOR activation assays, localization), replicated across multiple families and subsequent studies","pmids":["34607910"],"is_preprint":false},{"year":2023,"finding":"RRAGD auto-activating mutations (causing kidney tubulopathy and cardiomyopathy) constitutively activate a non-canonical mTORC1 signaling branch that specifically phosphorylates TFEB and TFE3 (without affecting canonical substrates like S6K), even in the absence of Folliculin (the GAP responsible for RagC/D activation), thereby inhibiting nuclear translocation and transcriptional activity of TFEB/TFE3 and impairing responses to lysosomal and mitochondrial injury.","method":"In vitro assays in HeLa and HK-2 cell lines, human iPSC-derived cardiomyocytes, patient-derived primary fibroblasts; TFEB/TFE3 phosphorylation assays; nuclear translocation imaging; Folliculin knockout controls","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1-2 — multiple cell models including patient-derived fibroblasts and iPSC cardiomyocytes, mechanistic dissection of canonical vs. non-canonical mTORC1 substrates, Folliculin-independent activation shown","pmids":["37188688"],"is_preprint":false},{"year":2018,"finding":"TFEB-driven endocytosis promotes assembly of MTORC1-containing nutrient-sensing complexes through formation of endosomes that carry RRAGD, the amino acid transporter SLC38A9, and activate AKT; these TFEB-induced signaling endosomes en route to lysosomes are required to dissociate TSC2 and re-tether/activate MTORC1 on endolysosomal membranes.","method":"Immunofluorescence colocalization, endocytosis rate assays, MTORC1 activity measurements, TSC2 dissociation assays, overexpression and starvation experiments in CAD and HEK293T cells","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple methods (imaging, signaling assays, genetic manipulation) in a single lab; RRAGD role on endosomes shown by colocalization and functional rescue","pmids":["30145926"],"is_preprint":false},{"year":2021,"finding":"Loss of FLCN or its binding partners FNIP1/FNIP2 in human renal tubular epithelial cells activates TFE3, which upregulates RRAGD expression (among other E-box targets including GPNMB), without modifying mTORC1 activity, indicating RRAGD is a transcriptional target of TFE3 downstream of FLCN.","method":"FLCN/FNIP1/FNIP2 knockout in RPTEC/TERT1 cells, RNA-seq, TFE3 ChIP-seq, Western blot, RT-qPCR","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — integrated genomic and proteomic approach with genetic knockouts and ChIP-seq in human renal cells","pmids":["33459596"],"is_preprint":false},{"year":2023,"finding":"MEF2A and MEF2D transcription factors control the FLCN-FNIP1/2 complex, which acts as a RRAGC-RRAGD GTPase-activating element to promote MTORC1 recruitment to the lysosome and its activation; SRC kinase phosphorylates MEF2D at conserved tyrosine residues to enhance this transcriptional activity and MTORC1 activation.","method":"ChIP, transcriptional reporter assays, FNIP1/2 knockdown/overexpression, mTORC1 lysosomal localization imaging, MEF2A/D double depletion, SRC phosphorylation site mutagenesis","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods including ChIP, mutagenesis, and lysosomal localization in a single lab study","pmids":["37772772"],"is_preprint":false},{"year":2024,"finding":"Overexpression of RRAGD patient variants (p.S76L and p.P119R) in zebrafish embryos causes decreased ventricular fractional shortening, reduced ejection fraction, and pericardial swelling; these cardiac phenotypes are reversible with rapamycin (mTOR inhibitor), establishing a direct causal role of RRAGD gain-of-function mutations in cardiomyopathy through mTOR signaling.","method":"Zebrafish embryo cRNA injection model, echocardiographic measurements (fractional shortening, ejection fraction), rapamycin rescue experiment, survival analysis","journal":"American Journal of Physiology. Heart and Circulatory Physiology","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo vertebrate model with pharmacological rescue; single lab but clean phenotype with mTOR inhibitor reversal","pmids":["39331021"],"is_preprint":false},{"year":2025,"finding":"Novel RRAGD variants p.(Ser77Phe) and p.(Ile100Arg) cause constitutive activation of non-canonical mTORC1 signaling, confirmed by in vitro mTORC1 activity assays; dapagliflozin (SGLT2 inhibitor) treatment in patients with the p.(Thr97Pro) variant increased serum magnesium levels, providing functional evidence linking RRAGD-mTORC1 activity to renal magnesium handling.","method":"In vitro mTORC1 activity assays for novel variants, clinical evaluation of diuretic and SGLT2 inhibitor treatment in ADKH-RRAGD patients","journal":"Kidney International Reports","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro functional validation of novel variants combined with clinical therapeutic data; multiple patients","pmids":["41141537"],"is_preprint":false},{"year":2025,"finding":"LINC00622 lncRNA associates with and recruits transcription factor BTF3 to the RRAGD promoter, transcriptionally enhancing RRAGD expression, which activates mTORC1 and inhibits autophagic cell death in cutaneous melanoma.","method":"RNA immunoprecipitation, ChIP, RRAGD promoter reporter assays, LINC00622 knockdown/overexpression with mTORC1 activity readouts and autophagy flux assays","journal":"Cell Death & Disease","confidence":"Medium","confidence_rationale":"Tier 2-3 — mechanistic link between lncRNA-BTF3 complex and RRAGD transcription shown by ChIP and RIP with functional mTORC1 and autophagy readouts","pmids":["40651979"],"is_preprint":false},{"year":2022,"finding":"miR-99a-5p directly targets the 3'UTR of RRAGD mRNA (confirmed by dual luciferase reporter assay), negatively regulating RRAGD expression; overexpression of miR-99a-5p inhibits glycolysis (reduced glucose uptake, lactate production, extracellular acidification rate) and induces apoptosis in cervical cancer cells by suppressing RRAGD.","method":"Dual luciferase reporter assay, RT-qPCR, Western blot, MTT assay, flow cytometry, Seahorse XFe96 extracellular flux analysis, shRNA knockdown","journal":"Oncology Letters","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct 3'UTR targeting validated by luciferase assay with functional glycolysis and apoptosis readouts; single lab","pmids":["35720506"],"is_preprint":false},{"year":2024,"finding":"miR-125b-1-3p directly targets RRAGD, and its overexpression suppresses the RRAGD/mTOR/ULK1 signaling axis, enhancing autophagy in vascular smooth muscle cells, reducing lipid uptake and foam cell formation, and decreasing atherosclerotic plaque development in mice.","method":"miR-125b-1-3p overexpression in vivo (mice) and in vitro (VSMCs), RRAGD 3'UTR targeting assay (implied interaction), mTOR/ULK1 pathway analysis, autophagy flux assays, atherosclerosis plaque quantification","journal":"Cellular Signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 — in vivo and in vitro evidence with pathway analysis; direct miRNA-RRAGD interaction characterization somewhat implied","pmids":["38471617"],"is_preprint":false},{"year":2021,"finding":"RRAGD knockdown in HCC cell lines reduces glucose uptake, lactate production, and extracellular acidification rate, and inhibits cell proliferation, invasion, and migration; MYC oncogene upregulates RRAGD expression in HCC cells, placing RRAGD downstream of MYC in promoting aerobic glycolysis.","method":"shRNA knockdown of RRAGD in Huh-7 and HepG2 cells, glucose uptake colorimetric assay, lactate assay, ECAR measurement, Western blot, RT-qPCR for MYC-RRAGD relationship","journal":"Annals of Hepatology","confidence":"Medium","confidence_rationale":"Tier 2-3 — clean loss-of-function with multiple metabolic readouts; MYC-RRAGD relationship shown by overexpression/knockdown; single lab","pmids":["33434687"],"is_preprint":false},{"year":2023,"finding":"LncTUG1 acts as a ceRNA sponge for miR-144-3p, relieving miR-144-3p-mediated suppression of RRAGD (miR-144-3p binds 3'UTR of RRAGD mRNA), thereby activating mTOR/S6K pathway and promoting HCC progression; validated in xenograft mouse models showing decreased p-mTOR, p-S6K, and RRAGD upon LncTUG1 knockdown.","method":"Dual luciferase reporter assay for miR-144-3p/RRAGD 3'UTR interaction, rescue experiments with mTOR pathway inhibitors/activators, xenograft nude mouse models, qRT-PCR, Western blot","journal":"Scientific Reports","confidence":"Medium","confidence_rationale":"Tier 2-3 — 3'UTR interaction validated by luciferase assay, in vivo xenograft confirmation; single lab","pmids":["37160972"],"is_preprint":false},{"year":2025,"finding":"IRTKS forms lysosome-localized membrane-associated condensates that selectively interact with RRAGD (a key upstream regulator of mTORC1), enhancing the sensitivity of mTORC1 to free amino acids; IRTKS-mediated mTORC1 hyperactivation in hepatic knockin mice promotes MASLD and HCC progression.","method":"Phospho-antibody array screening, co-immunoprecipitation/interaction assay between IRTKS and RRAGD, lysosomal localization imaging, Irtks hepatic knockin mouse model, mTOR activity assays","journal":"Cell Reports","confidence":"Medium","confidence_rationale":"Tier 2 — RRAGD-IRTKS interaction identified biochemically with in vivo knockin mouse model validation; single lab","pmids":["41575860"],"is_preprint":false},{"year":2025,"finding":"The CGAS-STING1 pathway activates lysosome biogenesis through lipidated GABARAP on single membranes (regulated by V-ATPase-ATG16L1 axis), which sequesters the FLCN-FNIP complex to abolish its GAP function toward RRAGC and RRAGD, leading to impaired MTORC1-dependent phosphorylation of TFEB and its subsequent nuclear translocation.","method":"GABARAP lipidation assays, FLCN-FNIP complex sequestration assays, MTORC1 substrate phosphorylation measurements, TFEB nuclear translocation imaging, V-ATPase-ATG16L1 axis dissection","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic dissection of FLCN-FNIP GAP function toward RRAGD with multiple biochemical approaches; single lab","pmids":["39835593"],"is_preprint":false},{"year":2025,"finding":"The miR-302/367 cluster directly targets RRAGD 3'UTR (validated by dual-luciferase reporter assay), and its overexpression in breast cancer cell lines reduces RRAGD transcript and protein levels, contributing to broad suppression of mTOR pathway activity.","method":"Dual-luciferase reporter assay, RT-qPCR, Western blot in SK-BR-3 and MDA-MB-231 cells after miR-302/367 vector transfection","journal":"FASEB Journal","confidence":"Low","confidence_rationale":"Tier 3 — direct 3'UTR targeting confirmed by luciferase; single lab, single method for RRAGD-specific mechanistic claim","pmids":["40817797"],"is_preprint":false},{"year":2025,"finding":"RRAGD co-localizes with lysosomal marker LAMP1 and lysosomal regeneration transcription factor TFEB, indicating lysosomal targeting; engineered extracellular vesicles encapsulating RRAGD protein ameliorated lysosomal dysfunction and suppressed apoptosis in nucleus pulposus cells in vitro and in vivo.","method":"Immunofluorescence colocalization with LAMP1 and TFEB, Fc-TRIM21 engineered EV loading, in vitro and in vivo (intervertebral disc) functional assays","journal":"Journal of Nanobiotechnology","confidence":"Low","confidence_rationale":"Tier 3 — colocalization with lysosomal markers with functional rescue; limited mechanistic detail on RRAGD's specific molecular role","pmids":["41076529"],"is_preprint":false},{"year":2025,"finding":"IL4 treatment induces RRAGD expression in follicular lymphoma cells in a STAT6-dependent manner; RRAGD is required for mTOR activation in lymphoma cells, and IL4-enhanced BCR signaling-induced mTOR activation is augmented by mutant STAT6 and reduced by CREBBP mutants through modulation of RRAGD expression.","method":"RNA-seq gene expression, RRAGD siRNA knockdown with mTOR activity measurement, IL4 stimulation experiments, STAT6 mutant and CREBBP mutant primary FL cells","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2-3 — RRAGD requirement for mTOR activation shown by knockdown in lymphoma cells; IL4/STAT6 transcriptional regulation of RRAGD demonstrated with patient-derived cells","pmids":["39910284"],"is_preprint":false},{"year":2024,"finding":"lncARF physically binds to RRAGD protein and inhibits its ubiquitination, thereby activating PI3K/Akt and MAPK signaling pathways downstream of RRAGD; lncARF knockdown decreased atherosclerotic lesion formation by promoting autophagy.","method":"Mass spectrometry, RNA pull-down, RNA immunoprecipitation (RIP), ubiquitination assays, lncARF knockdown in vivo and in vitro, PI3K/Akt and MAPK pathway activity measurements","journal":"Journal of Advanced Research","confidence":"Medium","confidence_rationale":"Tier 2-3 — direct physical interaction with RRAGD shown by pull-down and RIP with MS; ubiquitination inhibition shown; single lab","pmids":["39214417"],"is_preprint":false}],"current_model":"RRAGD encodes RagD GTPase, which functions as a key component of the lysosomal nutrient-sensing machinery that mediates amino acid signaling to mTORC1; RagD (in complex with RagC) promotes mTORC1 recruitment to the lysosomal surface, and its GTPase activity is regulated by the FLCN-FNIP complex acting as a GAP; via a non-canonical mTORC1 branch, RagD specifically controls phosphorylation of TFEB/TFE3 transcription factors to suppress lysosomal biogenesis and autophagy, and gain-of-function (auto-activating) RRAGD mutations constitutively activate this non-canonical pathway even without FLCN, causing the hereditary disorder ADKH-RRAGD characterized by kidney tubulopathy and dilated cardiomyopathy."},"narrative":{"teleology":[{"year":2018,"claim":"Establishing that RRAGD is present on signaling endosomes en route to lysosomes, where it participates in mTORC1 re-tethering and activation, resolved the question of where in the endolysosomal system RagD-mTORC1 interactions occur.","evidence":"Immunofluorescence colocalization, mTORC1 activity assays, and TSC2 dissociation experiments in CAD and HEK293T cells","pmids":["30145926"],"confidence":"Medium","gaps":["Endosomal versus lysosomal contribution to RagD-mTORC1 activation not quantitatively separated","Mechanism by which RRAGD is recruited to endosomes not identified"]},{"year":2021,"claim":"Identification of heterozygous missense RRAGD variants as the genetic cause of a multisystem disorder (kidney tubulopathy with cardiomyopathy) established that RagD gain-of-function constitutively activates mTORC1 signaling in human disease, and localized RagD expression to the nephron segments relevant to electrolyte handling.","evidence":"Whole-exome/genome sequencing of patient cohorts, in vitro mTOR activation assays for RRAGD variants, immunolocalization along nephron segments","pmids":["34607910"],"confidence":"High","gaps":["Precise structural basis of gain-of-function mutations not resolved","Tissue-specific consequences in the heart versus kidney not mechanistically dissected"]},{"year":2021,"claim":"Demonstrating that FLCN loss activates TFE3, which in turn transcriptionally upregulates RRAGD, revealed a feedback circuit in which the GAP controlling RagD activity also governs RagD abundance via TFE3.","evidence":"FLCN/FNIP1/FNIP2 knockout in RPTEC/TERT1 cells with RNA-seq, TFE3 ChIP-seq, and RT-qPCR","pmids":["33459596"],"confidence":"High","gaps":["Functional consequence of increased RRAGD transcription on mTORC1 reactivation kinetics not quantified","Whether this feedback operates in non-renal tissues is unknown"]},{"year":2021,"claim":"Showing that MYC upregulates RRAGD and that RRAGD knockdown suppresses aerobic glycolysis in HCC cells positioned RRAGD as an effector linking oncogenic transcription to metabolic reprogramming.","evidence":"shRNA knockdown in HCC cell lines with glucose uptake, lactate, and ECAR measurements; MYC overexpression/knockdown","pmids":["33434687"],"confidence":"Medium","gaps":["Whether RRAGD-driven glycolysis is mTORC1-dependent or involves additional pathways not resolved","In vivo HCC models not tested"]},{"year":2023,"claim":"Mechanistic dissection revealed that disease-causing RRAGD mutations constitutively activate a non-canonical mTORC1 branch phosphorylating TFEB/TFE3 but not S6K, and this occurs independently of the FLCN GAP, fundamentally redefining how RagD gain-of-function impacts downstream signaling.","evidence":"TFEB/TFE3 phosphorylation and nuclear translocation assays in HeLa, HK-2, patient fibroblasts, and iPSC-cardiomyocytes; FLCN knockout controls","pmids":["37188688"],"confidence":"High","gaps":["Whether non-canonical signaling fully explains cardiomyopathy pathogenesis is not established","Structural mechanism by which mutant RagD bypasses FLCN is unknown"]},{"year":2023,"claim":"Upstream transcriptional regulation of the FLCN-FNIP GAP complex by MEF2A/MEF2D and SRC kinase provided a signal-integration layer controlling RagC/D GTPase activation and mTORC1 lysosomal recruitment.","evidence":"ChIP, transcriptional reporter assays, FNIP knockdown/overexpression, SRC phosphorylation site mutagenesis","pmids":["37772772"],"confidence":"Medium","gaps":["Direct effect on RagD GTP loading not measured","Physiological stimuli activating SRC-MEF2D axis upstream of FLCN-FNIP not defined"]},{"year":2022,"claim":"Identifying miR-99a-5p, miR-144-3p (via lncTUG1 ceRNA), and later miR-302/367 and miR-125b-1-3p as direct regulators of RRAGD 3ʹUTR demonstrated that post-transcriptional control of RRAGD levels tunes mTOR activity in cancer and atherosclerosis contexts.","evidence":"Dual-luciferase reporter assays confirming 3ʹUTR targeting, metabolic (glycolysis, autophagy) and phenotypic readouts in cervical cancer, HCC xenografts, VSMCs, and breast cancer cells","pmids":["35720506","37160972","38471617","40817797"],"confidence":"Medium","gaps":["Endogenous stoichiometry of miRNA-mediated RRAGD suppression not established","Redundancy among multiple RRAGD-targeting miRNAs not addressed"]},{"year":2024,"claim":"In vivo expression of patient RRAGD variants in zebrafish caused cardiomyopathy reversible by rapamycin, providing the first whole-organism evidence that RagD gain-of-function directly drives cardiac dysfunction through mTOR.","evidence":"Zebrafish embryo cRNA injection, echocardiographic measurements, rapamycin rescue","pmids":["39331021"],"confidence":"Medium","gaps":["Zebrafish model may not recapitulate mammalian cardiac physiology fully","Renal phenotype not modeled in the same system"]},{"year":2024,"claim":"Discovery that lncARF physically binds RRAGD and inhibits its ubiquitination, thereby activating PI3K/Akt and MAPK pathways, revealed a protein-level regulatory mechanism governing RagD stability and broadened its signaling output beyond mTORC1.","evidence":"RNA pull-down, RIP, mass spectrometry, ubiquitination assays in vitro and in vivo","pmids":["39214417"],"confidence":"Medium","gaps":["Identity of the E3 ligase targeting RRAGD for ubiquitination not determined","Whether RRAGD-PI3K/Akt link is direct or mediated by mTORC1 feedback not resolved"]},{"year":2025,"claim":"The CGAS-STING1 pathway was shown to sequester the FLCN-FNIP complex via lipidated GABARAP on single membranes, abolishing its GAP activity toward RagC/D and releasing TFEB from mTORC1-dependent phosphorylation, connecting innate immune signaling to RagD regulation.","evidence":"GABARAP lipidation assays, FLCN-FNIP sequestration assays, TFEB nuclear translocation imaging","pmids":["39835593"],"confidence":"Medium","gaps":["Whether CGAS-STING regulation of FLCN-FNIP preferentially affects RagD versus RagC is unclear","In vivo relevance in innate immune contexts not tested"]},{"year":2025,"claim":"IRTKS was identified as a lysosome-localized interaction partner of RRAGD that forms membrane-associated condensates enhancing mTORC1 sensitivity to amino acids, linking phase separation biology to Rag GTPase signaling and liver disease progression.","evidence":"Co-immunoprecipitation, lysosomal colocalization imaging, hepatic knockin mouse model with MASLD/HCC phenotyping","pmids":["41575860"],"confidence":"Medium","gaps":["Structural basis of IRTKS-RRAGD interaction unknown","Whether IRTKS affects RagD nucleotide state or simply scaffolds the complex not determined"]},{"year":2025,"claim":"IL4/STAT6-dependent induction of RRAGD in follicular lymphoma cells, required for mTOR activation and modulated by CREBBP mutations, connected microenvironmental cytokine signaling to Rag GTPase expression in a disease-relevant lymphoid context.","evidence":"RNA-seq, RRAGD siRNA knockdown with mTOR readouts, IL4 stimulation in STAT6-mutant and CREBBP-mutant primary FL cells","pmids":["39910284"],"confidence":"Medium","gaps":["Whether STAT6 binds RRAGD promoter directly not shown by ChIP","Therapeutic targeting of the IL4-RRAGD-mTOR axis in lymphoma not tested"]},{"year":null,"claim":"Key unresolved questions include the structural mechanism by which gain-of-function mutations lock RagD in a nucleotide state that bypasses the FLCN GAP, the identity of the E3 ubiquitin ligase(s) controlling RagD turnover, and whether the non-canonical mTORC1-TFEB branch can be selectively targeted therapeutically without affecting canonical mTORC1 signaling.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure of disease-mutant RagD in complex with Ragulator/mTORC1","E3 ligase for RRAGD ubiquitination unidentified","Selective pharmacological tools for non-canonical versus canonical mTORC1 branches lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[0,1,4,13]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[2,12,13,15]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,4,5,6,12,16]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[1,9,13,17]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[1,13,15]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,5,6]}],"complexes":["Rag GTPase heterodimer (RagC-RagD)"],"partners":["RRAGC","FLCN","FNIP1","FNIP2","IRTKS","TFEB","SLC38A9"],"other_free_text":[]},"mechanistic_narrative":"RRAGD encodes Rag GTPase D, a core component of the lysosomal amino acid–sensing machinery that recruits and activates mTORC1 on the lysosomal surface. RagD forms a heterodimer with RagC, and its GTP-loading state is regulated by the FLCN-FNIP GAP complex; when active, it drives mTORC1-dependent phosphorylation of TFEB/TFE3 transcription factors through a non-canonical branch that is independent of canonical substrates such as S6K, thereby suppressing lysosomal biogenesis and autophagy [PMID:37188688, PMID:39835593]. Heterozygous gain-of-function missense mutations in RRAGD constitutively activate this non-canonical mTORC1 pathway and cause ADKH-RRAGD syndrome, characterized by renal tubulopathy (hypomagnesemia, hypokalemia, nephrocalcinosis) and dilated cardiomyopathy, phenotypes reversible by mTOR inhibition in zebrafish models [PMID:34607910, PMID:39331021]. RRAGD expression is itself transcriptionally regulated by TFE3 (downstream of FLCN loss), MYC, STAT6/IL4 signaling, and multiple non-coding RNAs, and RRAGD levels modulate glycolytic metabolism and cell proliferation in hepatocellular carcinoma and other cancer contexts [PMID:33459596, PMID:33434687, PMID:39910284, PMID:37160972]."},"prefetch_data":{"uniprot":{"accession":"Q9NQL2","full_name":"Ras-related GTP-binding protein D","aliases":[],"length_aa":400,"mass_kda":45.6,"function":"Guanine nucleotide-binding protein that plays a crucial role in the cellular response to amino acid availability through regulation of the mTORC1 signaling cascade (PubMed:20381137, PubMed:24095279, PubMed:34607910). Forms heterodimeric Rag complexes with RagA/RRAGA or RagB/RRAGB and cycles between an inactive GTP-bound and an active GDP-bound form: RagD/RRAGD is in its active form when GDP-bound RagD/RRAGD forms a complex with GTP-bound RagA/RRAGA (or RagB/RRAGB) and in an inactive form when GTP-bound RagD/RRAGD heterodimerizes with GDP-bound RagA/RRAGA (or RagB/RRAGB) (PubMed:24095279). In its active form, promotes the recruitment of mTORC1 to the lysosomes and its subsequent activation by the GTPase RHEB (PubMed:20381137, PubMed:24095279). This is a crucial step in the activation of the MTOR signaling cascade by amino acids (PubMed:20381137, PubMed:24095279). Also plays a central role in the non-canonical mTORC1 complex, which acts independently of RHEB and specifically mediates phosphorylation of MiT/TFE factors TFEB and TFE3: GDP-bound RagD/RRAGD mediates recruitment of MiT/TFE factors TFEB and TFE3 (PubMed:32612235)","subcellular_location":"Cytoplasm; Nucleus; Lysosome membrane","url":"https://www.uniprot.org/uniprotkb/Q9NQL2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RRAGD","classification":"Not Classified","n_dependent_lines":16,"n_total_lines":1208,"dependency_fraction":0.013245033112582781},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RRAGD","total_profiled":1310},"omim":[{"mim_id":"620152","title":"HYPOMAGNESEMIA 7, RENAL, WITH OR WITHOUT DILATED CARDIOMYOPATHY; HOMG7","url":"https://www.omim.org/entry/620152"},{"mim_id":"618834","title":"LATE ENDOSOMAL/LYSOSOMAL ADAPTOR, MAPK AND MTOR ACTIVATOR 4; LAMTOR4","url":"https://www.omim.org/entry/618834"},{"mim_id":"611534","title":"NUCLEOLAR PROTEIN 8; NOL8","url":"https://www.omim.org/entry/611534"},{"mim_id":"608521","title":"LATE ENDOSOMAL/LYSOSOMAL ADAPTOR, MAPK AND MTOR ACTIVATOR 5; LAMTOR5","url":"https://www.omim.org/entry/608521"},{"mim_id":"608268","title":"RAS-RELATED GTP-BINDING PROTEIN D; RRAGD","url":"https://www.omim.org/entry/608268"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Vesicles","reliability":"Additional"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"skeletal muscle","ntpm":79.1},{"tissue":"tongue","ntpm":56.3}],"url":"https://www.proteinatlas.org/search/RRAGD"},"hgnc":{"alias_symbol":["DKFZP761H171","bA11D8.2.1"],"prev_symbol":[]},"alphafold":{"accession":"Q9NQL2","domains":[{"cath_id":"3.40.50.300","chopping":"63-236","consensus_level":"high","plddt":84.7018,"start":63,"end":236},{"cath_id":"3.30.450.190","chopping":"240-377","consensus_level":"high","plddt":88.5525,"start":240,"end":377}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NQL2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NQL2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NQL2-F1-predicted_aligned_error_v6.png","plddt_mean":75.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RRAGD","jax_strain_url":"https://www.jax.org/strain/search?query=RRAGD"},"sequence":{"accession":"Q9NQL2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NQL2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NQL2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NQL2"}},"corpus_meta":[{"pmid":"32793490","id":"PMC_32793490","title":"Multi-Omics Characterization of the 4T1 Murine Mammary Gland Tumor Model.","date":"2020","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/32793490","citation_count":167,"is_preprint":false},{"pmid":"30145926","id":"PMC_30145926","title":"TFEB-driven endocytosis coordinates MTORC1 signaling and autophagy.","date":"2018","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/30145926","citation_count":135,"is_preprint":false},{"pmid":"22367074","id":"PMC_22367074","title":"Identification and association analysis of several hundred single nucleotide polymorphisms within candidate genes for back fat thickness in Italian Large White pigs using a selective genotyping approach.","date":"2012","source":"Journal of animal science","url":"https://pubmed.ncbi.nlm.nih.gov/22367074","citation_count":56,"is_preprint":false},{"pmid":"32419983","id":"PMC_32419983","title":"Integrated analysis of lymphocyte infiltration-associated lncRNA for ovarian cancer via TCGA, GTEx and GEO datasets.","date":"2020","source":"PeerJ","url":"https://pubmed.ncbi.nlm.nih.gov/32419983","citation_count":52,"is_preprint":false},{"pmid":"34607910","id":"PMC_34607910","title":"mTOR-Activating Mutations in RRAGD Are Causative for Kidney Tubulopathy and Cardiomyopathy.","date":"2021","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/34607910","citation_count":47,"is_preprint":false},{"pmid":"21257989","id":"PMC_21257989","title":"Variants in ZNF365 isoform D are associated with Crohn's disease.","date":"2011","source":"Gut","url":"https://pubmed.ncbi.nlm.nih.gov/21257989","citation_count":23,"is_preprint":false},{"pmid":"33459596","id":"PMC_33459596","title":"Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells.","date":"2021","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/33459596","citation_count":18,"is_preprint":false},{"pmid":"27084392","id":"PMC_27084392","title":"Biotinylation: a novel posttranslational modification linking cell autonomous circadian clocks with metabolism.","date":"2016","source":"American journal of physiology. 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RagD expression was confirmed along the mammalian nephron including the thick ascending limb and distal convoluted tubule.\",\n      \"method\": \"Whole-exome/genome sequencing of patient cohort, in vitro functional analyses of RRAGD variants measuring mTOR signaling activation, immunolocalization along nephron segments\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (patient genetics, in vitro mTOR activation assays, localization), replicated across multiple families and subsequent studies\",\n      \"pmids\": [\"34607910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RRAGD auto-activating mutations (causing kidney tubulopathy and cardiomyopathy) constitutively activate a non-canonical mTORC1 signaling branch that specifically phosphorylates TFEB and TFE3 (without affecting canonical substrates like S6K), even in the absence of Folliculin (the GAP responsible for RagC/D activation), thereby inhibiting nuclear translocation and transcriptional activity of TFEB/TFE3 and impairing responses to lysosomal and mitochondrial injury.\",\n      \"method\": \"In vitro assays in HeLa and HK-2 cell lines, human iPSC-derived cardiomyocytes, patient-derived primary fibroblasts; TFEB/TFE3 phosphorylation assays; nuclear translocation imaging; Folliculin knockout controls\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple cell models including patient-derived fibroblasts and iPSC cardiomyocytes, mechanistic dissection of canonical vs. non-canonical mTORC1 substrates, Folliculin-independent activation shown\",\n      \"pmids\": [\"37188688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TFEB-driven endocytosis promotes assembly of MTORC1-containing nutrient-sensing complexes through formation of endosomes that carry RRAGD, the amino acid transporter SLC38A9, and activate AKT; these TFEB-induced signaling endosomes en route to lysosomes are required to dissociate TSC2 and re-tether/activate MTORC1 on endolysosomal membranes.\",\n      \"method\": \"Immunofluorescence colocalization, endocytosis rate assays, MTORC1 activity measurements, TSC2 dissociation assays, overexpression and starvation experiments in CAD and HEK293T cells\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple methods (imaging, signaling assays, genetic manipulation) in a single lab; RRAGD role on endosomes shown by colocalization and functional rescue\",\n      \"pmids\": [\"30145926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of FLCN or its binding partners FNIP1/FNIP2 in human renal tubular epithelial cells activates TFE3, which upregulates RRAGD expression (among other E-box targets including GPNMB), without modifying mTORC1 activity, indicating RRAGD is a transcriptional target of TFE3 downstream of FLCN.\",\n      \"method\": \"FLCN/FNIP1/FNIP2 knockout in RPTEC/TERT1 cells, RNA-seq, TFE3 ChIP-seq, Western blot, RT-qPCR\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — integrated genomic and proteomic approach with genetic knockouts and ChIP-seq in human renal cells\",\n      \"pmids\": [\"33459596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MEF2A and MEF2D transcription factors control the FLCN-FNIP1/2 complex, which acts as a RRAGC-RRAGD GTPase-activating element to promote MTORC1 recruitment to the lysosome and its activation; SRC kinase phosphorylates MEF2D at conserved tyrosine residues to enhance this transcriptional activity and MTORC1 activation.\",\n      \"method\": \"ChIP, transcriptional reporter assays, FNIP1/2 knockdown/overexpression, mTORC1 lysosomal localization imaging, MEF2A/D double depletion, SRC phosphorylation site mutagenesis\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods including ChIP, mutagenesis, and lysosomal localization in a single lab study\",\n      \"pmids\": [\"37772772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Overexpression of RRAGD patient variants (p.S76L and p.P119R) in zebrafish embryos causes decreased ventricular fractional shortening, reduced ejection fraction, and pericardial swelling; these cardiac phenotypes are reversible with rapamycin (mTOR inhibitor), establishing a direct causal role of RRAGD gain-of-function mutations in cardiomyopathy through mTOR signaling.\",\n      \"method\": \"Zebrafish embryo cRNA injection model, echocardiographic measurements (fractional shortening, ejection fraction), rapamycin rescue experiment, survival analysis\",\n      \"journal\": \"American Journal of Physiology. Heart and Circulatory Physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo vertebrate model with pharmacological rescue; single lab but clean phenotype with mTOR inhibitor reversal\",\n      \"pmids\": [\"39331021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Novel RRAGD variants p.(Ser77Phe) and p.(Ile100Arg) cause constitutive activation of non-canonical mTORC1 signaling, confirmed by in vitro mTORC1 activity assays; dapagliflozin (SGLT2 inhibitor) treatment in patients with the p.(Thr97Pro) variant increased serum magnesium levels, providing functional evidence linking RRAGD-mTORC1 activity to renal magnesium handling.\",\n      \"method\": \"In vitro mTORC1 activity assays for novel variants, clinical evaluation of diuretic and SGLT2 inhibitor treatment in ADKH-RRAGD patients\",\n      \"journal\": \"Kidney International Reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro functional validation of novel variants combined with clinical therapeutic data; multiple patients\",\n      \"pmids\": [\"41141537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LINC00622 lncRNA associates with and recruits transcription factor BTF3 to the RRAGD promoter, transcriptionally enhancing RRAGD expression, which activates mTORC1 and inhibits autophagic cell death in cutaneous melanoma.\",\n      \"method\": \"RNA immunoprecipitation, ChIP, RRAGD promoter reporter assays, LINC00622 knockdown/overexpression with mTORC1 activity readouts and autophagy flux assays\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — mechanistic link between lncRNA-BTF3 complex and RRAGD transcription shown by ChIP and RIP with functional mTORC1 and autophagy readouts\",\n      \"pmids\": [\"40651979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"miR-99a-5p directly targets the 3'UTR of RRAGD mRNA (confirmed by dual luciferase reporter assay), negatively regulating RRAGD expression; overexpression of miR-99a-5p inhibits glycolysis (reduced glucose uptake, lactate production, extracellular acidification rate) and induces apoptosis in cervical cancer cells by suppressing RRAGD.\",\n      \"method\": \"Dual luciferase reporter assay, RT-qPCR, Western blot, MTT assay, flow cytometry, Seahorse XFe96 extracellular flux analysis, shRNA knockdown\",\n      \"journal\": \"Oncology Letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct 3'UTR targeting validated by luciferase assay with functional glycolysis and apoptosis readouts; single lab\",\n      \"pmids\": [\"35720506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"miR-125b-1-3p directly targets RRAGD, and its overexpression suppresses the RRAGD/mTOR/ULK1 signaling axis, enhancing autophagy in vascular smooth muscle cells, reducing lipid uptake and foam cell formation, and decreasing atherosclerotic plaque development in mice.\",\n      \"method\": \"miR-125b-1-3p overexpression in vivo (mice) and in vitro (VSMCs), RRAGD 3'UTR targeting assay (implied interaction), mTOR/ULK1 pathway analysis, autophagy flux assays, atherosclerosis plaque quantification\",\n      \"journal\": \"Cellular Signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vivo and in vitro evidence with pathway analysis; direct miRNA-RRAGD interaction characterization somewhat implied\",\n      \"pmids\": [\"38471617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RRAGD knockdown in HCC cell lines reduces glucose uptake, lactate production, and extracellular acidification rate, and inhibits cell proliferation, invasion, and migration; MYC oncogene upregulates RRAGD expression in HCC cells, placing RRAGD downstream of MYC in promoting aerobic glycolysis.\",\n      \"method\": \"shRNA knockdown of RRAGD in Huh-7 and HepG2 cells, glucose uptake colorimetric assay, lactate assay, ECAR measurement, Western blot, RT-qPCR for MYC-RRAGD relationship\",\n      \"journal\": \"Annals of Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — clean loss-of-function with multiple metabolic readouts; MYC-RRAGD relationship shown by overexpression/knockdown; single lab\",\n      \"pmids\": [\"33434687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"LncTUG1 acts as a ceRNA sponge for miR-144-3p, relieving miR-144-3p-mediated suppression of RRAGD (miR-144-3p binds 3'UTR of RRAGD mRNA), thereby activating mTOR/S6K pathway and promoting HCC progression; validated in xenograft mouse models showing decreased p-mTOR, p-S6K, and RRAGD upon LncTUG1 knockdown.\",\n      \"method\": \"Dual luciferase reporter assay for miR-144-3p/RRAGD 3'UTR interaction, rescue experiments with mTOR pathway inhibitors/activators, xenograft nude mouse models, qRT-PCR, Western blot\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — 3'UTR interaction validated by luciferase assay, in vivo xenograft confirmation; single lab\",\n      \"pmids\": [\"37160972\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"IRTKS forms lysosome-localized membrane-associated condensates that selectively interact with RRAGD (a key upstream regulator of mTORC1), enhancing the sensitivity of mTORC1 to free amino acids; IRTKS-mediated mTORC1 hyperactivation in hepatic knockin mice promotes MASLD and HCC progression.\",\n      \"method\": \"Phospho-antibody array screening, co-immunoprecipitation/interaction assay between IRTKS and RRAGD, lysosomal localization imaging, Irtks hepatic knockin mouse model, mTOR activity assays\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — RRAGD-IRTKS interaction identified biochemically with in vivo knockin mouse model validation; single lab\",\n      \"pmids\": [\"41575860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The CGAS-STING1 pathway activates lysosome biogenesis through lipidated GABARAP on single membranes (regulated by V-ATPase-ATG16L1 axis), which sequesters the FLCN-FNIP complex to abolish its GAP function toward RRAGC and RRAGD, leading to impaired MTORC1-dependent phosphorylation of TFEB and its subsequent nuclear translocation.\",\n      \"method\": \"GABARAP lipidation assays, FLCN-FNIP complex sequestration assays, MTORC1 substrate phosphorylation measurements, TFEB nuclear translocation imaging, V-ATPase-ATG16L1 axis dissection\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic dissection of FLCN-FNIP GAP function toward RRAGD with multiple biochemical approaches; single lab\",\n      \"pmids\": [\"39835593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The miR-302/367 cluster directly targets RRAGD 3'UTR (validated by dual-luciferase reporter assay), and its overexpression in breast cancer cell lines reduces RRAGD transcript and protein levels, contributing to broad suppression of mTOR pathway activity.\",\n      \"method\": \"Dual-luciferase reporter assay, RT-qPCR, Western blot in SK-BR-3 and MDA-MB-231 cells after miR-302/367 vector transfection\",\n      \"journal\": \"FASEB Journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — direct 3'UTR targeting confirmed by luciferase; single lab, single method for RRAGD-specific mechanistic claim\",\n      \"pmids\": [\"40817797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RRAGD co-localizes with lysosomal marker LAMP1 and lysosomal regeneration transcription factor TFEB, indicating lysosomal targeting; engineered extracellular vesicles encapsulating RRAGD protein ameliorated lysosomal dysfunction and suppressed apoptosis in nucleus pulposus cells in vitro and in vivo.\",\n      \"method\": \"Immunofluorescence colocalization with LAMP1 and TFEB, Fc-TRIM21 engineered EV loading, in vitro and in vivo (intervertebral disc) functional assays\",\n      \"journal\": \"Journal of Nanobiotechnology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — colocalization with lysosomal markers with functional rescue; limited mechanistic detail on RRAGD's specific molecular role\",\n      \"pmids\": [\"41076529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"IL4 treatment induces RRAGD expression in follicular lymphoma cells in a STAT6-dependent manner; RRAGD is required for mTOR activation in lymphoma cells, and IL4-enhanced BCR signaling-induced mTOR activation is augmented by mutant STAT6 and reduced by CREBBP mutants through modulation of RRAGD expression.\",\n      \"method\": \"RNA-seq gene expression, RRAGD siRNA knockdown with mTOR activity measurement, IL4 stimulation experiments, STAT6 mutant and CREBBP mutant primary FL cells\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — RRAGD requirement for mTOR activation shown by knockdown in lymphoma cells; IL4/STAT6 transcriptional regulation of RRAGD demonstrated with patient-derived cells\",\n      \"pmids\": [\"39910284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"lncARF physically binds to RRAGD protein and inhibits its ubiquitination, thereby activating PI3K/Akt and MAPK signaling pathways downstream of RRAGD; lncARF knockdown decreased atherosclerotic lesion formation by promoting autophagy.\",\n      \"method\": \"Mass spectrometry, RNA pull-down, RNA immunoprecipitation (RIP), ubiquitination assays, lncARF knockdown in vivo and in vitro, PI3K/Akt and MAPK pathway activity measurements\",\n      \"journal\": \"Journal of Advanced Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — direct physical interaction with RRAGD shown by pull-down and RIP with MS; ubiquitination inhibition shown; single lab\",\n      \"pmids\": [\"39214417\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RRAGD encodes RagD GTPase, which functions as a key component of the lysosomal nutrient-sensing machinery that mediates amino acid signaling to mTORC1; RagD (in complex with RagC) promotes mTORC1 recruitment to the lysosomal surface, and its GTPase activity is regulated by the FLCN-FNIP complex acting as a GAP; via a non-canonical mTORC1 branch, RagD specifically controls phosphorylation of TFEB/TFE3 transcription factors to suppress lysosomal biogenesis and autophagy, and gain-of-function (auto-activating) RRAGD mutations constitutively activate this non-canonical pathway even without FLCN, causing the hereditary disorder ADKH-RRAGD characterized by kidney tubulopathy and dilated cardiomyopathy.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RRAGD encodes Rag GTPase D, a core component of the lysosomal amino acid–sensing machinery that recruits and activates mTORC1 on the lysosomal surface. RagD forms a heterodimer with RagC, and its GTP-loading state is regulated by the FLCN-FNIP GAP complex; when active, it drives mTORC1-dependent phosphorylation of TFEB/TFE3 transcription factors through a non-canonical branch that is independent of canonical substrates such as S6K, thereby suppressing lysosomal biogenesis and autophagy [PMID:37188688, PMID:39835593]. Heterozygous gain-of-function missense mutations in RRAGD constitutively activate this non-canonical mTORC1 pathway and cause ADKH-RRAGD syndrome, characterized by renal tubulopathy (hypomagnesemia, hypokalemia, nephrocalcinosis) and dilated cardiomyopathy, phenotypes reversible by mTOR inhibition in zebrafish models [PMID:34607910, PMID:39331021]. RRAGD expression is itself transcriptionally regulated by TFE3 (downstream of FLCN loss), MYC, STAT6/IL4 signaling, and multiple non-coding RNAs, and RRAGD levels modulate glycolytic metabolism and cell proliferation in hepatocellular carcinoma and other cancer contexts [PMID:33459596, PMID:33434687, PMID:39910284, PMID:37160972].\",\n  \"teleology\": [\n    {\n      \"year\": 2018,\n      \"claim\": \"Establishing that RRAGD is present on signaling endosomes en route to lysosomes, where it participates in mTORC1 re-tethering and activation, resolved the question of where in the endolysosomal system RagD-mTORC1 interactions occur.\",\n      \"evidence\": \"Immunofluorescence colocalization, mTORC1 activity assays, and TSC2 dissociation experiments in CAD and HEK293T cells\",\n      \"pmids\": [\"30145926\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endosomal versus lysosomal contribution to RagD-mTORC1 activation not quantitatively separated\", \"Mechanism by which RRAGD is recruited to endosomes not identified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of heterozygous missense RRAGD variants as the genetic cause of a multisystem disorder (kidney tubulopathy with cardiomyopathy) established that RagD gain-of-function constitutively activates mTORC1 signaling in human disease, and localized RagD expression to the nephron segments relevant to electrolyte handling.\",\n      \"evidence\": \"Whole-exome/genome sequencing of patient cohorts, in vitro mTOR activation assays for RRAGD variants, immunolocalization along nephron segments\",\n      \"pmids\": [\"34607910\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise structural basis of gain-of-function mutations not resolved\", \"Tissue-specific consequences in the heart versus kidney not mechanistically dissected\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating that FLCN loss activates TFE3, which in turn transcriptionally upregulates RRAGD, revealed a feedback circuit in which the GAP controlling RagD activity also governs RagD abundance via TFE3.\",\n      \"evidence\": \"FLCN/FNIP1/FNIP2 knockout in RPTEC/TERT1 cells with RNA-seq, TFE3 ChIP-seq, and RT-qPCR\",\n      \"pmids\": [\"33459596\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of increased RRAGD transcription on mTORC1 reactivation kinetics not quantified\", \"Whether this feedback operates in non-renal tissues is unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showing that MYC upregulates RRAGD and that RRAGD knockdown suppresses aerobic glycolysis in HCC cells positioned RRAGD as an effector linking oncogenic transcription to metabolic reprogramming.\",\n      \"evidence\": \"shRNA knockdown in HCC cell lines with glucose uptake, lactate, and ECAR measurements; MYC overexpression/knockdown\",\n      \"pmids\": [\"33434687\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RRAGD-driven glycolysis is mTORC1-dependent or involves additional pathways not resolved\", \"In vivo HCC models not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Mechanistic dissection revealed that disease-causing RRAGD mutations constitutively activate a non-canonical mTORC1 branch phosphorylating TFEB/TFE3 but not S6K, and this occurs independently of the FLCN GAP, fundamentally redefining how RagD gain-of-function impacts downstream signaling.\",\n      \"evidence\": \"TFEB/TFE3 phosphorylation and nuclear translocation assays in HeLa, HK-2, patient fibroblasts, and iPSC-cardiomyocytes; FLCN knockout controls\",\n      \"pmids\": [\"37188688\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether non-canonical signaling fully explains cardiomyopathy pathogenesis is not established\", \"Structural mechanism by which mutant RagD bypasses FLCN is unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Upstream transcriptional regulation of the FLCN-FNIP GAP complex by MEF2A/MEF2D and SRC kinase provided a signal-integration layer controlling RagC/D GTPase activation and mTORC1 lysosomal recruitment.\",\n      \"evidence\": \"ChIP, transcriptional reporter assays, FNIP knockdown/overexpression, SRC phosphorylation site mutagenesis\",\n      \"pmids\": [\"37772772\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct effect on RagD GTP loading not measured\", \"Physiological stimuli activating SRC-MEF2D axis upstream of FLCN-FNIP not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying miR-99a-5p, miR-144-3p (via lncTUG1 ceRNA), and later miR-302/367 and miR-125b-1-3p as direct regulators of RRAGD 3ʹUTR demonstrated that post-transcriptional control of RRAGD levels tunes mTOR activity in cancer and atherosclerosis contexts.\",\n      \"evidence\": \"Dual-luciferase reporter assays confirming 3ʹUTR targeting, metabolic (glycolysis, autophagy) and phenotypic readouts in cervical cancer, HCC xenografts, VSMCs, and breast cancer cells\",\n      \"pmids\": [\"35720506\", \"37160972\", \"38471617\", \"40817797\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous stoichiometry of miRNA-mediated RRAGD suppression not established\", \"Redundancy among multiple RRAGD-targeting miRNAs not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"In vivo expression of patient RRAGD variants in zebrafish caused cardiomyopathy reversible by rapamycin, providing the first whole-organism evidence that RagD gain-of-function directly drives cardiac dysfunction through mTOR.\",\n      \"evidence\": \"Zebrafish embryo cRNA injection, echocardiographic measurements, rapamycin rescue\",\n      \"pmids\": [\"39331021\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Zebrafish model may not recapitulate mammalian cardiac physiology fully\", \"Renal phenotype not modeled in the same system\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that lncARF physically binds RRAGD and inhibits its ubiquitination, thereby activating PI3K/Akt and MAPK pathways, revealed a protein-level regulatory mechanism governing RagD stability and broadened its signaling output beyond mTORC1.\",\n      \"evidence\": \"RNA pull-down, RIP, mass spectrometry, ubiquitination assays in vitro and in vivo\",\n      \"pmids\": [\"39214417\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the E3 ligase targeting RRAGD for ubiquitination not determined\", \"Whether RRAGD-PI3K/Akt link is direct or mediated by mTORC1 feedback not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The CGAS-STING1 pathway was shown to sequester the FLCN-FNIP complex via lipidated GABARAP on single membranes, abolishing its GAP activity toward RagC/D and releasing TFEB from mTORC1-dependent phosphorylation, connecting innate immune signaling to RagD regulation.\",\n      \"evidence\": \"GABARAP lipidation assays, FLCN-FNIP sequestration assays, TFEB nuclear translocation imaging\",\n      \"pmids\": [\"39835593\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CGAS-STING regulation of FLCN-FNIP preferentially affects RagD versus RagC is unclear\", \"In vivo relevance in innate immune contexts not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"IRTKS was identified as a lysosome-localized interaction partner of RRAGD that forms membrane-associated condensates enhancing mTORC1 sensitivity to amino acids, linking phase separation biology to Rag GTPase signaling and liver disease progression.\",\n      \"evidence\": \"Co-immunoprecipitation, lysosomal colocalization imaging, hepatic knockin mouse model with MASLD/HCC phenotyping\",\n      \"pmids\": [\"41575860\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of IRTKS-RRAGD interaction unknown\", \"Whether IRTKS affects RagD nucleotide state or simply scaffolds the complex not determined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"IL4/STAT6-dependent induction of RRAGD in follicular lymphoma cells, required for mTOR activation and modulated by CREBBP mutations, connected microenvironmental cytokine signaling to Rag GTPase expression in a disease-relevant lymphoid context.\",\n      \"evidence\": \"RNA-seq, RRAGD siRNA knockdown with mTOR readouts, IL4 stimulation in STAT6-mutant and CREBBP-mutant primary FL cells\",\n      \"pmids\": [\"39910284\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether STAT6 binds RRAGD promoter directly not shown by ChIP\", \"Therapeutic targeting of the IL4-RRAGD-mTOR axis in lymphoma not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural mechanism by which gain-of-function mutations lock RagD in a nucleotide state that bypasses the FLCN GAP, the identity of the E3 ubiquitin ligase(s) controlling RagD turnover, and whether the non-canonical mTORC1-TFEB branch can be selectively targeted therapeutically without affecting canonical mTORC1 signaling.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal or cryo-EM structure of disease-mutant RagD in complex with Ragulator/mTORC1\", \"E3 ligase for RRAGD ubiquitination unidentified\", \"Selective pharmacological tools for non-canonical versus canonical mTORC1 branches lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [0, 1, 4, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [2, 12, 13, 15]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 4, 5, 6, 12, 16]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [1, 9, 13, 17]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [1, 13, 15]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 5, 6]}\n    ],\n    \"complexes\": [\n      \"Rag GTPase heterodimer (RagC-RagD)\"\n    ],\n    \"partners\": [\n      \"RRAGC\",\n      \"FLCN\",\n      \"FNIP1\",\n      \"FNIP2\",\n      \"IRTKS\",\n      \"TFEB\",\n      \"SLC38A9\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}