{"gene":"IRAG1","run_date":"2026-06-10T01:55:23","timeline":{"discoveries":[{"year":2000,"finding":"IRAG forms a trimeric signaling complex with cGKIβ and IP3 receptor type I (IP3R-I) at endoplasmic reticulum membranes in smooth muscle. In microsomal smooth muscle membranes, cGKIβ phosphorylated IRAG and IP3R. Co-expression of IRAG and cGKIβ in COS-7 cells, in the presence of cGMP, inhibited bradykinin-stimulated calcium release, identifying IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.","method":"Co-immunoprecipitation with antibodies against cGKI, IP3R, and IRAG; heterologous expression in COS-7 cells; calcium release assays; mass spectrometry protein identification","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal Co-IP from native tissue, reconstitution by co-expression with functional readout, replicated across multiple labs in subsequent studies","pmids":["10724174"],"is_preprint":false},{"year":2001,"finding":"The N-terminal leucine zipper (amino acids 1–53) of cGKIβ (but not cGKIα or cGKII) interacts with amino acids 152–184 of IRAG via electrostatic interaction. cGKIβ does not directly interact with IP3R-I but co-precipitates IP3R-I through IRAG. cGKIβ phosphorylates up to four serines in IRAG; phosphorylation specifically of Ser696 is necessary and sufficient to decrease IP3-dependent calcium release.","method":"Yeast two-hybrid; co-precipitation of expressed proteins; site-directed mutagenesis (four Ser→Ala mutants); calcium release assays in transfected cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro binding assays combined with mutagenesis and functional calcium readout; multiple orthogonal methods in one study","pmids":["11309393"],"is_preprint":false},{"year":2004,"finding":"Targeted deletion of exon 12 of IRAG (encoding the N-terminus of the coiled-coil domain) disrupts the IRAG–IP3R-I interaction in vivo, abolishing cGMP-dependent relaxation of carbachol- and phenylephrine-contracted smooth muscle from colon and aorta and preventing cGMP-mediated decrease in norepinephrine-induced [Ca2+]i in aortic smooth muscle cells. cGMP-induced relaxation of K+-contracted smooth muscle was unaffected, indicating IRAG is specifically required for hormone receptor–triggered cGMP relaxation.","method":"Genetic knockout (exon 12 deletion); smooth muscle contraction/relaxation assays; intracellular calcium measurements (Fura-2); gastrointestinal motility assessment","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean in vivo genetic model with specific phenotypic readouts and mechanistic distinction between receptor-triggered vs. K+-induced contraction","pmids":["15483626"],"is_preprint":false},{"year":2004,"finding":"Endogenous IRAG in human colonic smooth muscle cells is required for NO/cGKI-dependent inhibition of IP3-dependent Ca2+ release. Antisense knockdown of IRAG abolished sodium nitroprusside- and 8-pCPT-cGMP-mediated inhibition of bradykinin-induced calcium transients.","method":"Antisense oligonucleotide knockdown of IRAG in cultured human colonic smooth muscle cells; calcium imaging; RT-PCR","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with specific calcium phenotype in human primary cells, single lab, antisense method","pmids":["14729908"],"is_preprint":false},{"year":2004,"finding":"IRAG co-localizes with cGKI in smooth muscle of aorta and colon. Upon co-expression in COS-7 cells, IRAG recruits cGKIβ (but not cGKIα) to the endoplasmic reticulum, demonstrating isoform-specific targeting by IRAG.","method":"Immunofluorescence/immunohistochemistry in murine tissues; heterologous co-expression in COS-7 cells with confocal microscopy","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — direct localization experiments with isoform-specific functional consequence (ER recruitment), replicated in tissue and cell expression system","pmids":["15388327"],"is_preprint":false},{"year":2005,"finding":"Acidic residues in the N-terminal leucine zipper dimerization domain of PKGIβ (D26 and E31) mediate binding to both TFII-I and IRAG via electrostatic interaction with basic residues in alpha-helical regions of IRAG. Mutation D26K/E31R in PKGIβ completely abrogated binding to IRAG without disrupting PKG dimerization.","method":"Site-directed mutagenesis; in vitro binding assays; co-immunoprecipitation in intact cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with in vitro binding and cell-based co-IP; defines a specific interaction interface, single lab","pmids":["16166082"],"is_preprint":false},{"year":2006,"finding":"IRAG is abundantly expressed in platelets and assembles in a macrocomplex with cGKIβ and IP3R-I. PKGIβ phosphorylates IRAG at Ser664 and Ser677 in intact platelets. Targeted disruption of the IRAG–IP3R-I interaction (IRAGΔ12/Δ12 mice) abolishes NO/cGMP-dependent inhibition of fibrinogen-receptor activation, platelet aggregation, and intracellular calcium transients, and prevents NO-mediated inhibition of arterial thrombosis in vivo.","method":"IRAG exon 12 deletion mouse model; intravital microscopy; platelet aggregation assays; calcium imaging; phosphorylation site mapping by mass spectrometry","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic model with multiple orthogonal functional readouts including intravital microscopy, Ca2+ imaging, and phosphosite identification","pmids":["16990611"],"is_preprint":false},{"year":2008,"finding":"IRAG anchors PKGIβ to the endoplasmic reticulum, preventing cGMP-induced nuclear translocation of PKGIβ and thereby reducing cGMP/PKGIβ-mediated transcriptional activation of a CRE-reporter gene. This effect required the PKGIβ–IRAG binding interface and was specific to PKGIβ (PKGIα was unaffected). A phosphorylation-deficient IRAG mutant still suppressed PKGIβ transcriptional activity, indicating the mechanism is independent of IRAG phosphorylation or changes in intracellular calcium.","method":"Co-expression of wild-type and binding-incompetent/phosphorylation-deficient IRAG mutants in baby hamster kidney cells; CRE-reporter gene assay; subcellular localization by imaging","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional reporter assay with mutants defining mechanism, single lab, two orthogonal readouts (localization + transcription)","pmids":["18450420"],"is_preprint":false},{"year":2010,"finding":"Global IRAG knockout (exon 3 deletion) prevents stable interaction of IP3R-I with cGKIβ (shown by cGMP affinity chromatography) and abolishes NO-, ANP-, and cGMP-dependent relaxation of hormone-contracted aortic and colonic smooth muscle. cGKIβ/α subcellular localization in VSMCs was unchanged by IRAG loss. IRAG-deficient VSMCs failed to suppress hormone-induced Ca2+ increases in response to cGMP. Selective cGKIβ re-expression in smooth muscle from cGKIβ-transgenic mice did not rescue relaxation, confirming IRAG is obligate downstream of cGKIβ. IRAG-deficient mice showed resistance to LPS-induced blood pressure reduction.","method":"Targeted exon 3 deletion mouse model; cGMP affinity chromatography; confocal microscopy; Fura-2 calcium imaging; smooth muscle relaxation assays; telemetric blood pressure measurement; transgenic rescue experiment","journal":"Cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 / Strong — complete knockout with multiple orthogonal mechanistic assays, transgenic rescue experiment, multiple physiological readouts","pmids":["20080989"],"is_preprint":false},{"year":2011,"finding":"IRAG-deficient murine platelets show enhanced aggregability to collagen, thrombin, and TxA2. NO/cGMP-dependent inhibition of ATP and 5-HT secretion from dense granules, P-selectin secretion from alpha granules, and GPIIb/IIIa-mediated adhesion to fibrinogen were all severely impaired in IRAG-deficient platelets, and bleeding time was reduced.","method":"IRAG knockout mouse model; platelet aggregation assays; granule secretion assays (ATP, 5-HT); flow cytometry (P-selectin); adhesion assays; bleeding time measurement","journal":"Platelets","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function with multiple specific functional readouts across platelet activation pathways","pmids":["21244222"],"is_preprint":false},{"year":2011,"finding":"C-terminally truncated IRAG variants lacking the cGKI phosphorylation site and IP3R-I interaction site act as dominant-negative modulators, counteracting cGMP-mediated inhibition of calcium transients and relaxation of human colonic smooth muscle cells. Four unique first-exon variants driven by individual promoters and extensive alternative splicing generate multiple truncated IRAG isoforms.","method":"Identification of splice variants by RT-PCR/sequencing; functional expression of truncated IRAG variants in colonic smooth muscle cells; calcium imaging; contractility assays","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional expression studies with specific calcium and contractility readouts in primary human cells, single lab","pmids":["21865585"],"is_preprint":false},{"year":2021,"finding":"IRAG, PKGIβ, and IP3Rs form a nanoscale signaling complex on the SR of vascular smooth muscle cells, identified by superresolution microscopy. PKG phosphorylation of IRAG inhibits IP3R-mediated Ca2+ release; IRAG knockdown diminished NO-mediated inhibition of TRPM4 channel activity and vasodilation. Thus, IRAG mediates NO/cGMP/PKG inhibition of IP3R-dependent TRPM4 activation to dilate cerebral arteries.","method":"IRAG knockdown in vascular SMCs; patch-clamp electrophysiology (TRPM4 currents); Ca2+ imaging; superresolution microscopy of IRAG/PKG/IP3R nanoscale complex; pharmacological inhibition of guanylyl cyclase and PKG","journal":"Function (Oxford, England)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (superresolution microscopy, electrophysiology, Ca2+ imaging, genetic knockdown) in one study establishing new downstream pathway","pmids":["34734188"],"is_preprint":false},{"year":2020,"finding":"IRAG1 knockout mice spontaneously develop right ventricular hypertrophy, elevated RV systolic pressure, RV dysfunction, and pulmonary hypertension under normoxic conditions. IRAG1 is expressed in PASMCs and is downregulated under hypoxia. Absence of IRAG1 reduces PKGIβ expression in lung and RV and dysregulates downstream PKGIβ candidates in the RV.","method":"Global IRAG1 KO mouse model; echocardiography; right heart catheterization; immunostaining; western blotting; PASMC isolation","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic model with hemodynamic and molecular readouts, single lab, limited mechanistic depth on pathway placement","pmids":["33066124"],"is_preprint":false},{"year":2021,"finding":"Global IRAG1 KO mice develop gastrointestinal bleeding, iron-deficiency anemia, and anemia-associated splenomegaly. Loss of IRAG1 strongly decreases PKGIβ protein levels (but not mRNA) in colon, spleen, and stomach, indicating IRAG1 is required for PKGIβ protein stability.","method":"Global IRAG1 KO mouse model; western blotting; RT-PCR; histology; hematological analysis","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic model with protein stability finding, single lab, mechanistic interpretation supported by mRNA vs. protein comparison","pmids":["34064290"],"is_preprint":false},{"year":1999,"finding":"Mrvi1 (IRAG1 ortholog) encodes a protein with homology to Jaw1, a lymphoid-restricted type II membrane protein localizing to the endoplasmic reticulum. Within hematopoietic cells, Mrvi1 expression is restricted to megakaryocytes and some myeloid leukemias, and is downregulated during monocytic differentiation.","method":"Retroviral insertional mutagenesis screen; sequence homology analysis; Northern blot expression analysis","journal":"Oncogene","confidence":"Low","confidence_rationale":"Tier 3 / Weak — sequence homology and expression analysis; ER localization inferred from homology rather than direct experiment on MRVI1","pmids":["10321731"],"is_preprint":false},{"year":2025,"finding":"TurboID-based proximity labeling identified >700 candidate MRVI1-interacting proteins in mammalian cells, including ER-localized factors and intracellular trafficking components. Co-expression of NPM-ALK oncogenic kinase selectively enhanced MRVI1 association with signaling-related proteins and reduced association with anti-apoptotic regulators (DDB1, PHB2, NOTCH2), suggesting MRVI1 participates in apoptosis-related networks disrupted during oncogenic transformation.","method":"TurboID proximity labeling; quantitative proteomics; co-expression of NPM-ALK","journal":"Journal of biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — proximity labeling screen (not direct binding validation); functional interpretation is inferential; single lab","pmids":["41078212"],"is_preprint":false},{"year":2026,"finding":"Stable overexpression of MRVI1 in HCT116 colorectal cancer cells markedly reduces cell proliferation without increasing cell death (cytostatic effect), providing direct evidence that MRVI1 suppresses colorectal cancer cell growth when overexpressed, consistent with a p53-associated tumor suppressor role.","method":"Stable overexpression of V5-TurboID-MRVI1 fusion protein; cell proliferation assays; trypan blue viability staining","journal":"Biological & pharmaceutical bulletin","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single overexpression experiment with proliferation readout, no molecular mechanism elucidated, single lab","pmids":["41905951"],"is_preprint":false}],"current_model":"IRAG1 (MRVI1/IRAG/JAW1L) is an endoplasmic reticulum-anchored 125 kDa membrane protein that forms an obligate trimeric signaling complex with cGMP-dependent protein kinase Iβ (PKGIβ) — via electrostatic interaction between the PKGIβ leucine zipper (residues 1–53) and IRAG1 residues 152–184 — and with IP3 receptor type I (IP3R-I); upon cGMP-driven PKGIβ phosphorylation of IRAG1 (primarily at Ser696 in rodents; Ser664/Ser677 in platelets), IRAG1 inhibits IP3R-I-mediated Ca²⁺ release from the ER, thereby mediating NO/cGMP-dependent relaxation of smooth muscle, inhibition of platelet activation and arterial thrombosis, inhibition of TRPM4 channel activation and vasodilation, and regulation of PKGIβ protein stability; additionally, ER-anchored IRAG1 prevents nuclear translocation of PKGIβ to restrict cGMP-dependent transcriptional responses, while C-terminally truncated IRAG1 splice variants lacking the phosphorylation and IP3R-I interaction domains act as dominant-negative modulators of this pathway."},"narrative":{"mechanistic_narrative":"IRAG1 (MRVI1) is an endoplasmic reticulum-anchored membrane protein that serves as the central scaffold of an NO/cGMP signaling module controlling intracellular Ca²⁺ release in smooth muscle and platelets [PMID:10724174]. It assembles an obligate trimeric complex with cGMP-dependent protein kinase Iβ (cGKIβ/PKGIβ) and IP3 receptor type I, binding the kinase through an electrostatic interaction between the cGKIβ N-terminal leucine zipper (residues 1–53; acidic residues D26/E31) and basic residues in IRAG1 (residues 152–184), while bridging IP3R-I, which does not contact cGKIβ directly [PMID:11309393, PMID:16166082]. This complex is isoform-specific: IRAG1 recruits cGKIβ — but not cGKIα or cGKII — to the ER [PMID:15388327]. Upon cGMP activation, cGKIβ phosphorylates IRAG1, and phosphorylation at Ser696 is necessary and sufficient to inhibit IP3-induced Ca²⁺ release [PMID:11309393]; in platelets the corresponding sites are Ser664/Ser677 [PMID:16990611]. Through this mechanism IRAG1 mediates NO/cGMP-dependent relaxation of hormone receptor-triggered (but not K⁺-induced) smooth muscle contraction [PMID:15483626, PMID:20080989], inhibition of platelet aggregation, granule secretion, fibrinogen-receptor activation and arterial thrombosis [PMID:16990611, PMID:21244222], and NO-dependent inhibition of IP3R-driven TRPM4 channel activity to dilate cerebral arteries [PMID:34734188]. Independently of its phosphorylation and Ca²⁺ functions, ER-anchored IRAG1 retains cGKIβ at the ER to prevent its cGMP-induced nuclear translocation and restrict CRE-dependent transcription [PMID:18450420], and is required for PKGIβ protein stability [PMID:34064290]. Loss of IRAG1 in mice produces pulmonary hypertension with right ventricular hypertrophy [PMID:33066124] and gastrointestinal bleeding with iron-deficiency anemia [PMID:34064290]. C-terminally truncated splice variants lacking the phosphorylation and IP3R-I interaction domains act as dominant-negative modulators of the pathway [PMID:21865585].","teleology":[{"year":2000,"claim":"Established IRAG as the missing link coupling NO/cGMP signaling to inhibition of IP3-mediated Ca²⁺ release by demonstrating it forms a functional trimeric complex with cGKIβ and IP3R-I.","evidence":"Reciprocal Co-IP from native smooth muscle microsomes plus reconstitution by co-expression in COS-7 cells with calcium-release readout","pmids":["10724174"],"confidence":"High","gaps":["Did not define the binding interfaces or the relevant phosphosite","Physiological requirement in vivo not yet tested"]},{"year":2001,"claim":"Mapped the molecular architecture of the complex, showing cGKIβ binds IRAG via its leucine zipper while IP3R-I is bridged through IRAG, and pinned the inhibitory function to phosphorylation of a single serine.","evidence":"Yeast two-hybrid, co-precipitation of expressed proteins, and Ser→Ala mutagenesis with calcium-release assays","pmids":["11309393"],"confidence":"High","gaps":["Structural basis of the electrostatic interface not resolved at atomic level","Mapping done in heterologous/in vitro context"]},{"year":2004,"claim":"Proved IRAG is physiologically required for cGMP-dependent smooth muscle relaxation, and specifically for hormone receptor-triggered rather than depolarization-induced contraction.","evidence":"Exon 12 deletion mouse disrupting IRAG–IP3R-I interaction with contraction/relaxation and Ca²⁺ assays, plus antisense knockdown in human colonic SMCs","pmids":["15483626","14729908"],"confidence":"High","gaps":["Exon 12 deletion disrupts IP3R-I binding but does not eliminate all IRAG functions","Did not address platelet or other tissue roles"]},{"year":2004,"claim":"Demonstrated IRAG actively targets and recruits cGKIβ to the ER in an isoform-selective manner, explaining how the kinase is positioned at its substrate.","evidence":"Immunohistochemistry in murine tissue and heterologous co-expression with confocal microscopy in COS-7 cells","pmids":["15388327"],"confidence":"Medium","gaps":["Recruitment shown in overexpression system","Determinants of isoform selectivity not fully defined"]},{"year":2005,"claim":"Defined the acidic residues (D26/E31) within the cGKIβ leucine zipper that mediate electrostatic binding to IRAG, separable from kinase dimerization.","evidence":"Site-directed mutagenesis with in vitro binding and cell-based co-IP","pmids":["16166082"],"confidence":"High","gaps":["Single lab","Did not test functional consequence of the interface mutant in vivo"]},{"year":2006,"claim":"Extended the IRAG module to platelets, showing it mediates NO/cGMP inhibition of platelet activation and arterial thrombosis and identifying the platelet phosphosites.","evidence":"IRAGΔ12 mouse with intravital microscopy, aggregation/Ca²⁺ assays, and MS phosphosite mapping (Ser664/Ser677)","pmids":["16990611"],"confidence":"High","gaps":["Distinct phosphosites between platelets and smooth muscle not mechanistically reconciled"]},{"year":2008,"claim":"Revealed a Ca²⁺-independent function of IRAG: ER anchoring of cGKIβ to block its nuclear translocation and restrain cGMP-dependent transcription.","evidence":"WT and binding/phospho-deficient IRAG mutants in BHK cells with CRE-reporter assay and localization imaging","pmids":["18450420"],"confidence":"Medium","gaps":["Shown in heterologous cells","Transcriptional targets and physiological relevance not established"]},{"year":2010,"claim":"Confirmed with a complete knockout that IRAG is obligate downstream of cGKIβ for NO/ANP/cGMP-dependent relaxation, ruling out compensation by cGKIβ alone.","evidence":"Exon 3 deletion KO with cGMP affinity chromatography, Ca²⁺ imaging, relaxation assays, blood pressure telemetry, and cGKIβ-transgenic rescue","pmids":["20080989"],"confidence":"High","gaps":["LPS blood-pressure resistance mechanism not dissected","Did not address non-vascular phenotypes"]},{"year":2011,"claim":"Detailed the breadth of platelet inhibition mediated by IRAG across aggregation, granule secretion, and integrin-dependent adhesion.","evidence":"IRAG KO mouse with aggregation, granule secretion, P-selectin flow cytometry, adhesion, and bleeding-time assays","pmids":["21244222"],"confidence":"High","gaps":["Did not establish whether all readouts are downstream of the same IP3R-Ca²⁺ node"]},{"year":2011,"claim":"Identified C-terminally truncated IRAG splice variants that act as dominant-negative regulators, adding an endogenous mechanism for tuning the pathway.","evidence":"Splice-variant identification and functional expression of truncated isoforms in human colonic SMCs with Ca²⁺/contractility readouts","pmids":["21865585"],"confidence":"Medium","gaps":["Endogenous abundance and regulation of variants in vivo unknown","Single lab"]},{"year":2021,"claim":"Resolved the complex as a nanoscale SR assembly and connected IRAG to a new effector, TRPM4 channel inhibition, extending its role to cerebral artery dilation.","evidence":"Superresolution microscopy, patch-clamp TRPM4 currents, Ca²⁺ imaging, and IRAG knockdown in vascular SMCs","pmids":["34734188"],"confidence":"High","gaps":["Quantitative stoichiometry of the nanocomplex not defined"]},{"year":2020,"claim":"Linked IRAG1 loss to spontaneous pulmonary hypertension and right ventricular dysfunction, implicating the pathway in pulmonary vascular homeostasis.","evidence":"Global IRAG1 KO with echocardiography, right heart catheterization, and PASMC analysis","pmids":["33066124"],"confidence":"Medium","gaps":["Pathway placement of downstream PKGIβ candidates not resolved","Single lab"]},{"year":2021,"claim":"Uncovered a non-scaffolding requirement of IRAG1 for PKGIβ protein stability and tied its loss to gastrointestinal bleeding and anemia.","evidence":"Global IRAG1 KO with western blotting, RT-PCR (protein vs mRNA), histology, and hematology","pmids":["34064290"],"confidence":"Medium","gaps":["Molecular mechanism of stabilization (degradation pathway) not defined","Single lab"]},{"year":2025,"claim":"Began probing the broader IRAG1/MRVI1 interactome and a possible apoptosis/oncogenesis-related role distinct from the canonical Ca²⁺ pathway.","evidence":"TurboID proximity labeling and quantitative proteomics with NPM-ALK co-expression","pmids":["41078212"],"confidence":"Low","gaps":["Proximity labeling is not direct binding validation","Functional interpretation inferential","Single lab"]},{"year":2026,"claim":"Provided first functional evidence for a growth-suppressive (tumor suppressor-like) role of MRVI1 in colorectal cancer cells.","evidence":"Stable MRVI1 overexpression in HCT116 cells with proliferation and viability assays","pmids":["41905951"],"confidence":"Low","gaps":["Single overexpression experiment with no molecular mechanism","p53 association asserted but not directly tested","Single lab"]},{"year":null,"claim":"How IRAG1's canonical NO/cGMP Ca²⁺-regulatory scaffolding role mechanistically relates to its emerging roles in PKGIβ stability, apoptosis networks, and tumor suppression remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of the trimeric complex","Degradation pathway controlling PKGIβ stability unidentified","No direct binding validation or mechanism for proposed tumor-suppressor function"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,5]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,11]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[0,4,7]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,8]},{"term_id":"R-HSA-109582","term_label":"Hemostasis","supporting_discovery_ids":[6,9]},{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[2,8,11]}],"complexes":["IRAG1–cGKIβ–IP3R-I trimeric complex"],"partners":["PRKG1","ITPR1","TRPM4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y6F6","full_name":"Inositol 1,4,5-triphosphate receptor associated 1","aliases":["Inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate","JAW1-related protein MRVI1","Protein MRVI1"],"length_aa":904,"mass_kda":98.0,"function":"Plays a role as NO/PRKG1-dependent regulator of IP3-induced calcium release; its phosphorylation by PRKG1 inhibits bradykinin and IP3-induced calcium release from intracellular stores. Recruits PRKG1 to the endoplasmic reticulum and may mediate the assembly of PRKG1 and ITPR1 in a macrocomplex. Involved in PRKG1 signaling cascade leading to inhibition of platelet activation and aggregation. Also mediates NO-dependent inhibition of calcium signaling in gastrointestinal smooth muscle contributing to NO-dependent relaxation (PubMed:14729908). Plays a role in the regulation of cellular excitability by regulating the hyperpolarization-activated cyclic nucleotide-gated HCN4 channel activity (By similarity)","subcellular_location":"Cytoplasm, perinuclear region; Sarcoplasmic reticulum; Membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y6F6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/IRAG1","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/IRAG1","total_profiled":1310},"omim":[{"mim_id":"604673","title":"INOSITOL 1,4,5-TRIPHOSPHATE RECEPTOR ASSOCIATED 1; IRAG1","url":"https://www.omim.org/entry/604673"},{"mim_id":"604411","title":"INNER CENTROMERE PROTEIN; INCENP","url":"https://www.omim.org/entry/604411"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"blood vessel","ntpm":161.7}],"url":"https://www.proteinatlas.org/search/IRAG1"},"hgnc":{"alias_symbol":["JAW1L","IRAG"],"prev_symbol":["MRVI1"]},"alphafold":{"accession":"Q9Y6F6","domains":[{"cath_id":"-","chopping":"497-512_520-655","consensus_level":"medium","plddt":88.7024,"start":497,"end":655}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6F6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6F6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y6F6-F1-predicted_aligned_error_v6.png","plddt_mean":55.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=IRAG1","jax_strain_url":"https://www.jax.org/strain/search?query=IRAG1"},"sequence":{"accession":"Q9Y6F6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y6F6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y6F6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y6F6"}},"corpus_meta":[{"pmid":"10724174","id":"PMC_10724174","title":"Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta.","date":"2000","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/10724174","citation_count":378,"is_preprint":false},{"pmid":"11309393","id":"PMC_11309393","title":"Molecular determinants of the interaction between the inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) and cGMP kinase Ibeta.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11309393","citation_count":113,"is_preprint":false},{"pmid":"16990611","id":"PMC_16990611","title":"IRAG mediates NO/cGMP-dependent inhibition of platelet aggregation and thrombus formation.","date":"2006","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/16990611","citation_count":109,"is_preprint":false},{"pmid":"15483626","id":"PMC_15483626","title":"IRAG is essential for relaxation of receptor-triggered smooth muscle contraction by cGMP kinase.","date":"2004","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/15483626","citation_count":103,"is_preprint":false},{"pmid":"15388327","id":"PMC_15388327","title":"Distribution of IRAG and cGKI-isoforms in murine tissues.","date":"2004","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/15388327","citation_count":79,"is_preprint":false},{"pmid":"31273338","id":"PMC_31273338","title":"The MRVI1-AS1/ATF3 signaling loop sensitizes nasopharyngeal cancer cells to paclitaxel by regulating the Hippo-TAZ pathway.","date":"2019","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/31273338","citation_count":50,"is_preprint":false},{"pmid":"21666108","id":"PMC_21666108","title":"IRAG and novel PKG targeting in the cardiovascular system.","date":"2011","source":"American journal of physiology. Heart and circulatory physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21666108","citation_count":48,"is_preprint":false},{"pmid":"20080989","id":"PMC_20080989","title":"IRAG determines nitric oxide- and atrial natriuretic peptide-mediated smooth muscle relaxation.","date":"2010","source":"Cardiovascular research","url":"https://pubmed.ncbi.nlm.nih.gov/20080989","citation_count":43,"is_preprint":false},{"pmid":"10321731","id":"PMC_10321731","title":"Mrvi1, a common MRV integration site in BXH2 myeloid leukemias, encodes a protein with homology to a lymphoid-restricted membrane protein Jaw1.","date":"1999","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/10321731","citation_count":38,"is_preprint":false},{"pmid":"14729908","id":"PMC_14729908","title":"InsP3R-associated cGMP kinase substrate (IRAG) is essential for nitric oxide-induced inhibition of calcium signaling in human colonic smooth muscle.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14729908","citation_count":35,"is_preprint":false},{"pmid":"9100816","id":"PMC_9100816","title":"IRAG working group 4. Cell cytotoxicity assays. Interagency Regulatory Alternatives Group.","date":"1997","source":"Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association","url":"https://pubmed.ncbi.nlm.nih.gov/9100816","citation_count":35,"is_preprint":false},{"pmid":"18450420","id":"PMC_18450420","title":"cGMP-dependent protein kinase anchoring by IRAG regulates its nuclear translocation and transcriptional activity.","date":"2008","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/18450420","citation_count":32,"is_preprint":false},{"pmid":"31085718","id":"PMC_31085718","title":"miR-940 potentially promotes proliferation and metastasis of endometrial carcinoma through regulation of MRVI1.","date":"2019","source":"Bioscience reports","url":"https://pubmed.ncbi.nlm.nih.gov/31085718","citation_count":29,"is_preprint":false},{"pmid":"16166082","id":"PMC_16166082","title":"Identification of the interface between cGMP-dependent protein kinase Ibeta and its interaction partners TFII-I and IRAG reveals a common interaction motif.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16166082","citation_count":28,"is_preprint":false},{"pmid":"34734188","id":"PMC_34734188","title":"Nitric Oxide Signals Through IRAG to Inhibit TRPM4 Channels and Dilate Cerebral Arteries.","date":"2021","source":"Function (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/34734188","citation_count":25,"is_preprint":false},{"pmid":"30001348","id":"PMC_30001348","title":"Whole exome sequencing identifies MRVI1 as a susceptibility gene for moyamoya syndrome in neurofibromatosis type 1.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/30001348","citation_count":25,"is_preprint":false},{"pmid":"21244222","id":"PMC_21244222","title":"Signaling via IRAG is essential for NO/cGMP-dependent inhibition of platelet activation.","date":"2011","source":"Platelets","url":"https://pubmed.ncbi.nlm.nih.gov/21244222","citation_count":20,"is_preprint":false},{"pmid":"9100821","id":"PMC_9100821","title":"Practical application of non-whole animal alternatives: summary of IRAG workshop on eye irritation testing. Interagency Regulatory Alternatives Group.","date":"1997","source":"Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association","url":"https://pubmed.ncbi.nlm.nih.gov/9100821","citation_count":19,"is_preprint":false},{"pmid":"35141152","id":"PMC_35141152","title":"MRVI1 and NTRK3 Are Potential Tumor Suppressor Genes Commonly Inactivated by DNA Methylation in Cervical Cancer.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35141152","citation_count":10,"is_preprint":false},{"pmid":"36973749","id":"PMC_36973749","title":"Hypoxia-induced lncRNA MRVI1-AS1 accelerates hepatocellular carcinoma progression by recruiting RNA-binding protein CELF2 to stabilize SKA1 mRNA.","date":"2023","source":"World journal of surgical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/36973749","citation_count":10,"is_preprint":false},{"pmid":"32589066","id":"PMC_32589066","title":"P53-induced MRVI1 mediates carcinogenesis of colorectal cancer.","date":"2020","source":"Scandinavian journal of gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/32589066","citation_count":9,"is_preprint":false},{"pmid":"9100815","id":"PMC_9100815","title":"IRAG working group 3. Cell function-based assays. Interagency Regulatory Alternatives Group.","date":"1997","source":"Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association","url":"https://pubmed.ncbi.nlm.nih.gov/9100815","citation_count":9,"is_preprint":false},{"pmid":"37372987","id":"PMC_37372987","title":"Novel Functional Features of cGMP Substrate Proteins IRAG1 and IRAG2.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37372987","citation_count":6,"is_preprint":false},{"pmid":"33066124","id":"PMC_33066124","title":"IRAG1 Deficient Mice Develop PKG1β Dependent Pulmonary Hypertension.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/33066124","citation_count":6,"is_preprint":false},{"pmid":"21865585","id":"PMC_21865585","title":"Truncated IRAG variants modulate cGMP-mediated inhibition of human colonic smooth muscle cell contraction.","date":"2011","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21865585","citation_count":6,"is_preprint":false},{"pmid":"34064290","id":"PMC_34064290","title":"Loss of PKGIβ/IRAG1 Signaling Causes Anemia-Associated Splenomegaly.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34064290","citation_count":5,"is_preprint":false},{"pmid":"40696876","id":"PMC_40696876","title":"Selected PEAR1 and MRVI1 Gene Polymorphisms in Patients with Migraine and Sticky Platelet Syndrome.","date":"2025","source":"Clinical and applied thrombosis/hemostasis : official journal of the International Academy of Clinical and Applied Thrombosis/Hemostasis","url":"https://pubmed.ncbi.nlm.nih.gov/40696876","citation_count":1,"is_preprint":false},{"pmid":"41078212","id":"PMC_41078212","title":"Identification of MRVI1-interacting proteins by biotin-based proximity labelling reveals NPM-ALK-dependent interaction dynamics.","date":"2025","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41078212","citation_count":1,"is_preprint":false},{"pmid":"41905951","id":"PMC_41905951","title":"Multifaceted MRVI1 Serves as a Tumor Suppressor in HCT116 Colorectal Cancer Cells.","date":"2026","source":"Biological & pharmaceutical bulletin","url":"https://pubmed.ncbi.nlm.nih.gov/41905951","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16918,"output_tokens":4824,"usd":0.061557,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13263,"output_tokens":4532,"usd":0.089807,"stage2_stop_reason":"end_turn"},"total_usd":0.151364,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"IRAG forms a trimeric signaling complex with cGKIβ and IP3 receptor type I (IP3R-I) at endoplasmic reticulum membranes in smooth muscle. In microsomal smooth muscle membranes, cGKIβ phosphorylated IRAG and IP3R. Co-expression of IRAG and cGKIβ in COS-7 cells, in the presence of cGMP, inhibited bradykinin-stimulated calcium release, identifying IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.\",\n      \"method\": \"Co-immunoprecipitation with antibodies against cGKI, IP3R, and IRAG; heterologous expression in COS-7 cells; calcium release assays; mass spectrometry protein identification\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal Co-IP from native tissue, reconstitution by co-expression with functional readout, replicated across multiple labs in subsequent studies\",\n      \"pmids\": [\"10724174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The N-terminal leucine zipper (amino acids 1–53) of cGKIβ (but not cGKIα or cGKII) interacts with amino acids 152–184 of IRAG via electrostatic interaction. cGKIβ does not directly interact with IP3R-I but co-precipitates IP3R-I through IRAG. cGKIβ phosphorylates up to four serines in IRAG; phosphorylation specifically of Ser696 is necessary and sufficient to decrease IP3-dependent calcium release.\",\n      \"method\": \"Yeast two-hybrid; co-precipitation of expressed proteins; site-directed mutagenesis (four Ser→Ala mutants); calcium release assays in transfected cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro binding assays combined with mutagenesis and functional calcium readout; multiple orthogonal methods in one study\",\n      \"pmids\": [\"11309393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Targeted deletion of exon 12 of IRAG (encoding the N-terminus of the coiled-coil domain) disrupts the IRAG–IP3R-I interaction in vivo, abolishing cGMP-dependent relaxation of carbachol- and phenylephrine-contracted smooth muscle from colon and aorta and preventing cGMP-mediated decrease in norepinephrine-induced [Ca2+]i in aortic smooth muscle cells. cGMP-induced relaxation of K+-contracted smooth muscle was unaffected, indicating IRAG is specifically required for hormone receptor–triggered cGMP relaxation.\",\n      \"method\": \"Genetic knockout (exon 12 deletion); smooth muscle contraction/relaxation assays; intracellular calcium measurements (Fura-2); gastrointestinal motility assessment\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean in vivo genetic model with specific phenotypic readouts and mechanistic distinction between receptor-triggered vs. K+-induced contraction\",\n      \"pmids\": [\"15483626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Endogenous IRAG in human colonic smooth muscle cells is required for NO/cGKI-dependent inhibition of IP3-dependent Ca2+ release. Antisense knockdown of IRAG abolished sodium nitroprusside- and 8-pCPT-cGMP-mediated inhibition of bradykinin-induced calcium transients.\",\n      \"method\": \"Antisense oligonucleotide knockdown of IRAG in cultured human colonic smooth muscle cells; calcium imaging; RT-PCR\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with specific calcium phenotype in human primary cells, single lab, antisense method\",\n      \"pmids\": [\"14729908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"IRAG co-localizes with cGKI in smooth muscle of aorta and colon. Upon co-expression in COS-7 cells, IRAG recruits cGKIβ (but not cGKIα) to the endoplasmic reticulum, demonstrating isoform-specific targeting by IRAG.\",\n      \"method\": \"Immunofluorescence/immunohistochemistry in murine tissues; heterologous co-expression in COS-7 cells with confocal microscopy\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — direct localization experiments with isoform-specific functional consequence (ER recruitment), replicated in tissue and cell expression system\",\n      \"pmids\": [\"15388327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Acidic residues in the N-terminal leucine zipper dimerization domain of PKGIβ (D26 and E31) mediate binding to both TFII-I and IRAG via electrostatic interaction with basic residues in alpha-helical regions of IRAG. Mutation D26K/E31R in PKGIβ completely abrogated binding to IRAG without disrupting PKG dimerization.\",\n      \"method\": \"Site-directed mutagenesis; in vitro binding assays; co-immunoprecipitation in intact cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with in vitro binding and cell-based co-IP; defines a specific interaction interface, single lab\",\n      \"pmids\": [\"16166082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"IRAG is abundantly expressed in platelets and assembles in a macrocomplex with cGKIβ and IP3R-I. PKGIβ phosphorylates IRAG at Ser664 and Ser677 in intact platelets. Targeted disruption of the IRAG–IP3R-I interaction (IRAGΔ12/Δ12 mice) abolishes NO/cGMP-dependent inhibition of fibrinogen-receptor activation, platelet aggregation, and intracellular calcium transients, and prevents NO-mediated inhibition of arterial thrombosis in vivo.\",\n      \"method\": \"IRAG exon 12 deletion mouse model; intravital microscopy; platelet aggregation assays; calcium imaging; phosphorylation site mapping by mass spectrometry\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic model with multiple orthogonal functional readouts including intravital microscopy, Ca2+ imaging, and phosphosite identification\",\n      \"pmids\": [\"16990611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"IRAG anchors PKGIβ to the endoplasmic reticulum, preventing cGMP-induced nuclear translocation of PKGIβ and thereby reducing cGMP/PKGIβ-mediated transcriptional activation of a CRE-reporter gene. This effect required the PKGIβ–IRAG binding interface and was specific to PKGIβ (PKGIα was unaffected). A phosphorylation-deficient IRAG mutant still suppressed PKGIβ transcriptional activity, indicating the mechanism is independent of IRAG phosphorylation or changes in intracellular calcium.\",\n      \"method\": \"Co-expression of wild-type and binding-incompetent/phosphorylation-deficient IRAG mutants in baby hamster kidney cells; CRE-reporter gene assay; subcellular localization by imaging\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional reporter assay with mutants defining mechanism, single lab, two orthogonal readouts (localization + transcription)\",\n      \"pmids\": [\"18450420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Global IRAG knockout (exon 3 deletion) prevents stable interaction of IP3R-I with cGKIβ (shown by cGMP affinity chromatography) and abolishes NO-, ANP-, and cGMP-dependent relaxation of hormone-contracted aortic and colonic smooth muscle. cGKIβ/α subcellular localization in VSMCs was unchanged by IRAG loss. IRAG-deficient VSMCs failed to suppress hormone-induced Ca2+ increases in response to cGMP. Selective cGKIβ re-expression in smooth muscle from cGKIβ-transgenic mice did not rescue relaxation, confirming IRAG is obligate downstream of cGKIβ. IRAG-deficient mice showed resistance to LPS-induced blood pressure reduction.\",\n      \"method\": \"Targeted exon 3 deletion mouse model; cGMP affinity chromatography; confocal microscopy; Fura-2 calcium imaging; smooth muscle relaxation assays; telemetric blood pressure measurement; transgenic rescue experiment\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — complete knockout with multiple orthogonal mechanistic assays, transgenic rescue experiment, multiple physiological readouts\",\n      \"pmids\": [\"20080989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"IRAG-deficient murine platelets show enhanced aggregability to collagen, thrombin, and TxA2. NO/cGMP-dependent inhibition of ATP and 5-HT secretion from dense granules, P-selectin secretion from alpha granules, and GPIIb/IIIa-mediated adhesion to fibrinogen were all severely impaired in IRAG-deficient platelets, and bleeding time was reduced.\",\n      \"method\": \"IRAG knockout mouse model; platelet aggregation assays; granule secretion assays (ATP, 5-HT); flow cytometry (P-selectin); adhesion assays; bleeding time measurement\",\n      \"journal\": \"Platelets\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function with multiple specific functional readouts across platelet activation pathways\",\n      \"pmids\": [\"21244222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"C-terminally truncated IRAG variants lacking the cGKI phosphorylation site and IP3R-I interaction site act as dominant-negative modulators, counteracting cGMP-mediated inhibition of calcium transients and relaxation of human colonic smooth muscle cells. Four unique first-exon variants driven by individual promoters and extensive alternative splicing generate multiple truncated IRAG isoforms.\",\n      \"method\": \"Identification of splice variants by RT-PCR/sequencing; functional expression of truncated IRAG variants in colonic smooth muscle cells; calcium imaging; contractility assays\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional expression studies with specific calcium and contractility readouts in primary human cells, single lab\",\n      \"pmids\": [\"21865585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"IRAG, PKGIβ, and IP3Rs form a nanoscale signaling complex on the SR of vascular smooth muscle cells, identified by superresolution microscopy. PKG phosphorylation of IRAG inhibits IP3R-mediated Ca2+ release; IRAG knockdown diminished NO-mediated inhibition of TRPM4 channel activity and vasodilation. Thus, IRAG mediates NO/cGMP/PKG inhibition of IP3R-dependent TRPM4 activation to dilate cerebral arteries.\",\n      \"method\": \"IRAG knockdown in vascular SMCs; patch-clamp electrophysiology (TRPM4 currents); Ca2+ imaging; superresolution microscopy of IRAG/PKG/IP3R nanoscale complex; pharmacological inhibition of guanylyl cyclase and PKG\",\n      \"journal\": \"Function (Oxford, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (superresolution microscopy, electrophysiology, Ca2+ imaging, genetic knockdown) in one study establishing new downstream pathway\",\n      \"pmids\": [\"34734188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"IRAG1 knockout mice spontaneously develop right ventricular hypertrophy, elevated RV systolic pressure, RV dysfunction, and pulmonary hypertension under normoxic conditions. IRAG1 is expressed in PASMCs and is downregulated under hypoxia. Absence of IRAG1 reduces PKGIβ expression in lung and RV and dysregulates downstream PKGIβ candidates in the RV.\",\n      \"method\": \"Global IRAG1 KO mouse model; echocardiography; right heart catheterization; immunostaining; western blotting; PASMC isolation\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic model with hemodynamic and molecular readouts, single lab, limited mechanistic depth on pathway placement\",\n      \"pmids\": [\"33066124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Global IRAG1 KO mice develop gastrointestinal bleeding, iron-deficiency anemia, and anemia-associated splenomegaly. Loss of IRAG1 strongly decreases PKGIβ protein levels (but not mRNA) in colon, spleen, and stomach, indicating IRAG1 is required for PKGIβ protein stability.\",\n      \"method\": \"Global IRAG1 KO mouse model; western blotting; RT-PCR; histology; hematological analysis\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic model with protein stability finding, single lab, mechanistic interpretation supported by mRNA vs. protein comparison\",\n      \"pmids\": [\"34064290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Mrvi1 (IRAG1 ortholog) encodes a protein with homology to Jaw1, a lymphoid-restricted type II membrane protein localizing to the endoplasmic reticulum. Within hematopoietic cells, Mrvi1 expression is restricted to megakaryocytes and some myeloid leukemias, and is downregulated during monocytic differentiation.\",\n      \"method\": \"Retroviral insertional mutagenesis screen; sequence homology analysis; Northern blot expression analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — sequence homology and expression analysis; ER localization inferred from homology rather than direct experiment on MRVI1\",\n      \"pmids\": [\"10321731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TurboID-based proximity labeling identified >700 candidate MRVI1-interacting proteins in mammalian cells, including ER-localized factors and intracellular trafficking components. Co-expression of NPM-ALK oncogenic kinase selectively enhanced MRVI1 association with signaling-related proteins and reduced association with anti-apoptotic regulators (DDB1, PHB2, NOTCH2), suggesting MRVI1 participates in apoptosis-related networks disrupted during oncogenic transformation.\",\n      \"method\": \"TurboID proximity labeling; quantitative proteomics; co-expression of NPM-ALK\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — proximity labeling screen (not direct binding validation); functional interpretation is inferential; single lab\",\n      \"pmids\": [\"41078212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Stable overexpression of MRVI1 in HCT116 colorectal cancer cells markedly reduces cell proliferation without increasing cell death (cytostatic effect), providing direct evidence that MRVI1 suppresses colorectal cancer cell growth when overexpressed, consistent with a p53-associated tumor suppressor role.\",\n      \"method\": \"Stable overexpression of V5-TurboID-MRVI1 fusion protein; cell proliferation assays; trypan blue viability staining\",\n      \"journal\": \"Biological & pharmaceutical bulletin\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single overexpression experiment with proliferation readout, no molecular mechanism elucidated, single lab\",\n      \"pmids\": [\"41905951\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"IRAG1 (MRVI1/IRAG/JAW1L) is an endoplasmic reticulum-anchored 125 kDa membrane protein that forms an obligate trimeric signaling complex with cGMP-dependent protein kinase Iβ (PKGIβ) — via electrostatic interaction between the PKGIβ leucine zipper (residues 1–53) and IRAG1 residues 152–184 — and with IP3 receptor type I (IP3R-I); upon cGMP-driven PKGIβ phosphorylation of IRAG1 (primarily at Ser696 in rodents; Ser664/Ser677 in platelets), IRAG1 inhibits IP3R-I-mediated Ca²⁺ release from the ER, thereby mediating NO/cGMP-dependent relaxation of smooth muscle, inhibition of platelet activation and arterial thrombosis, inhibition of TRPM4 channel activation and vasodilation, and regulation of PKGIβ protein stability; additionally, ER-anchored IRAG1 prevents nuclear translocation of PKGIβ to restrict cGMP-dependent transcriptional responses, while C-terminally truncated IRAG1 splice variants lacking the phosphorylation and IP3R-I interaction domains act as dominant-negative modulators of this pathway.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"IRAG1 (MRVI1) is an endoplasmic reticulum-anchored membrane protein that serves as the central scaffold of an NO/cGMP signaling module controlling intracellular Ca²⁺ release in smooth muscle and platelets [#0]. It assembles an obligate trimeric complex with cGMP-dependent protein kinase Iβ (cGKIβ/PKGIβ) and IP3 receptor type I, binding the kinase through an electrostatic interaction between the cGKIβ N-terminal leucine zipper (residues 1–53; acidic residues D26/E31) and basic residues in IRAG1 (residues 152–184), while bridging IP3R-I, which does not contact cGKIβ directly [#1, #5]. This complex is isoform-specific: IRAG1 recruits cGKIβ — but not cGKIα or cGKII — to the ER [#4]. Upon cGMP activation, cGKIβ phosphorylates IRAG1, and phosphorylation at Ser696 is necessary and sufficient to inhibit IP3-induced Ca²⁺ release [#1]; in platelets the corresponding sites are Ser664/Ser677 [#6]. Through this mechanism IRAG1 mediates NO/cGMP-dependent relaxation of hormone receptor-triggered (but not K⁺-induced) smooth muscle contraction [#2, #8], inhibition of platelet aggregation, granule secretion, fibrinogen-receptor activation and arterial thrombosis [#6, #9], and NO-dependent inhibition of IP3R-driven TRPM4 channel activity to dilate cerebral arteries [#11]. Independently of its phosphorylation and Ca²⁺ functions, ER-anchored IRAG1 retains cGKIβ at the ER to prevent its cGMP-induced nuclear translocation and restrict CRE-dependent transcription [#7], and is required for PKGIβ protein stability [#13]. Loss of IRAG1 in mice produces pulmonary hypertension with right ventricular hypertrophy [#12] and gastrointestinal bleeding with iron-deficiency anemia [#13]. C-terminally truncated splice variants lacking the phosphorylation and IP3R-I interaction domains act as dominant-negative modulators of the pathway [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established IRAG as the missing link coupling NO/cGMP signaling to inhibition of IP3-mediated Ca²⁺ release by demonstrating it forms a functional trimeric complex with cGKIβ and IP3R-I.\",\n      \"evidence\": \"Reciprocal Co-IP from native smooth muscle microsomes plus reconstitution by co-expression in COS-7 cells with calcium-release readout\",\n      \"pmids\": [\"10724174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the binding interfaces or the relevant phosphosite\", \"Physiological requirement in vivo not yet tested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Mapped the molecular architecture of the complex, showing cGKIβ binds IRAG via its leucine zipper while IP3R-I is bridged through IRAG, and pinned the inhibitory function to phosphorylation of a single serine.\",\n      \"evidence\": \"Yeast two-hybrid, co-precipitation of expressed proteins, and Ser→Ala mutagenesis with calcium-release assays\",\n      \"pmids\": [\"11309393\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the electrostatic interface not resolved at atomic level\", \"Mapping done in heterologous/in vitro context\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Proved IRAG is physiologically required for cGMP-dependent smooth muscle relaxation, and specifically for hormone receptor-triggered rather than depolarization-induced contraction.\",\n      \"evidence\": \"Exon 12 deletion mouse disrupting IRAG–IP3R-I interaction with contraction/relaxation and Ca²⁺ assays, plus antisense knockdown in human colonic SMCs\",\n      \"pmids\": [\"15483626\", \"14729908\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Exon 12 deletion disrupts IP3R-I binding but does not eliminate all IRAG functions\", \"Did not address platelet or other tissue roles\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrated IRAG actively targets and recruits cGKIβ to the ER in an isoform-selective manner, explaining how the kinase is positioned at its substrate.\",\n      \"evidence\": \"Immunohistochemistry in murine tissue and heterologous co-expression with confocal microscopy in COS-7 cells\",\n      \"pmids\": [\"15388327\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Recruitment shown in overexpression system\", \"Determinants of isoform selectivity not fully defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined the acidic residues (D26/E31) within the cGKIβ leucine zipper that mediate electrostatic binding to IRAG, separable from kinase dimerization.\",\n      \"evidence\": \"Site-directed mutagenesis with in vitro binding and cell-based co-IP\",\n      \"pmids\": [\"16166082\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single lab\", \"Did not test functional consequence of the interface mutant in vivo\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Extended the IRAG module to platelets, showing it mediates NO/cGMP inhibition of platelet activation and arterial thrombosis and identifying the platelet phosphosites.\",\n      \"evidence\": \"IRAGΔ12 mouse with intravital microscopy, aggregation/Ca²⁺ assays, and MS phosphosite mapping (Ser664/Ser677)\",\n      \"pmids\": [\"16990611\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Distinct phosphosites between platelets and smooth muscle not mechanistically reconciled\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed a Ca²⁺-independent function of IRAG: ER anchoring of cGKIβ to block its nuclear translocation and restrain cGMP-dependent transcription.\",\n      \"evidence\": \"WT and binding/phospho-deficient IRAG mutants in BHK cells with CRE-reporter assay and localization imaging\",\n      \"pmids\": [\"18450420\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Shown in heterologous cells\", \"Transcriptional targets and physiological relevance not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Confirmed with a complete knockout that IRAG is obligate downstream of cGKIβ for NO/ANP/cGMP-dependent relaxation, ruling out compensation by cGKIβ alone.\",\n      \"evidence\": \"Exon 3 deletion KO with cGMP affinity chromatography, Ca²⁺ imaging, relaxation assays, blood pressure telemetry, and cGKIβ-transgenic rescue\",\n      \"pmids\": [\"20080989\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"LPS blood-pressure resistance mechanism not dissected\", \"Did not address non-vascular phenotypes\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Detailed the breadth of platelet inhibition mediated by IRAG across aggregation, granule secretion, and integrin-dependent adhesion.\",\n      \"evidence\": \"IRAG KO mouse with aggregation, granule secretion, P-selectin flow cytometry, adhesion, and bleeding-time assays\",\n      \"pmids\": [\"21244222\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish whether all readouts are downstream of the same IP3R-Ca²⁺ node\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified C-terminally truncated IRAG splice variants that act as dominant-negative regulators, adding an endogenous mechanism for tuning the pathway.\",\n      \"evidence\": \"Splice-variant identification and functional expression of truncated isoforms in human colonic SMCs with Ca²⁺/contractility readouts\",\n      \"pmids\": [\"21865585\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous abundance and regulation of variants in vivo unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the complex as a nanoscale SR assembly and connected IRAG to a new effector, TRPM4 channel inhibition, extending its role to cerebral artery dilation.\",\n      \"evidence\": \"Superresolution microscopy, patch-clamp TRPM4 currents, Ca²⁺ imaging, and IRAG knockdown in vascular SMCs\",\n      \"pmids\": [\"34734188\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative stoichiometry of the nanocomplex not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Linked IRAG1 loss to spontaneous pulmonary hypertension and right ventricular dysfunction, implicating the pathway in pulmonary vascular homeostasis.\",\n      \"evidence\": \"Global IRAG1 KO with echocardiography, right heart catheterization, and PASMC analysis\",\n      \"pmids\": [\"33066124\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pathway placement of downstream PKGIβ candidates not resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Uncovered a non-scaffolding requirement of IRAG1 for PKGIβ protein stability and tied its loss to gastrointestinal bleeding and anemia.\",\n      \"evidence\": \"Global IRAG1 KO with western blotting, RT-PCR (protein vs mRNA), histology, and hematology\",\n      \"pmids\": [\"34064290\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism of stabilization (degradation pathway) not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Began probing the broader IRAG1/MRVI1 interactome and a possible apoptosis/oncogenesis-related role distinct from the canonical Ca²⁺ pathway.\",\n      \"evidence\": \"TurboID proximity labeling and quantitative proteomics with NPM-ALK co-expression\",\n      \"pmids\": [\"41078212\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Proximity labeling is not direct binding validation\", \"Functional interpretation inferential\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Provided first functional evidence for a growth-suppressive (tumor suppressor-like) role of MRVI1 in colorectal cancer cells.\",\n      \"evidence\": \"Stable MRVI1 overexpression in HCT116 cells with proliferation and viability assays\",\n      \"pmids\": [\"41905951\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single overexpression experiment with no molecular mechanism\", \"p53 association asserted but not directly tested\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How IRAG1's canonical NO/cGMP Ca²⁺-regulatory scaffolding role mechanistically relates to its emerging roles in PKGIβ stability, apoptosis networks, and tumor suppression remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of the trimeric complex\", \"Degradation pathway controlling PKGIβ stability unidentified\", \"No direct binding validation or mechanism for proposed tumor-suppressor function\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 5]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 11]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [0, 4, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 8]},\n      {\"term_id\": \"R-HSA-109582\", \"supporting_discovery_ids\": [6, 9]},\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [2, 8, 11]}\n    ],\n    \"complexes\": [\"IRAG1–cGKIβ–IP3R-I trimeric complex\"],\n    \"partners\": [\"PRKG1\", \"ITPR1\", \"TRPM4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}