{"gene":"AHCYL1","run_date":"2026-06-09T22:02:42","timeline":{"discoveries":[{"year":2003,"finding":"IRBIT (AHCYL1) was identified as a novel IP3 receptor type 1 (IP3R1)-binding protein that is released from IP3R1 upon IP3 binding. The N-terminal region of IRBIT (the 104 aa appendage) is essential for interaction with IP3R1, and the IRBIT binding region maps to the IP3-binding core of IP3R1. Alkaline phosphatase treatment abolished the interaction, demonstrating that both IP3 and phosphorylation dually regulate the interaction.","method":"Affinity purification from rat brain microsomes using immobilized IP3R1 N-terminal domain, IP3 elution, in vitro binding experiments, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal biochemical purification, in vitro binding, co-IP, replicated across multiple orthogonal methods in a single rigorous study","pmids":["12525476"],"is_preprint":false},{"year":2006,"finding":"IRBIT suppresses IP3R activation by competing with IP3 for the same binding site on IP3R. Multiserine phosphorylation of IRBIT is essential for binding; 10 of 12 key IP3R residues for IP3 recognition also participate in IRBIT binding. IRBIT acts as an endogenous 'pseudoligand' of IP3R whose inhibitory activity is modulated by its phosphorylation status.","method":"[3H]IP3 binding competition assay, in vitro Ca2+ release assay, Ca2+ imaging in intact cells, mutagenesis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal functional assays (radioligand binding, in vitro Ca2+ release, live-cell imaging) plus mutagenesis in a single study","pmids":["16793548"],"is_preprint":false},{"year":2006,"finding":"IRBIT directly binds to the IP3 receptor (both the suppressor domain and the IP3-binding core are required for strong interaction). A PEST motif, a PDZ-ligand, and Asp-73 on IRBIT are critical for this interaction. IRBIT inhibits both IP3 binding and IP3-induced Ca2+ release from IP3R.","method":"Direct in vitro binding assays, mutagenesis, [3H]IP3 binding inhibition, Ca2+ release assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct binding demonstrated, mutagenesis of critical residues, functional Ca2+ release assay, single lab but multiple orthogonal methods","pmids":["16527252"],"is_preprint":false},{"year":2006,"finding":"IRBIT specifically binds to the pancreas-type splicing variant of NBC1 (pNBC1) but not to kidney-type NBC1 (kNBC1). IRBIT binds the N-terminal pNBC1-specific domain, and binding depends on phosphorylation of multiple serine residues in IRBIT. Electrophysiological analysis in Xenopus oocytes showed that pNBC1 requires IRBIT coexpression to manifest substantial transport activity.","method":"Co-immunoprecipitation, two-electrode voltage-clamp in Xenopus oocytes, mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct electrophysiology in Xenopus oocytes with co-IP and mutagenesis, multiple orthogonal methods","pmids":["16769890"],"is_preprint":false},{"year":2007,"finding":"Protein phosphatase-1 (PP1) binds to a conserved docking site on IRBIT preceding the PEST domain and mediates dephosphorylation of IRBIT Ser68. Phosphorylation of Ser68 is required for subsequent phosphorylation of Ser71 and Ser74, whereas phosphorylation of Ser71 and Ser74 (but not Ser68 alone) is sufficient to enable IRBIT inhibition of IP3 binding to IP3R. Mutational inactivation of the PP1 docking site on IRBIT increased IRBIT affinity for IP3R.","method":"In vitro PP1 binding assay, co-immunoprecipitation, site-directed mutagenesis, [3H]IP3 binding assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro binding, mutagenesis of phosphorylation sites, functional IP3 binding assay, multiple orthogonal methods in one study","pmids":["17635105"],"is_preprint":false},{"year":2008,"finding":"IRBIT binds to the C-terminal domain of NHE3 (Na+/H+ exchanger 3) and activates NHE3 activity in a Ca2+-dependent manner. IRBIT-dependent activation involves exocytic trafficking of NHE3 to the plasma membrane, and this activation is blocked by inhibition of calmodulin or CaM-dependent kinase II. Co-expression of IRBIT reverses NHERF2-dependent inhibition of NHE3.","method":"Co-immunoprecipitation, NHE3 activity assay, siRNA knockdown, cell surface biotinylation, pharmacological inhibition of CaM/CaMKII","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP, functional transport assay, siRNA knockdown, surface trafficking assay, multiple orthogonal methods in single lab","pmids":["18829453"],"is_preprint":false},{"year":2008,"finding":"IRBIT activates both basolateral pNBC1 and luminal CFTR to coordinate fluid and HCO3- secretion in the murine pancreatic duct. siRNA knockdown of IRBIT markedly inhibited ductal pNBC1 and CFTR activities, and fluid and HCO3- secretion. Activation of pNBC1 required only the IRBIT PEST domain, while activation of CFTR required multiple IRBIT domains, indicating distinct mechanisms. Single-channel measurements showed IRBIT regulated CFTR by reducing channel mean close time.","method":"Video microscopy of sealed intralobular pancreatic ducts, ion-selective microelectrodes, siRNA knockdown, single-channel patch clamp, domain deletion constructs in HEK cells","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal functional assays (fluid secretion, ion concentrations, single-channel recording), siRNA knockdown with specific phenotypic rescue, domain mapping","pmids":["19033647"],"is_preprint":false},{"year":2009,"finding":"IRBIT associates with the cleavage and polyadenylation specificity factor (CPSF) complex in a phosphorylation-dependent manner. The main IRBIT target within CPSF is the Fip1 subunit. Phosphorylation of the serine-rich region of IRBIT is required for association with Fip1 in vitro and for redistribution of Fip1 to the cytoplasm. IRBIT also interacts with poly(A) polymerase (PAP) and inhibits PAP activity in a phosphorylation-dependent manner. Oxidative stress (tBHQ) increases IRBIT phosphorylation, enhances IRBIT-CPSF interaction, and promotes cytoplasmic distribution of Fip1.","method":"Co-immunoprecipitation, in vitro binding with Fip1, in vitro PAP activity assay, RNA recruitment assay, immunofluorescence, phosphorylation analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro PAP activity assay, direct binding to Fip1, co-IP, localization, multiple orthogonal methods in single lab","pmids":["19224921"],"is_preprint":false},{"year":2010,"finding":"IRBIT mediates activation of NHE3 by angiotensin II (ANG II) via a CaMKII-dependent pathway. ANG II transiently increases IRBIT-NHE3 binding, which precedes increased NHE3 surface expression and activity. Inhibition of CaMKII blocks ANG II-induced IRBIT-NHE3 binding and NHE3 surface abundance. Mutations of IRBIT Ser-68, Ser-71, and Ser-74 decreased IRBIT binding to NHE3 and reduced ANG II-stimulated NHE3 activity.","method":"Co-immunoprecipitation, NHE3 activity assay (fluorometric intracellular pH), cell surface biotinylation, CaMKII pharmacological inhibition, siRNA knockdown, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP, functional transport assay, surface trafficking, mutagenesis of phosphorylation sites, multiple methods in single lab","pmids":["20584908"],"is_preprint":false},{"year":2011,"finding":"IRBIT opposes the WNK/SPAK kinase pathway by recruiting PP1 to the CFTR/NBCe1-B complex to dephosphorylate these transporters and restore their cell surface expression. WNK kinases act as scaffolds recruiting SPAK, which phosphorylates CFTR and NBCe1-B to reduce their surface expression. IRBIT opposes this effect and also directly stimulates transporter activity. Silencing of both SPAK and IRBIT in the same ducts rescued ductal secretion caused by IRBIT silencing alone (epistasis).","method":"siRNA knockdown, genetic epistasis (double silencing), cell surface expression assay, fluid secretion measurement in sealed pancreatic ducts, co-immunoprecipitation, phosphorylation analysis","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis by double silencing, co-IP, functional fluid secretion assay, surface expression assay, multiple orthogonal methods","pmids":["21317537"],"is_preprint":false},{"year":2011,"finding":"IRBIT relieves the autoinhibitory domain (AID) of NBCe1-B to activate it, but the stimulatory effect of IRBIT cannot be explained solely by masking autoinhibitory determinants. A mutant IRBIT resistant to protein phosphatase-1 action stimulates NBCe1-B to an extent 50% greater than removal of the AID alone. An NBCe1-B construct lacking residues 2-16 is fully autoinhibited but cannot be stimulated by IRBIT, indicating that autoinhibitory and IRBIT-binding determinants are not identical.","method":"Two-electrode voltage-clamp on Xenopus oocytes, IRBIT and NBCe1-B deletion/mutation constructs","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct electrophysiology with mutagenesis constructs, quantitative comparison of multiple NBCe1-B variants, single lab","pmids":["22012331"],"is_preprint":false},{"year":2013,"finding":"IRBIT mediates synergistic activation of CFTR and Slc26a6 by Ca2+ and cAMP signaling pathways. In resting cells, IRBIT is sequestered by IP3Rs in the ER. cAMP-dependent phosphorylation of IP3Rs reduces their affinity for IRBIT, enabling IP3-induced dissociation and translocation of IRBIT to CFTR and Slc26a6 at the plasma membrane. This synergistic mechanism was absent in Irbit-/- mice and in mice with IP3Rs bearing mutations preventing PKA phosphorylation.","method":"Knockout mice (Irbit-/- and Slc26a6-/-), co-immunoprecipitation, CFTR Cl- current measurement, intracellular pH assay (Slc26a6 activity), fluid secretion measurement, site-directed mutagenesis of IP3R PKA sites","journal":"Gastroenterology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO mice plus multiple orthogonal functional assays and mechanistic mutagenesis, independently validated in two gland types","pmids":["23542070"],"is_preprint":false},{"year":2013,"finding":"A positively charged module within NBCe1-B (residues 37-65) is required for interaction with and activation by IRBIT and for regulation by PIP2. Phosphorylation of Thr49 is required for regulation by both IRBIT and SPAK. A homologous sequence exists in the CFTR R domain and Slc26a6 STAS domain, both of which bind IRBIT; the R domain is required for CFTR activation by IRBIT.","method":"Co-immunoprecipitation, mutagenesis, electrophysiology in Xenopus oocytes, domain deletion analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — functional electrophysiology, co-IP, systematic mutagenesis identifying critical phosphorylation sites and binding domains, multiple transporters tested","pmids":["23431199"],"is_preprint":false},{"year":2014,"finding":"IRBIT forms a dATP-dependent complex with ribonucleotide reductase (RNR), stabilizes dATP in the activity site of RNR, and thereby inhibits RNR enzymatic activity. Formation of the RNR-IRBIT complex is regulated by IRBIT phosphorylation. Ablation of IRBIT in HeLa cells causes imbalanced dNTP pools and altered cell cycle progression.","method":"Co-immunoprecipitation of RNR-IRBIT complex, RNR enzymatic activity assay, IRBIT knockdown in HeLa cells with dNTP pool measurement, cell cycle analysis","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro RNR activity assay, co-IP of complex, functional cellular readout (dNTP pools, cell cycle), multiple orthogonal methods in single lab","pmids":["25237103"],"is_preprint":false},{"year":2015,"finding":"IRBIT binds to CaMKIIα and suppresses its kinase activity by inhibiting the binding of calmodulin to CaMKIIα. In IRBIT-deficient mice, phosphorylation of tyrosine hydroxylase (TH) by CaMKIIα is significantly increased in the ventral tegmental area, leading to elevated catecholamine levels and behavioral abnormalities.","method":"Co-immunoprecipitation, CaMKIIα kinase activity assay, IRBIT knockout mice, TH phosphorylation analysis by immunoblot, behavioral assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP, in vitro/cellular kinase assay, KO mouse with specific biochemical (phospho-TH) and behavioral phenotypes, multiple orthogonal methods","pmids":["25922519"],"is_preprint":false},{"year":2015,"finding":"IRBIT forms signaling complexes with phosphatidylinositol phosphate kinases (PIPKs), specifically interacting with PIPKIα and PIPKIIα in mouse cerebellum. Two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are required for interaction with IRBIT. Phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfere with the interaction, indicating IRBIT interacts with the catalytic core. Mutations in the serine-rich region of IRBIT affect selectivity for PIPKIα vs PIPKIIα. IRBIT and PIPKIα interact with NBCe1-B.","method":"Co-immunoprecipitation from mouse cerebellum, heterologous expression binding assays, site-directed mutagenesis, immunocytochemistry, in vitro binding","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — co-IP from tissue and heterologous system, mutagenesis of catalytic residues, localization, single lab","pmids":["26509711"],"is_preprint":false},{"year":2016,"finding":"IRBIT interacts with the Bcl-2 homolog Bcl2l10 and together they exert additive inhibition of IP3R. Both proteins associate in mitochondria-associated membranes (MAMs). Upon apoptotic stress, IRBIT is dephosphorylated and becomes an inhibitor of Bcl2l10. IRBIT promotes ER-mitochondria contact. By inhibiting Bcl2l10 and promoting ER-mitochondria contact, IRBIT facilitates Ca2+ transfer to mitochondria and promotes apoptosis.","method":"Co-immunoprecipitation, subcellular fractionation (MAM isolation), Ca2+ imaging, cell death assays, dephosphorylation experiments, IP3R activity assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP, subcellular fractionation, functional Ca2+ and apoptosis assays, mechanistic model supported by multiple methods in single lab","pmids":["27995898"],"is_preprint":false},{"year":2016,"finding":"NHERF1, NHE3, and IRBIT form a macrocomplex. ANG II induces a concomitant increase in NHERF1 interactions with NHE3 and IRBIT requiring the NHERF1 PDZ1 domain. IRBIT is indispensable for ANG II-provoked increase in NHERF1-NHE3 interactions, and phosphorylation of IRBIT at Ser68 is necessary for assembly of the NHERF1-IRBIT-NHE3 complex and NHE3 activation.","method":"Co-immunoprecipitation, dominant negative PDZ1 overexpression, siRNA knockdown of IRBIT, cell surface biotinylation, fluorescence imaging","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — co-IP, dominant negative, knockdown, surface trafficking assay, single lab with multiple methods","pmids":["27279487"],"is_preprint":false},{"year":2017,"finding":"IRBIT family proteins form homo- and heteromultimers. N-terminal splicing of Long-IRBIT changes protein stability and selectivity for target molecules. Different IRBIT family members exhibit distinct mRNA expression patterns and different target selectivities determined by N-terminal diversity and multimer combinations.","method":"Co-immunoprecipitation, protein stability assays, target binding assays with multiple IRBIT family constructs, RT-PCR expression analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP for multimerization, binding selectivity assays, single lab with multiple constructs","pmids":["28348216"],"is_preprint":false},{"year":2018,"finding":"IRBIT controls five phosphorylation sites in NBCe1-B that determine active vs. inactive transporter conformation and regulate sensitivity to intracellular Cl-. IRBIT recruits PP1 and SPAK to control phosphorylation of Ser65 (affecting Cl- sensing via 32GXXXP36 motif), and recruits calcineurin and CaMKII to control phosphorylation of Ser12 (affecting Cl- sensing via 194GXXXP198 motif). Phosphorylation status of Ser232, Ser233, Ser235 determines active/inactive conformations of NBCe1-B.","method":"Phosphoproteomic analysis, mutagenesis, electrophysiology, co-immunoprecipitation of recruited kinases/phosphatases","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — phosphoproteomics plus systematic mutagenesis plus electrophysiology plus co-IP of kinase/phosphatase recruitment, multiple orthogonal methods","pmids":["30377224"],"is_preprint":false},{"year":2020,"finding":"IRBIT activates NBCe1-B by releasing the autoinhibitory domain (AID, acting as a brake) from the transmembrane domain via competitive binding. The AID (residues 40-85) binds to the transmembrane domain via electrostatic attraction to slow NBCe1-B. IRBIT binding domain spans residues 1-52 with two arms (negatively charged residues 1-24 and positively charged residues 40-52). Negatively charged Asp/Glu residues plus Ser/Thr residues in the IRBIT PEST domain are required for NBCe1-B interaction.","method":"Systematic mutagenesis of NBCe1-B and IRBIT, two-electrode voltage-clamp in Xenopus oocytes, co-immunoprecipitation","journal":"The Journal of physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis combined with quantitative electrophysiology, mechanistic model validated, single lab","pmids":["33237573"],"is_preprint":false},{"year":2020,"finding":"IRBIT expression in intestinal stem cell progeny in the Drosophila midgut regulates their differentiation into enterocytes via suppression of RNR activity. Disruption of the IRBIT-RNR regulatory circuit causes premature loss of intestinal tissue integrity, and age-related dysplasia can be reversed by suppression of RNR activity in ISC progeny.","method":"Drosophila genetics (IRBIT loss-of-function, RNR inhibition), histology of midgut epithelium, intestinal stem cell differentiation assays","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function in Drosophila with specific differentiation phenotype and epistasis with RNR, replicated by independent RNR suppression","pmids":["32179478"],"is_preprint":false},{"year":2021,"finding":"AHCYL1 (IRBIT) acts as a SAH (S-adenosyl-l-homocysteine) sensor to inhibit autophagy through PIK3C3. The C-terminus of AHCYL1 interacts with SAH specifically, and this SAH binding promotes the N-terminus to bind the catalytic domain of PIK3C3 and inhibit it, independently of MTORC1.","method":"Co-immunoprecipitation, PIK3C3 activity assay, autophagy flux assay, AHCYL1 deletion constructs, in vivo validation","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — enzymatic activity assay of PIK3C3, domain dissection by co-IP, in vivo validation, multiple orthogonal methods in single lab","pmids":["33993848"],"is_preprint":false},{"year":2021,"finding":"Both IRBIT and Long-IRBIT interact with anion exchanger AE2 through the conserved AHCY-homologous domain of IRBIT/L-IRBIT binding the N-terminal cytoplasmic region of AE2. L-IRBIT KO reduces AE2 activity and protein expression, while IRBIT KO does not. IRBIT binding facilitates lysosomal degradation of AE2, which is inhibited by coexisting L-IRBIT, revealing opposing regulatory roles.","method":"Co-immunoprecipitation, AE2 activity assay (intracellular pH), IRBIT/L-IRBIT KO cells, bafilomycin A1 treatment (lysosomal inhibition), cell volume regulation assay, cell migration assay","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO cells, functional AE2 assay, lysosomal inhibitor rescue, co-IP, multiple orthogonal methods identifying opposing roles of two paralogs","pmids":["33727633"],"is_preprint":false},{"year":2022,"finding":"Endogenous tau physically interacts with AHCYL1/IRBIT in brain tissues and cultured cells. Tau overexpression modifies the close localization of AHCYL1/IRBIT to IP3R at the endoplasmic reticulum.","method":"Protein microarray binding assay with recombinant tau, co-immunoprecipitation from brain tissue and cultured cells, proximity ligation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP from tissue, proximity ligation assay for localization change, multiple methods but no functional readout of the interaction","pmids":["35218773"],"is_preprint":false},{"year":2025,"finding":"IRBIT overexpression in mice using AAV9 causes cardiac hypertrophy with impaired systolic function. IRBIT overexpression decreases Ca2+ transient amplitude, slows Ca2+ transient rise, reduces Ca2+ wave propagation velocity, increases the dyssynchrony index, and reduces nuclear envelope invaginations in isolated cardiomyocytes. IP3R mRNA levels are decreased in IRBIT-overexpressing hearts. AAV9-shRNA reduction of IRBIT expression does not alter heart morphometric parameters.","method":"AAV9-mediated overexpression and shRNA knockdown in mice, echocardiography, isolated cardiomyocyte Ca2+ imaging, fluorescence microscopy","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo AAV overexpression model with functional cardiac and Ca2+ imaging readouts, single lab","pmids":["39929439"],"is_preprint":false},{"year":2025,"finding":"AHCYL1 interacts with PREX2 (a Rac1 GEF) and enhances PREX2 GEF activity by alleviating mutual inhibition between PREX2 and PTEN, leading to increased RAC1 activation and NSCLC cell growth.","method":"Pull-down assay with LC-MS/MS identification, co-immunoprecipitation, in vitro GEF activity assay, active RAC1 pull-down assay, PREX2/AHCYL1 knockdown, xenograft mouse model","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro GEF assay, pull-down identification, co-IP, functional RAC1 activation assay, single lab","pmids":["40365293"],"is_preprint":false},{"year":2025,"finding":"IRBIT and LIMA1 associate with the Cl-/HCO3- exchanger SLC26A3 (DRA) under basal conditions, and this association increases with cAMP/ATP stimulation. KD of IRBIT or LIMA1 reduces cAMP/Ca2+-stimulated DRA activity without altering basal activity. Maximum ATP (but not maximal forskolin) stimulation of DRA is IRBIT-dependent. cAMP/ATP-induced elevation of intracellular Ca2+ is also IRBIT-dependent. cAMP/ATP stimulation increases coprecipitation of LIMA1 with both IRBIT and DRA.","method":"Co-immunoprecipitation, siRNA knockdown, DRA activity assay (intracellular pH), Ca2+ imaging, cell surface biotinylation","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — co-IP, knockdown with functional assay, Ca2+ imaging, multiple methods in single lab","pmids":["40569378"],"is_preprint":false},{"year":2026,"finding":"Phosphorylation of IRBIT S80, S84, and S85 (by casein kinase 2) provides binding sites for the IP3-binding core (IBC) of IP3R and is sufficient to compete with IP3 for the IP3-binding pocket. S68 is the predominant phosphorylation site but is not required for IP3R binding. PKA phosphorylates S62/S64/S66; CK2 phosphorylates S80/T82/S84/S85. IRBIT-S68A/S80D/S84D/S85D was sufficient to inhibit IP3R-mediated Ca2+ release in living cells.","method":"In vitro kinase assay (PKA, CK2), pulldown assay with IBC domain, computational binding estimation, Ca2+ imaging in living cells, site-directed mutagenesis","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, direct binding pulldown with defined phosphorylation mutants, functional Ca2+ imaging, computational binding model, multiple orthogonal methods","pmids":["42032162"],"is_preprint":false},{"year":2025,"finding":"NAD modulates NBCe1-B activation by serving as a cofactor of IRBIT or L-IRBIT. Blocking the NAD salvage pathway decreases NBCe1-B activation by the IRBITs. The oxidized form NAD+ enhances, while the reduced form NADH decreases, NBCe1-B activity, linking cellular redox state to IRBIT-dependent pH regulation.","method":"Electrophysiology (Xenopus oocyte or mammalian cell NBCe1-B activity assay), NAD salvage pathway inhibition, NAD+/NADH administration, IRBIT constructs","journal":"Science China. Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional electrophysiology with pharmacological manipulation of NAD redox state, single lab","pmids":["39985648"],"is_preprint":false}],"current_model":"AHCYL1/IRBIT is a multifunctional scaffold protein that acts as a phosphorylation-dependent pseudoligand of the IP3 receptor (competing with IP3 at its binding core to inhibit Ca2+ release), and, upon release from IP3R in response to IP3, translocates to activate multiple ion transporters (pNBC1/NBCe1-B, NHE3, CFTR, Slc26a6, AE2, DRA/SLC26A3) by relieving autoinhibitory domains and recruiting kinases (SPAK, CaMKII) and phosphatases (PP1, calcineurin) to fine-tune transporter phosphorylation; it also inhibits ribonucleotide reductase (RNR) in a dATP-dependent manner to regulate dNTP pools and cell cycle progression, suppresses CaMKIIα activity to control catecholamine homeostasis, inhibits autophagy by sensing SAH and blocking PIK3C3, promotes apoptosis via MAM association with Bcl2l10 and ER-mitochondria contact, and modulates mRNA polyadenylation by binding CPSF/Fip1 and inhibiting poly(A) polymerase—with all these activities gated by multisite serine phosphorylation in its N-terminal PEST domain and regulated by NAD redox state as a cofactor."},"narrative":{"mechanistic_narrative":"AHCYL1/IRBIT is a phosphorylation-gated scaffold and pseudoligand that couples IP3 receptor (IP3R) Ca2+ signaling to the activation of epithelial ion transporters [PMID:12525476, PMID:16793548, PMID:23542070]. In resting cells it is sequestered on the IP3R, where multisite serine phosphorylation in its N-terminal PEST region lets it occupy the IP3-binding core and competitively suppress IP3 binding and Ca2+ release, acting as an endogenous pseudoligand [PMID:16793548, PMID:16527252, PMID:42032162]. Phosphorylation of IRBIT is set by a PP1 docking site and an ordered serine cascade, with PKA and CK2 phosphorylating distinct N-terminal serines that license IP3R binding [PMID:17635105, PMID:42032162]. cAMP/PKA phosphorylation of the IP3R lowers its affinity for IRBIT, so IP3 releases IRBIT to translocate to the plasma membrane and activate bicarbonate/chloride transporters—pNBC1/NBCe1-B, CFTR, Slc26a6, and DRA/SLC26A3—thereby integrating Ca2+ and cAMP signals to drive fluid and HCO3- secretion [PMID:19033647, PMID:23542070, PMID:25237103, PMID:40569378]. Mechanistically, IRBIT activates NBCe1-B by competitively relieving its autoinhibitory domain and by recruiting opposing kinases and phosphatases (SPAK and CaMKII versus PP1 and calcineurin) to control a defined set of transporter phosphosites that also tune intracellular Cl- sensing [PMID:21317537, PMID:22012331, PMID:30377224, PMID:33237573]; it activates NHE3 through Ca2+/CaMKII-dependent surface trafficking within a NHERF1 macrocomplex [PMID:18829453, PMID:20584908, PMID:27279487]. Beyond transport, IRBIT moonlights in nucleotide metabolism by forming a dATP-dependent complex with ribonucleotide reductase to inhibit it and balance dNTP pools and cell-cycle progression [PMID:25237103], suppresses CaMKIIα by blocking calmodulin binding to control tyrosine hydroxylase phosphorylation and catecholamine homeostasis [PMID:25922519], senses S-adenosylhomocysteine to inhibit PIK3C3 and autophagy [PMID:33993848], and promotes apoptosis through Bcl2l10 inhibition and ER–mitochondria Ca2+ transfer at mitochondria-associated membranes [PMID:27995898]. Its activities are further shaped by NAD redox state acting as a cofactor and by homo/heteromultimerization with the Long-IRBIT paralog, which can confer opposing regulatory outcomes such as lysosomal versus stabilized AE2 [PMID:33727633, PMID:39985648, PMID:28348216].","teleology":[{"year":2003,"claim":"Established IRBIT as a physical partner of the IP3R whose binding is governed jointly by IP3 occupancy and phosphorylation, defining it as a regulated IP3R-associated protein.","evidence":"Affinity purification from rat brain microsomes with IP3 elution, in vitro binding and co-IP","pmids":["12525476"],"confidence":"High","gaps":["Functional consequence of the interaction not yet defined","Identity of the kinases setting the phospho-dependence unknown"]},{"year":2006,"claim":"Defined the inhibitory mechanism: IRBIT is a phosphorylation-dependent pseudoligand that competes with IP3 at the same recognition residues to suppress Ca2+ release.","evidence":"[3H]IP3 competition, in vitro Ca2+ release, live-cell Ca2+ imaging, mutagenesis; direct binding mapping to suppressor and IP3-binding core domains","pmids":["16793548","16527252"],"confidence":"High","gaps":["Which serines and kinases drive the competent phospho-state not yet resolved","Stoichiometry of IRBIT:IP3R unknown"]},{"year":2006,"claim":"Showed IRBIT is not solely an IP3R inhibitor but also a required activator of an epithelial transporter, revealing a translocation-based dual function.","evidence":"Co-IP and two-electrode voltage-clamp in Xenopus oocytes showing pNBC1 requires IRBIT for activity","pmids":["16769890"],"confidence":"High","gaps":["Mechanism of transporter activation not yet defined","Trigger linking IP3R release to transporter binding unknown"]},{"year":2007,"claim":"Mapped the phospho-regulatory logic: a PP1 docking site and an ordered Ser68→Ser71/Ser74 cascade set IRBIT affinity for the IP3R.","evidence":"In vitro PP1 binding, co-IP, phosphosite mutagenesis, [3H]IP3 binding assay","pmids":["17635105"],"confidence":"High","gaps":["Upstream kinases for each serine not identified here","How dephosphorylation is triggered physiologically unknown"]},{"year":2008,"claim":"Extended IRBIT's activator role to NHE3 and to coordinated CFTR/pNBC1 secretion, establishing it as a hub for epithelial fluid and HCO3- transport.","evidence":"Co-IP, NHE3 transport and surface biotinylation, siRNA, single-channel patch clamp and fluid secretion in sealed pancreatic ducts, domain mapping","pmids":["18829453","19033647"],"confidence":"High","gaps":["Distinct domain requirements across transporters mechanistically unexplained","How a single IRBIT pool coordinates basolateral and luminal targets unknown"]},{"year":2009,"claim":"Revealed a transcription-independent moonlighting role: phospho-IRBIT binds CPSF/Fip1 and inhibits poly(A) polymerase, linking IRBIT phosphorylation and oxidative stress to mRNA polyadenylation.","evidence":"Co-IP, in vitro Fip1 binding, in vitro PAP activity assay, Fip1 redistribution imaging, tBHQ stress","pmids":["19224921"],"confidence":"High","gaps":["Physiological mRNA targets not identified","Relationship to IRBIT's membrane functions unclear"]},{"year":2011,"claim":"Defined the activation mechanism for NBCe1-B as relief of an autoinhibitory domain combined with recruited phosphatase action, and placed IRBIT in opposition to the WNK/SPAK kinase pathway.","evidence":"Electrophysiology with NBCe1-B/IRBIT mutants; siRNA epistasis (SPAK/IRBIT double silencing), co-IP and fluid secretion in ducts","pmids":["22012331","21317537"],"confidence":"High","gaps":["The extra stimulatory component beyond AID masking not fully explained","Generalizability of PP1 recruitment to other transporters not yet tested here"]},{"year":2013,"claim":"Established the Ca2+/cAMP integration model: PKA phosphorylation of IP3R releases IRBIT for synergistic activation of CFTR and Slc26a6, validated in knockout mice.","evidence":"Irbit-/- and Slc26a6-/- mice, CFTR currents, Slc26a6 pH assay, fluid secretion, IP3R PKA-site mutagenesis; shared positively charged module mapped across NBCe1-B/CFTR R domain/Slc26a6 STAS","pmids":["23542070","23431199"],"confidence":"High","gaps":["In vivo relevance to human secretory disease not addressed","How Thr49/Ser65 phospho-states are dynamically controlled in vivo unknown"]},{"year":2014,"claim":"Uncovered a metabolic function: IRBIT inhibits ribonucleotide reductase in a dATP-dependent manner, coupling it to dNTP pool balance and cell-cycle progression.","evidence":"Co-IP of RNR-IRBIT complex, RNR activity assay, IRBIT knockdown in HeLa with dNTP and cell-cycle readouts","pmids":["25237103"],"confidence":"High","gaps":["Structural basis of dATP stabilization not resolved","Coordination with IRBIT's signaling roles unknown"]},{"year":2015,"claim":"Broadened IRBIT's regulatory reach to CaMKIIα suppression and catecholamine control, and to interactions with lipid-kinase catalytic cores.","evidence":"Co-IP, CaMKIIα kinase assay and IRBIT KO mice with phospho-TH and behavioral phenotypes; co-IP with PIPKIα/PIPKIIα catalytic residues from cerebellum","pmids":["25922519","26509711"],"confidence":"High","gaps":["PIPK interaction lacks an in vivo functional readout","How CaMKII suppression is balanced against CaMKII-dependent transporter activation unclear"]},{"year":2016,"claim":"Showed IRBIT operates at ER-mitochondria contacts, where dephosphorylated IRBIT inhibits Bcl2l10 and promotes pro-apoptotic Ca2+ transfer.","evidence":"Co-IP, MAM fractionation, Ca2+ imaging, cell death assays, dephosphorylation experiments; NHERF1-IRBIT-NHE3 macrocomplex assembly via Ser68","pmids":["27995898","27279487"],"confidence":"High","gaps":["Trigger coupling apoptotic dephosphorylation to MAM localization not defined","Stoichiometry within the NHERF1-IRBIT-NHE3 complex unknown"]},{"year":2017,"claim":"Established that IRBIT family members multimerize and that N-terminal splicing dictates stability and target selectivity, providing a combinatorial code for IRBIT function.","evidence":"Co-IP for multimerization, protein stability and target-binding selectivity assays across family constructs, expression analysis","pmids":["28348216"],"confidence":"Medium","gaps":["Physiological abundance of specific multimer combinations unknown","Single-lab characterization without in vivo validation"]},{"year":2018,"claim":"Resolved the full phospho-regulatory circuit by which IRBIT controls NBCe1-B conformation and Cl- sensitivity through recruited kinases and phosphatases at defined sites.","evidence":"Phosphoproteomics, mutagenesis, electrophysiology, co-IP of recruited SPAK/CaMKII/PP1/calcineurin","pmids":["30377224"],"confidence":"High","gaps":["Dynamic switching between recruited enzymes in vivo not directly observed","Generalization of the multi-enzyme recruitment model to other transporters untested"]},{"year":2020,"claim":"Defined the structural mechanism of NBCe1-B activation as competitive electrostatic displacement of the autoinhibitory domain by IRBIT's bipartite binding arms, and linked IRBIT-RNR control to tissue homeostasis in vivo.","evidence":"Systematic NBCe1-B/IRBIT mutagenesis with voltage-clamp electrophysiology and co-IP; Drosophila midgut genetics with RNR epistasis","pmids":["33237573","32179478"],"confidence":"High","gaps":["Drosophila finding is Medium-confidence model-organism genetics","Direct structural data on the IRBIT-NBCe1-B interface lacking"]},{"year":2021,"claim":"Identified two new ligand-sensing and adaptor functions: SAH-sensing inhibition of PIK3C3/autophagy, and paralog-specific control of AE2 stability and activity.","evidence":"Co-IP, PIK3C3 activity and autophagy flux assays with domain dissection and in vivo validation; AE2 activity assays in IRBIT/L-IRBIT KO cells with bafilomycin rescue","pmids":["33993848","33727633"],"confidence":"High","gaps":["Physiological conditions raising SAH to engage IRBIT not defined","Structural basis of opposing IRBIT vs L-IRBIT effects on AE2 unknown"]},{"year":2022,"claim":"Linked IRBIT to neurodegeneration-relevant biology by showing tau interacts with IRBIT and alters its proximity to the IP3R.","evidence":"Protein microarray, co-IP from brain and cells, proximity ligation assay","pmids":["35218773"],"confidence":"Medium","gaps":["No functional consequence of the tau-IRBIT interaction measured","Disease relevance not established"]},{"year":2025,"claim":"Connected IRBIT dosage to cardiac and redox physiology and to a cancer growth pathway, expanding its in vivo and disease-associated roles.","evidence":"AAV9 IRBIT overexpression/knockdown in mice with echocardiography and cardiomyocyte Ca2+ imaging; NAD salvage/redox electrophysiology on NBCe1-B; PREX2-AHCYL1 GEF assays, RAC1 activation, and NSCLC xenografts; IRBIT/LIMA1/SLC26A3 co-IP and activity assays","pmids":["39929439","39985648","40365293","40569378"],"confidence":"Medium","gaps":["Single-lab in vivo models for cardiac and cancer roles","Mechanism linking NAD redox cofactor binding to IRBIT activation not structurally defined","Whether PREX2/RAC1 role intersects IRBIT's IP3R functions unknown"]},{"year":2026,"claim":"Refined the pseudoligand mechanism by mapping the specific CK2 and PKA phosphosites that create the IP3-binding-core docking surface sufficient to inhibit Ca2+ release.","evidence":"In vitro PKA/CK2 kinase assays, IBC pulldown with defined phospho-mutants, computational binding estimation, Ca2+ imaging","pmids":["42032162"],"confidence":"High","gaps":["In vivo kinase regulation of these sites not demonstrated","How these sites coordinate with transporter-activation phospho-states unclear"]},{"year":null,"claim":"How IRBIT's many parallel functions—IP3R suppression, transporter activation, RNR inhibition, CaMKII/PIK3C3/PREX2 regulation, and apoptosis—are partitioned spatially and temporally within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of full-length IRBIT in any complex","Quantitative competition between competing binding partners untested","Mechanism selecting which function dominates under a given stimulus unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,2,16,22,26]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[9,17,19,27]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[1,2,28]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[22,29]}],"localization":[{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[11,16,24]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,5,6,11]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7,13]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[16]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[3,6,11,13,23,27]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,11,26]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[16]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[22]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[7]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[13]}],"complexes":["CPSF (Fip1)","ribonucleotide reductase (RNR) complex","NHERF1-IRBIT-NHE3 macrocomplex"],"partners":["ITPR1","NBCE1-B/SLC4A4 (PNBC1)","CFTR","SLC26A6","SLC26A3 (DRA)","NHE3/SLC9A3","CAMK2A","PIK3C3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43865","full_name":"S-adenosylhomocysteine hydrolase-like protein 1","aliases":["DC-expressed AHCY-like molecule","IP(3)Rs binding protein released with IP(3)","IRBIT","Putative adenosylhomocysteinase 2","S-adenosyl-L-homocysteine hydrolase 2","AdoHcyase 2"],"length_aa":530,"mass_kda":59.0,"function":"Multifaceted cellular regulator which coordinates several essential cellular functions including regulation of epithelial HCO3(-) and fluid secretion, mRNA processing and DNA replication. Regulates ITPR1 sensitivity to inositol 1,4,5-trisphosphate, competing for the common binding site and acting as endogenous 'pseudoligand' whose inhibitory activity can be modulated by its phosphorylation status. Promotes the formation of contact points between the endoplasmic reticulum (ER) and mitochondria, facilitating transfer of Ca(2+) from the ER to mitochondria (PubMed:27995898). Under normal cellular conditions, functions cooperatively with BCL2L10 to limit ITPR1-mediated Ca(2+) release but, under apoptotic stress conditions, dephosphorylated which promotes dissociation of both AHCYL1 and BCL2L10 from mitochondria-associated endoplasmic reticulum membranes, inhibits BCL2L10 interaction with ITPR1 and leads to increased Ca(2+) transfer to mitochondria which promotes apoptosis (PubMed:27995898). In the pancreatic and salivary ducts, at resting state, attenuates inositol 1,4,5-trisphosphate-induced calcium release by interacting with ITPR1 (PubMed:16793548). When extracellular stimuli induce ITPR1 phosphorylation or inositol 1,4,5-trisphosphate production, dissociates from ITPR1 to interact with CFTR and SLC26A6, mediating their synergistic activation by calcium and cAMP that stimulates the epithelial secretion of electrolytes and fluid (By similarity). Also activates basolateral SLC4A4 isoform 1 to coordinate fluid and HCO3(-) secretion (PubMed:16769890). Inhibits the effect of STK39 on SLC4A4 and CFTR by recruiting PP1 phosphatase which activates SLC4A4, SLC26A6 and CFTR through dephosphorylation (By similarity). Mediates the induction of SLC9A3 surface expression produced by Angiotensin-2 (PubMed:20584908). Depending on the cell type, activates SLC9A3 in response to calcium or reverses SLC9A3R2-dependent calcium inhibition (PubMed:18829453). May modulate the polyadenylation state of specific mRNAs, both by controlling the subcellular location of FIP1L1 and by inhibiting PAPOLA activity, in response to a stimulus that alters its phosphorylation state (PubMed:19224921). Acts as a (dATP)-dependent inhibitor of ribonucleotide reductase large subunit RRM1, controlling the endogenous dNTP pool and ensuring normal cell cycle progression (PubMed:25237103). In vitro does not exhibit any S-adenosyl-L-homocysteine hydrolase activity (By similarity)","subcellular_location":"Endoplasmic reticulum; Cytoplasm, cytosol; Apical cell membrane; Microsome","url":"https://www.uniprot.org/uniprotkb/O43865/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AHCYL1","classification":"Not Classified","n_dependent_lines":447,"n_total_lines":1208,"dependency_fraction":0.37003311258278143},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PIP4K2A","stoichiometry":10.0},{"gene":"AGAP3","stoichiometry":0.2},{"gene":"CANX","stoichiometry":0.2},{"gene":"MIF","stoichiometry":0.2},{"gene":"PIP5K2B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/AHCYL1","total_profiled":1310},"omim":[{"mim_id":"616520","title":"ADENOSYLHOMOCYSTEINASE-LIKE 2; AHCYL2","url":"https://www.omim.org/entry/616520"},{"mim_id":"607826","title":"ADENOSYLHOMOCYSTEINASE-LIKE 1; AHCYL1","url":"https://www.omim.org/entry/607826"},{"mim_id":"603345","title":"SOLUTE CARRIER FAMILY 4 (SODIUM BICARBONATE COTRANSPORTER), MEMBER 4; SLC4A4","url":"https://www.omim.org/entry/603345"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/AHCYL1"},"hgnc":{"alias_symbol":["XPVKONA","IRBIT","PPP1R78"],"prev_symbol":[]},"alphafold":{"accession":"O43865","domains":[{"cath_id":"3.40.50.1480","chopping":"91-283_450-505","consensus_level":"medium","plddt":95.2241,"start":91,"end":505},{"cath_id":"3.40.50.720","chopping":"290-446","consensus_level":"medium","plddt":95.7598,"start":290,"end":446}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43865","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43865-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43865-F1-predicted_aligned_error_v6.png","plddt_mean":85.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AHCYL1","jax_strain_url":"https://www.jax.org/strain/search?query=AHCYL1"},"sequence":{"accession":"O43865","fasta_url":"https://rest.uniprot.org/uniprotkb/O43865.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43865/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43865"}},"corpus_meta":[{"pmid":"12525476","id":"PMC_12525476","title":"IRBIT, 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IRBIT is a novel regulator of ribonucleotide reductase in higher eukaryotes.","date":"2014","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/25237103","citation_count":45,"is_preprint":false},{"pmid":"24518248","id":"PMC_24518248","title":"IRBIT: a regulator of ion channels and ion transporters.","date":"2014","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/24518248","citation_count":40,"is_preprint":false},{"pmid":"25922519","id":"PMC_25922519","title":"IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/25922519","citation_count":38,"is_preprint":false},{"pmid":"16527252","id":"PMC_16527252","title":"Binding of IRBIT to the IP3 receptor: determinants and functional effects.","date":"2006","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/16527252","citation_count":38,"is_preprint":false},{"pmid":"18536033","id":"PMC_18536033","title":"The IRBIT domain adds new functions to the AHCY family.","date":"2008","source":"BioEssays : news and reviews in molecular, cellular and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/18536033","citation_count":36,"is_preprint":false},{"pmid":"23026752","id":"PMC_23026752","title":"The WNK/SPAK and IRBIT/PP1 pathways in epithelial fluid and electrolyte transport.","date":"2012","source":"Physiology (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/23026752","citation_count":31,"is_preprint":false},{"pmid":"19224921","id":"PMC_19224921","title":"Inositol 1,4,5-triphosphate receptor-binding protein released with inositol 1,4,5-triphosphate (IRBIT) associates with components of the mRNA 3' processing machinery in a phosphorylation-dependent manner and inhibits polyadenylation.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19224921","citation_count":31,"is_preprint":false},{"pmid":"27062503","id":"PMC_27062503","title":"Mutations in the IRBIT domain of ITPR1 are a frequent cause of autosomal dominant nonprogressive congenital ataxia.","date":"2016","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/27062503","citation_count":31,"is_preprint":false},{"pmid":"23145124","id":"PMC_23145124","title":"AHCYL1 is mediated by estrogen-induced ERK1/2 MAPK cell signaling and microRNA regulation to effect functional aspects of the avian oviduct.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23145124","citation_count":29,"is_preprint":false},{"pmid":"33993848","id":"PMC_33993848","title":"AHCYL1 senses SAH to inhibit autophagy through interaction with PIK3C3 in an MTORC1-independent manner.","date":"2021","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/33993848","citation_count":28,"is_preprint":false},{"pmid":"16754674","id":"PMC_16754674","title":"Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development: a possible role for AHCYL1 in inositol phospholipid signaling.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16754674","citation_count":27,"is_preprint":false},{"pmid":"22929228","id":"PMC_22929228","title":"ROCK-phosphorylated vimentin modifies mutant huntingtin aggregation via sequestration of IRBIT.","date":"2012","source":"Molecular neurodegeneration","url":"https://pubmed.ncbi.nlm.nih.gov/22929228","citation_count":24,"is_preprint":false},{"pmid":"21152975","id":"PMC_21152975","title":"IRBIT: it is everywhere.","date":"2010","source":"Neurochemical research","url":"https://pubmed.ncbi.nlm.nih.gov/21152975","citation_count":22,"is_preprint":false},{"pmid":"19220705","id":"PMC_19220705","title":"An IRBIT homologue lacks binding activity to inositol 1,4,5-trisphosphate receptor due to the unique N-terminal appendage.","date":"2009","source":"Journal of neurochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19220705","citation_count":22,"is_preprint":false},{"pmid":"22826361","id":"PMC_22826361","title":"Paradoxical expression of AHCYL1 affecting ovarian carcinogenesis between chickens and women.","date":"2012","source":"Experimental biology and medicine (Maywood, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/22826361","citation_count":21,"is_preprint":false},{"pmid":"27279487","id":"PMC_27279487","title":"The NHERF1 PDZ1 domain and IRBIT interact and mediate the activation of Na+/H+ exchanger 3 by ANG II.","date":"2016","source":"American journal of physiology. 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/35218773","citation_count":10,"is_preprint":false},{"pmid":"26509711","id":"PMC_26509711","title":"IRBIT Interacts with the Catalytic Core of Phosphatidylinositol Phosphate Kinase Type Iα and IIα through Conserved Catalytic Aspartate Residues.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26509711","citation_count":10,"is_preprint":false},{"pmid":"32359081","id":"PMC_32359081","title":"Multiple acid-base and electrolyte disturbances upregulate NBCn1, NBCn2, IRBIT and L-IRBIT in the mTAL.","date":"2020","source":"The Journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/32359081","citation_count":8,"is_preprint":false},{"pmid":"23769829","id":"PMC_23769829","title":"IRBIT plays an important role in NHE3-mediated pHi regulation in HSG cells.","date":"2013","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/23769829","citation_count":8,"is_preprint":false},{"pmid":"38294692","id":"PMC_38294692","title":"NRAS Mutant Dictates AHCYL1-Governed ER Calcium Homeostasis for Melanoma Tumor Growth.","date":"2024","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/38294692","citation_count":7,"is_preprint":false},{"pmid":"33727633","id":"PMC_33727633","title":"Both IRBIT and long-IRBIT bind to and coordinately regulate Cl-/HCO3- exchanger AE2 activity through modulating the lysosomal degradation of AE2.","date":"2021","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/33727633","citation_count":7,"is_preprint":false},{"pmid":"31432119","id":"PMC_31432119","title":"MAP‑1B, PACS‑2 and AHCYL1 are regulated by miR‑34A/B/C and miR‑449 in neuroplasticity following traumatic spinal cord injury in rats: Preliminary explorative results from microarray data.","date":"2019","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/31432119","citation_count":7,"is_preprint":false},{"pmid":"31366495","id":"PMC_31366495","title":"Low IRBIT Levels Are Associated With Chemo-resistance in Gastric Cancer Patients.","date":"2019","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/31366495","citation_count":6,"is_preprint":false},{"pmid":"35351819","id":"PMC_35351819","title":"Synaptic plasticity in hippocampal CA1 neurons of mice lacking inositol-1,4,5-trisphosphate receptor-binding protein released with IP3 (IRBIT).","date":"2022","source":"Learning & memory (Cold Spring Harbor, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/35351819","citation_count":6,"is_preprint":false},{"pmid":"36872327","id":"PMC_36872327","title":"S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) inhibits lung cancer tumorigenesis by regulating cell plasticity.","date":"2023","source":"Biology direct","url":"https://pubmed.ncbi.nlm.nih.gov/36872327","citation_count":5,"is_preprint":false},{"pmid":"22453947","id":"PMC_22453947","title":"The discovery and structural investigation of the IP₃ receptor and the associated IRBIT protein.","date":"2012","source":"Advances in experimental medicine and biology","url":"https://pubmed.ncbi.nlm.nih.gov/22453947","citation_count":5,"is_preprint":false},{"pmid":"39929439","id":"PMC_39929439","title":"Cardiac hypertrophy induced by overexpression of IP3-released inositol 1, 4, 5-trisphosphate receptor-binding protein (IRBIT).","date":"2025","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/39929439","citation_count":4,"is_preprint":false},{"pmid":"37082243","id":"PMC_37082243","title":"Dietary sodium enhances the expression of SLC4 family transporters, IRBIT, L-IRBIT, and PP1 in rat kidney: Insights into the molecular mechanism for renal sodium handling.","date":"2023","source":"Frontiers in physiology","url":"https://pubmed.ncbi.nlm.nih.gov/37082243","citation_count":4,"is_preprint":false},{"pmid":"39985648","id":"PMC_39985648","title":"Redox state of NAD modulates the activation of Na-bicarbonate cotransporter NBCe1-B via IRBIT and L-IRBIT.","date":"2025","source":"Science China. Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/39985648","citation_count":3,"is_preprint":false},{"pmid":"34923880","id":"PMC_34923880","title":"Genetic variants in SCNN1B and AHCYL1 are associated with eggshell quality in Chinese domestic laying ducks (Anas platyrhynchos).","date":"2022","source":"British poultry science","url":"https://pubmed.ncbi.nlm.nih.gov/34923880","citation_count":2,"is_preprint":false},{"pmid":"40365293","id":"PMC_40365293","title":"AHCYL1 mediates the tumor-promoting effect of PREX2 in non-small cell lung carcinoma.","date":"2025","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/40365293","citation_count":1,"is_preprint":false},{"pmid":"40569378","id":"PMC_40569378","title":"IRBIT and LIMA1 associate with and are necessary for epithelial cell SLC26A3 (DRA) stimulation by cAMP/ATP.","date":"2025","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/40569378","citation_count":0,"is_preprint":false},{"pmid":"42032162","id":"PMC_42032162","title":"Deciphering the multi-site phos-code of IRBIT underlying its binding to IP3R.","date":"2026","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/42032162","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.08.15.670264","title":"Deployment of non-canonical splicing in tunicate genomes is mediated by divergent U2AF function and re-patterning of snRNA m6A modification","date":"2025-08-17","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.15.670264","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":30977,"output_tokens":8777,"usd":0.112293,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18692,"output_tokens":6376,"usd":0.12643,"stage2_stop_reason":"end_turn"},"total_usd":0.238723,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"IRBIT (AHCYL1) was identified as a novel IP3 receptor type 1 (IP3R1)-binding protein that is released from IP3R1 upon IP3 binding. The N-terminal region of IRBIT (the 104 aa appendage) is essential for interaction with IP3R1, and the IRBIT binding region maps to the IP3-binding core of IP3R1. Alkaline phosphatase treatment abolished the interaction, demonstrating that both IP3 and phosphorylation dually regulate the interaction.\",\n      \"method\": \"Affinity purification from rat brain microsomes using immobilized IP3R1 N-terminal domain, IP3 elution, in vitro binding experiments, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal biochemical purification, in vitro binding, co-IP, replicated across multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"12525476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"IRBIT suppresses IP3R activation by competing with IP3 for the same binding site on IP3R. Multiserine phosphorylation of IRBIT is essential for binding; 10 of 12 key IP3R residues for IP3 recognition also participate in IRBIT binding. IRBIT acts as an endogenous 'pseudoligand' of IP3R whose inhibitory activity is modulated by its phosphorylation status.\",\n      \"method\": \"[3H]IP3 binding competition assay, in vitro Ca2+ release assay, Ca2+ imaging in intact cells, mutagenesis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal functional assays (radioligand binding, in vitro Ca2+ release, live-cell imaging) plus mutagenesis in a single study\",\n      \"pmids\": [\"16793548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"IRBIT directly binds to the IP3 receptor (both the suppressor domain and the IP3-binding core are required for strong interaction). A PEST motif, a PDZ-ligand, and Asp-73 on IRBIT are critical for this interaction. IRBIT inhibits both IP3 binding and IP3-induced Ca2+ release from IP3R.\",\n      \"method\": \"Direct in vitro binding assays, mutagenesis, [3H]IP3 binding inhibition, Ca2+ release assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct binding demonstrated, mutagenesis of critical residues, functional Ca2+ release assay, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16527252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"IRBIT specifically binds to the pancreas-type splicing variant of NBC1 (pNBC1) but not to kidney-type NBC1 (kNBC1). IRBIT binds the N-terminal pNBC1-specific domain, and binding depends on phosphorylation of multiple serine residues in IRBIT. Electrophysiological analysis in Xenopus oocytes showed that pNBC1 requires IRBIT coexpression to manifest substantial transport activity.\",\n      \"method\": \"Co-immunoprecipitation, two-electrode voltage-clamp in Xenopus oocytes, mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct electrophysiology in Xenopus oocytes with co-IP and mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"16769890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Protein phosphatase-1 (PP1) binds to a conserved docking site on IRBIT preceding the PEST domain and mediates dephosphorylation of IRBIT Ser68. Phosphorylation of Ser68 is required for subsequent phosphorylation of Ser71 and Ser74, whereas phosphorylation of Ser71 and Ser74 (but not Ser68 alone) is sufficient to enable IRBIT inhibition of IP3 binding to IP3R. Mutational inactivation of the PP1 docking site on IRBIT increased IRBIT affinity for IP3R.\",\n      \"method\": \"In vitro PP1 binding assay, co-immunoprecipitation, site-directed mutagenesis, [3H]IP3 binding assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro binding, mutagenesis of phosphorylation sites, functional IP3 binding assay, multiple orthogonal methods in one study\",\n      \"pmids\": [\"17635105\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"IRBIT binds to the C-terminal domain of NHE3 (Na+/H+ exchanger 3) and activates NHE3 activity in a Ca2+-dependent manner. IRBIT-dependent activation involves exocytic trafficking of NHE3 to the plasma membrane, and this activation is blocked by inhibition of calmodulin or CaM-dependent kinase II. Co-expression of IRBIT reverses NHERF2-dependent inhibition of NHE3.\",\n      \"method\": \"Co-immunoprecipitation, NHE3 activity assay, siRNA knockdown, cell surface biotinylation, pharmacological inhibition of CaM/CaMKII\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, functional transport assay, siRNA knockdown, surface trafficking assay, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"18829453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"IRBIT activates both basolateral pNBC1 and luminal CFTR to coordinate fluid and HCO3- secretion in the murine pancreatic duct. siRNA knockdown of IRBIT markedly inhibited ductal pNBC1 and CFTR activities, and fluid and HCO3- secretion. Activation of pNBC1 required only the IRBIT PEST domain, while activation of CFTR required multiple IRBIT domains, indicating distinct mechanisms. Single-channel measurements showed IRBIT regulated CFTR by reducing channel mean close time.\",\n      \"method\": \"Video microscopy of sealed intralobular pancreatic ducts, ion-selective microelectrodes, siRNA knockdown, single-channel patch clamp, domain deletion constructs in HEK cells\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal functional assays (fluid secretion, ion concentrations, single-channel recording), siRNA knockdown with specific phenotypic rescue, domain mapping\",\n      \"pmids\": [\"19033647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"IRBIT associates with the cleavage and polyadenylation specificity factor (CPSF) complex in a phosphorylation-dependent manner. The main IRBIT target within CPSF is the Fip1 subunit. Phosphorylation of the serine-rich region of IRBIT is required for association with Fip1 in vitro and for redistribution of Fip1 to the cytoplasm. IRBIT also interacts with poly(A) polymerase (PAP) and inhibits PAP activity in a phosphorylation-dependent manner. Oxidative stress (tBHQ) increases IRBIT phosphorylation, enhances IRBIT-CPSF interaction, and promotes cytoplasmic distribution of Fip1.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding with Fip1, in vitro PAP activity assay, RNA recruitment assay, immunofluorescence, phosphorylation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro PAP activity assay, direct binding to Fip1, co-IP, localization, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"19224921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"IRBIT mediates activation of NHE3 by angiotensin II (ANG II) via a CaMKII-dependent pathway. ANG II transiently increases IRBIT-NHE3 binding, which precedes increased NHE3 surface expression and activity. Inhibition of CaMKII blocks ANG II-induced IRBIT-NHE3 binding and NHE3 surface abundance. Mutations of IRBIT Ser-68, Ser-71, and Ser-74 decreased IRBIT binding to NHE3 and reduced ANG II-stimulated NHE3 activity.\",\n      \"method\": \"Co-immunoprecipitation, NHE3 activity assay (fluorometric intracellular pH), cell surface biotinylation, CaMKII pharmacological inhibition, siRNA knockdown, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, functional transport assay, surface trafficking, mutagenesis of phosphorylation sites, multiple methods in single lab\",\n      \"pmids\": [\"20584908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"IRBIT opposes the WNK/SPAK kinase pathway by recruiting PP1 to the CFTR/NBCe1-B complex to dephosphorylate these transporters and restore their cell surface expression. WNK kinases act as scaffolds recruiting SPAK, which phosphorylates CFTR and NBCe1-B to reduce their surface expression. IRBIT opposes this effect and also directly stimulates transporter activity. Silencing of both SPAK and IRBIT in the same ducts rescued ductal secretion caused by IRBIT silencing alone (epistasis).\",\n      \"method\": \"siRNA knockdown, genetic epistasis (double silencing), cell surface expression assay, fluid secretion measurement in sealed pancreatic ducts, co-immunoprecipitation, phosphorylation analysis\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis by double silencing, co-IP, functional fluid secretion assay, surface expression assay, multiple orthogonal methods\",\n      \"pmids\": [\"21317537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"IRBIT relieves the autoinhibitory domain (AID) of NBCe1-B to activate it, but the stimulatory effect of IRBIT cannot be explained solely by masking autoinhibitory determinants. A mutant IRBIT resistant to protein phosphatase-1 action stimulates NBCe1-B to an extent 50% greater than removal of the AID alone. An NBCe1-B construct lacking residues 2-16 is fully autoinhibited but cannot be stimulated by IRBIT, indicating that autoinhibitory and IRBIT-binding determinants are not identical.\",\n      \"method\": \"Two-electrode voltage-clamp on Xenopus oocytes, IRBIT and NBCe1-B deletion/mutation constructs\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct electrophysiology with mutagenesis constructs, quantitative comparison of multiple NBCe1-B variants, single lab\",\n      \"pmids\": [\"22012331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"IRBIT mediates synergistic activation of CFTR and Slc26a6 by Ca2+ and cAMP signaling pathways. In resting cells, IRBIT is sequestered by IP3Rs in the ER. cAMP-dependent phosphorylation of IP3Rs reduces their affinity for IRBIT, enabling IP3-induced dissociation and translocation of IRBIT to CFTR and Slc26a6 at the plasma membrane. This synergistic mechanism was absent in Irbit-/- mice and in mice with IP3Rs bearing mutations preventing PKA phosphorylation.\",\n      \"method\": \"Knockout mice (Irbit-/- and Slc26a6-/-), co-immunoprecipitation, CFTR Cl- current measurement, intracellular pH assay (Slc26a6 activity), fluid secretion measurement, site-directed mutagenesis of IP3R PKA sites\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO mice plus multiple orthogonal functional assays and mechanistic mutagenesis, independently validated in two gland types\",\n      \"pmids\": [\"23542070\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A positively charged module within NBCe1-B (residues 37-65) is required for interaction with and activation by IRBIT and for regulation by PIP2. Phosphorylation of Thr49 is required for regulation by both IRBIT and SPAK. A homologous sequence exists in the CFTR R domain and Slc26a6 STAS domain, both of which bind IRBIT; the R domain is required for CFTR activation by IRBIT.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis, electrophysiology in Xenopus oocytes, domain deletion analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — functional electrophysiology, co-IP, systematic mutagenesis identifying critical phosphorylation sites and binding domains, multiple transporters tested\",\n      \"pmids\": [\"23431199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"IRBIT forms a dATP-dependent complex with ribonucleotide reductase (RNR), stabilizes dATP in the activity site of RNR, and thereby inhibits RNR enzymatic activity. Formation of the RNR-IRBIT complex is regulated by IRBIT phosphorylation. Ablation of IRBIT in HeLa cells causes imbalanced dNTP pools and altered cell cycle progression.\",\n      \"method\": \"Co-immunoprecipitation of RNR-IRBIT complex, RNR enzymatic activity assay, IRBIT knockdown in HeLa cells with dNTP pool measurement, cell cycle analysis\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro RNR activity assay, co-IP of complex, functional cellular readout (dNTP pools, cell cycle), multiple orthogonal methods in single lab\",\n      \"pmids\": [\"25237103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"IRBIT binds to CaMKIIα and suppresses its kinase activity by inhibiting the binding of calmodulin to CaMKIIα. In IRBIT-deficient mice, phosphorylation of tyrosine hydroxylase (TH) by CaMKIIα is significantly increased in the ventral tegmental area, leading to elevated catecholamine levels and behavioral abnormalities.\",\n      \"method\": \"Co-immunoprecipitation, CaMKIIα kinase activity assay, IRBIT knockout mice, TH phosphorylation analysis by immunoblot, behavioral assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, in vitro/cellular kinase assay, KO mouse with specific biochemical (phospho-TH) and behavioral phenotypes, multiple orthogonal methods\",\n      \"pmids\": [\"25922519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"IRBIT forms signaling complexes with phosphatidylinositol phosphate kinases (PIPKs), specifically interacting with PIPKIα and PIPKIIα in mouse cerebellum. Two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are required for interaction with IRBIT. Phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfere with the interaction, indicating IRBIT interacts with the catalytic core. Mutations in the serine-rich region of IRBIT affect selectivity for PIPKIα vs PIPKIIα. IRBIT and PIPKIα interact with NBCe1-B.\",\n      \"method\": \"Co-immunoprecipitation from mouse cerebellum, heterologous expression binding assays, site-directed mutagenesis, immunocytochemistry, in vitro binding\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — co-IP from tissue and heterologous system, mutagenesis of catalytic residues, localization, single lab\",\n      \"pmids\": [\"26509711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"IRBIT interacts with the Bcl-2 homolog Bcl2l10 and together they exert additive inhibition of IP3R. Both proteins associate in mitochondria-associated membranes (MAMs). Upon apoptotic stress, IRBIT is dephosphorylated and becomes an inhibitor of Bcl2l10. IRBIT promotes ER-mitochondria contact. By inhibiting Bcl2l10 and promoting ER-mitochondria contact, IRBIT facilitates Ca2+ transfer to mitochondria and promotes apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation (MAM isolation), Ca2+ imaging, cell death assays, dephosphorylation experiments, IP3R activity assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, subcellular fractionation, functional Ca2+ and apoptosis assays, mechanistic model supported by multiple methods in single lab\",\n      \"pmids\": [\"27995898\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NHERF1, NHE3, and IRBIT form a macrocomplex. ANG II induces a concomitant increase in NHERF1 interactions with NHE3 and IRBIT requiring the NHERF1 PDZ1 domain. IRBIT is indispensable for ANG II-provoked increase in NHERF1-NHE3 interactions, and phosphorylation of IRBIT at Ser68 is necessary for assembly of the NHERF1-IRBIT-NHE3 complex and NHE3 activation.\",\n      \"method\": \"Co-immunoprecipitation, dominant negative PDZ1 overexpression, siRNA knockdown of IRBIT, cell surface biotinylation, fluorescence imaging\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — co-IP, dominant negative, knockdown, surface trafficking assay, single lab with multiple methods\",\n      \"pmids\": [\"27279487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"IRBIT family proteins form homo- and heteromultimers. N-terminal splicing of Long-IRBIT changes protein stability and selectivity for target molecules. Different IRBIT family members exhibit distinct mRNA expression patterns and different target selectivities determined by N-terminal diversity and multimer combinations.\",\n      \"method\": \"Co-immunoprecipitation, protein stability assays, target binding assays with multiple IRBIT family constructs, RT-PCR expression analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP for multimerization, binding selectivity assays, single lab with multiple constructs\",\n      \"pmids\": [\"28348216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"IRBIT controls five phosphorylation sites in NBCe1-B that determine active vs. inactive transporter conformation and regulate sensitivity to intracellular Cl-. IRBIT recruits PP1 and SPAK to control phosphorylation of Ser65 (affecting Cl- sensing via 32GXXXP36 motif), and recruits calcineurin and CaMKII to control phosphorylation of Ser12 (affecting Cl- sensing via 194GXXXP198 motif). Phosphorylation status of Ser232, Ser233, Ser235 determines active/inactive conformations of NBCe1-B.\",\n      \"method\": \"Phosphoproteomic analysis, mutagenesis, electrophysiology, co-immunoprecipitation of recruited kinases/phosphatases\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — phosphoproteomics plus systematic mutagenesis plus electrophysiology plus co-IP of kinase/phosphatase recruitment, multiple orthogonal methods\",\n      \"pmids\": [\"30377224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"IRBIT activates NBCe1-B by releasing the autoinhibitory domain (AID, acting as a brake) from the transmembrane domain via competitive binding. The AID (residues 40-85) binds to the transmembrane domain via electrostatic attraction to slow NBCe1-B. IRBIT binding domain spans residues 1-52 with two arms (negatively charged residues 1-24 and positively charged residues 40-52). Negatively charged Asp/Glu residues plus Ser/Thr residues in the IRBIT PEST domain are required for NBCe1-B interaction.\",\n      \"method\": \"Systematic mutagenesis of NBCe1-B and IRBIT, two-electrode voltage-clamp in Xenopus oocytes, co-immunoprecipitation\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis combined with quantitative electrophysiology, mechanistic model validated, single lab\",\n      \"pmids\": [\"33237573\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"IRBIT expression in intestinal stem cell progeny in the Drosophila midgut regulates their differentiation into enterocytes via suppression of RNR activity. Disruption of the IRBIT-RNR regulatory circuit causes premature loss of intestinal tissue integrity, and age-related dysplasia can be reversed by suppression of RNR activity in ISC progeny.\",\n      \"method\": \"Drosophila genetics (IRBIT loss-of-function, RNR inhibition), histology of midgut epithelium, intestinal stem cell differentiation assays\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function in Drosophila with specific differentiation phenotype and epistasis with RNR, replicated by independent RNR suppression\",\n      \"pmids\": [\"32179478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AHCYL1 (IRBIT) acts as a SAH (S-adenosyl-l-homocysteine) sensor to inhibit autophagy through PIK3C3. The C-terminus of AHCYL1 interacts with SAH specifically, and this SAH binding promotes the N-terminus to bind the catalytic domain of PIK3C3 and inhibit it, independently of MTORC1.\",\n      \"method\": \"Co-immunoprecipitation, PIK3C3 activity assay, autophagy flux assay, AHCYL1 deletion constructs, in vivo validation\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — enzymatic activity assay of PIK3C3, domain dissection by co-IP, in vivo validation, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"33993848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Both IRBIT and Long-IRBIT interact with anion exchanger AE2 through the conserved AHCY-homologous domain of IRBIT/L-IRBIT binding the N-terminal cytoplasmic region of AE2. L-IRBIT KO reduces AE2 activity and protein expression, while IRBIT KO does not. IRBIT binding facilitates lysosomal degradation of AE2, which is inhibited by coexisting L-IRBIT, revealing opposing regulatory roles.\",\n      \"method\": \"Co-immunoprecipitation, AE2 activity assay (intracellular pH), IRBIT/L-IRBIT KO cells, bafilomycin A1 treatment (lysosomal inhibition), cell volume regulation assay, cell migration assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cells, functional AE2 assay, lysosomal inhibitor rescue, co-IP, multiple orthogonal methods identifying opposing roles of two paralogs\",\n      \"pmids\": [\"33727633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Endogenous tau physically interacts with AHCYL1/IRBIT in brain tissues and cultured cells. Tau overexpression modifies the close localization of AHCYL1/IRBIT to IP3R at the endoplasmic reticulum.\",\n      \"method\": \"Protein microarray binding assay with recombinant tau, co-immunoprecipitation from brain tissue and cultured cells, proximity ligation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP from tissue, proximity ligation assay for localization change, multiple methods but no functional readout of the interaction\",\n      \"pmids\": [\"35218773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"IRBIT overexpression in mice using AAV9 causes cardiac hypertrophy with impaired systolic function. IRBIT overexpression decreases Ca2+ transient amplitude, slows Ca2+ transient rise, reduces Ca2+ wave propagation velocity, increases the dyssynchrony index, and reduces nuclear envelope invaginations in isolated cardiomyocytes. IP3R mRNA levels are decreased in IRBIT-overexpressing hearts. AAV9-shRNA reduction of IRBIT expression does not alter heart morphometric parameters.\",\n      \"method\": \"AAV9-mediated overexpression and shRNA knockdown in mice, echocardiography, isolated cardiomyocyte Ca2+ imaging, fluorescence microscopy\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo AAV overexpression model with functional cardiac and Ca2+ imaging readouts, single lab\",\n      \"pmids\": [\"39929439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"AHCYL1 interacts with PREX2 (a Rac1 GEF) and enhances PREX2 GEF activity by alleviating mutual inhibition between PREX2 and PTEN, leading to increased RAC1 activation and NSCLC cell growth.\",\n      \"method\": \"Pull-down assay with LC-MS/MS identification, co-immunoprecipitation, in vitro GEF activity assay, active RAC1 pull-down assay, PREX2/AHCYL1 knockdown, xenograft mouse model\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro GEF assay, pull-down identification, co-IP, functional RAC1 activation assay, single lab\",\n      \"pmids\": [\"40365293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"IRBIT and LIMA1 associate with the Cl-/HCO3- exchanger SLC26A3 (DRA) under basal conditions, and this association increases with cAMP/ATP stimulation. KD of IRBIT or LIMA1 reduces cAMP/Ca2+-stimulated DRA activity without altering basal activity. Maximum ATP (but not maximal forskolin) stimulation of DRA is IRBIT-dependent. cAMP/ATP-induced elevation of intracellular Ca2+ is also IRBIT-dependent. cAMP/ATP stimulation increases coprecipitation of LIMA1 with both IRBIT and DRA.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, DRA activity assay (intracellular pH), Ca2+ imaging, cell surface biotinylation\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — co-IP, knockdown with functional assay, Ca2+ imaging, multiple methods in single lab\",\n      \"pmids\": [\"40569378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Phosphorylation of IRBIT S80, S84, and S85 (by casein kinase 2) provides binding sites for the IP3-binding core (IBC) of IP3R and is sufficient to compete with IP3 for the IP3-binding pocket. S68 is the predominant phosphorylation site but is not required for IP3R binding. PKA phosphorylates S62/S64/S66; CK2 phosphorylates S80/T82/S84/S85. IRBIT-S68A/S80D/S84D/S85D was sufficient to inhibit IP3R-mediated Ca2+ release in living cells.\",\n      \"method\": \"In vitro kinase assay (PKA, CK2), pulldown assay with IBC domain, computational binding estimation, Ca2+ imaging in living cells, site-directed mutagenesis\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, direct binding pulldown with defined phosphorylation mutants, functional Ca2+ imaging, computational binding model, multiple orthogonal methods\",\n      \"pmids\": [\"42032162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NAD modulates NBCe1-B activation by serving as a cofactor of IRBIT or L-IRBIT. Blocking the NAD salvage pathway decreases NBCe1-B activation by the IRBITs. The oxidized form NAD+ enhances, while the reduced form NADH decreases, NBCe1-B activity, linking cellular redox state to IRBIT-dependent pH regulation.\",\n      \"method\": \"Electrophysiology (Xenopus oocyte or mammalian cell NBCe1-B activity assay), NAD salvage pathway inhibition, NAD+/NADH administration, IRBIT constructs\",\n      \"journal\": \"Science China. Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional electrophysiology with pharmacological manipulation of NAD redox state, single lab\",\n      \"pmids\": [\"39985648\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AHCYL1/IRBIT is a multifunctional scaffold protein that acts as a phosphorylation-dependent pseudoligand of the IP3 receptor (competing with IP3 at its binding core to inhibit Ca2+ release), and, upon release from IP3R in response to IP3, translocates to activate multiple ion transporters (pNBC1/NBCe1-B, NHE3, CFTR, Slc26a6, AE2, DRA/SLC26A3) by relieving autoinhibitory domains and recruiting kinases (SPAK, CaMKII) and phosphatases (PP1, calcineurin) to fine-tune transporter phosphorylation; it also inhibits ribonucleotide reductase (RNR) in a dATP-dependent manner to regulate dNTP pools and cell cycle progression, suppresses CaMKIIα activity to control catecholamine homeostasis, inhibits autophagy by sensing SAH and blocking PIK3C3, promotes apoptosis via MAM association with Bcl2l10 and ER-mitochondria contact, and modulates mRNA polyadenylation by binding CPSF/Fip1 and inhibiting poly(A) polymerase—with all these activities gated by multisite serine phosphorylation in its N-terminal PEST domain and regulated by NAD redox state as a cofactor.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AHCYL1/IRBIT is a phosphorylation-gated scaffold and pseudoligand that couples IP3 receptor (IP3R) Ca2+ signaling to the activation of epithelial ion transporters [#0, #1, #11]. In resting cells it is sequestered on the IP3R, where multisite serine phosphorylation in its N-terminal PEST region lets it occupy the IP3-binding core and competitively suppress IP3 binding and Ca2+ release, acting as an endogenous pseudoligand [#1, #2, #28]. Phosphorylation of IRBIT is set by a PP1 docking site and an ordered serine cascade, with PKA and CK2 phosphorylating distinct N-terminal serines that license IP3R binding [#4, #28]. cAMP/PKA phosphorylation of the IP3R lowers its affinity for IRBIT, so IP3 releases IRBIT to translocate to the plasma membrane and activate bicarbonate/chloride transporters\\u2014pNBC1/NBCe1-B, CFTR, Slc26a6, and DRA/SLC26A3\\u2014thereby integrating Ca2+ and cAMP signals to drive fluid and HCO3- secretion [#6, #11, #13, #27]. Mechanistically, IRBIT activates NBCe1-B by competitively relieving its autoinhibitory domain and by recruiting opposing kinases and phosphatases (SPAK and CaMKII versus PP1 and calcineurin) to control a defined set of transporter phosphosites that also tune intracellular Cl- sensing [#9, #10, #19, #20]; it activates NHE3 through Ca2+/CaMKII-dependent surface trafficking within a NHERF1 macrocomplex [#5, #8, #17]. Beyond transport, IRBIT moonlights in nucleotide metabolism by forming a dATP-dependent complex with ribonucleotide reductase to inhibit it and balance dNTP pools and cell-cycle progression [#13], suppresses CaMKII\\u03b1 by blocking calmodulin binding to control tyrosine hydroxylase phosphorylation and catecholamine homeostasis [#14], senses S-adenosylhomocysteine to inhibit PIK3C3 and autophagy [#22], and promotes apoptosis through Bcl2l10 inhibition and ER\\u2013mitochondria Ca2+ transfer at mitochondria-associated membranes [#16]. Its activities are further shaped by NAD redox state acting as a cofactor and by homo/heteromultimerization with the Long-IRBIT paralog, which can confer opposing regulatory outcomes such as lysosomal versus stabilized AE2 [#23, #29, #18].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established IRBIT as a physical partner of the IP3R whose binding is governed jointly by IP3 occupancy and phosphorylation, defining it as a regulated IP3R-associated protein.\",\n      \"evidence\": \"Affinity purification from rat brain microsomes with IP3 elution, in vitro binding and co-IP\",\n      \"pmids\": [\"12525476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the interaction not yet defined\", \"Identity of the kinases setting the phospho-dependence unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the inhibitory mechanism: IRBIT is a phosphorylation-dependent pseudoligand that competes with IP3 at the same recognition residues to suppress Ca2+ release.\",\n      \"evidence\": \"[3H]IP3 competition, in vitro Ca2+ release, live-cell Ca2+ imaging, mutagenesis; direct binding mapping to suppressor and IP3-binding core domains\",\n      \"pmids\": [\"16793548\", \"16527252\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which serines and kinases drive the competent phospho-state not yet resolved\", \"Stoichiometry of IRBIT:IP3R unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed IRBIT is not solely an IP3R inhibitor but also a required activator of an epithelial transporter, revealing a translocation-based dual function.\",\n      \"evidence\": \"Co-IP and two-electrode voltage-clamp in Xenopus oocytes showing pNBC1 requires IRBIT for activity\",\n      \"pmids\": [\"16769890\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of transporter activation not yet defined\", \"Trigger linking IP3R release to transporter binding unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapped the phospho-regulatory logic: a PP1 docking site and an ordered Ser68\\u2192Ser71/Ser74 cascade set IRBIT affinity for the IP3R.\",\n      \"evidence\": \"In vitro PP1 binding, co-IP, phosphosite mutagenesis, [3H]IP3 binding assay\",\n      \"pmids\": [\"17635105\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream kinases for each serine not identified here\", \"How dephosphorylation is triggered physiologically unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Extended IRBIT's activator role to NHE3 and to coordinated CFTR/pNBC1 secretion, establishing it as a hub for epithelial fluid and HCO3- transport.\",\n      \"evidence\": \"Co-IP, NHE3 transport and surface biotinylation, siRNA, single-channel patch clamp and fluid secretion in sealed pancreatic ducts, domain mapping\",\n      \"pmids\": [\"18829453\", \"19033647\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Distinct domain requirements across transporters mechanistically unexplained\", \"How a single IRBIT pool coordinates basolateral and luminal targets unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Revealed a transcription-independent moonlighting role: phospho-IRBIT binds CPSF/Fip1 and inhibits poly(A) polymerase, linking IRBIT phosphorylation and oxidative stress to mRNA polyadenylation.\",\n      \"evidence\": \"Co-IP, in vitro Fip1 binding, in vitro PAP activity assay, Fip1 redistribution imaging, tBHQ stress\",\n      \"pmids\": [\"19224921\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological mRNA targets not identified\", \"Relationship to IRBIT's membrane functions unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined the activation mechanism for NBCe1-B as relief of an autoinhibitory domain combined with recruited phosphatase action, and placed IRBIT in opposition to the WNK/SPAK kinase pathway.\",\n      \"evidence\": \"Electrophysiology with NBCe1-B/IRBIT mutants; siRNA epistasis (SPAK/IRBIT double silencing), co-IP and fluid secretion in ducts\",\n      \"pmids\": [\"22012331\", \"21317537\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The extra stimulatory component beyond AID masking not fully explained\", \"Generalizability of PP1 recruitment to other transporters not yet tested here\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established the Ca2+/cAMP integration model: PKA phosphorylation of IP3R releases IRBIT for synergistic activation of CFTR and Slc26a6, validated in knockout mice.\",\n      \"evidence\": \"Irbit-/- and Slc26a6-/- mice, CFTR currents, Slc26a6 pH assay, fluid secretion, IP3R PKA-site mutagenesis; shared positively charged module mapped across NBCe1-B/CFTR R domain/Slc26a6 STAS\",\n      \"pmids\": [\"23542070\", \"23431199\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance to human secretory disease not addressed\", \"How Thr49/Ser65 phospho-states are dynamically controlled in vivo unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Uncovered a metabolic function: IRBIT inhibits ribonucleotide reductase in a dATP-dependent manner, coupling it to dNTP pool balance and cell-cycle progression.\",\n      \"evidence\": \"Co-IP of RNR-IRBIT complex, RNR activity assay, IRBIT knockdown in HeLa with dNTP and cell-cycle readouts\",\n      \"pmids\": [\"25237103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of dATP stabilization not resolved\", \"Coordination with IRBIT's signaling roles unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Broadened IRBIT's regulatory reach to CaMKII\\u03b1 suppression and catecholamine control, and to interactions with lipid-kinase catalytic cores.\",\n      \"evidence\": \"Co-IP, CaMKII\\u03b1 kinase assay and IRBIT KO mice with phospho-TH and behavioral phenotypes; co-IP with PIPKI\\u03b1/PIPKII\\u03b1 catalytic residues from cerebellum\",\n      \"pmids\": [\"25922519\", \"26509711\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PIPK interaction lacks an in vivo functional readout\", \"How CaMKII suppression is balanced against CaMKII-dependent transporter activation unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed IRBIT operates at ER-mitochondria contacts, where dephosphorylated IRBIT inhibits Bcl2l10 and promotes pro-apoptotic Ca2+ transfer.\",\n      \"evidence\": \"Co-IP, MAM fractionation, Ca2+ imaging, cell death assays, dephosphorylation experiments; NHERF1-IRBIT-NHE3 macrocomplex assembly via Ser68\",\n      \"pmids\": [\"27995898\", \"27279487\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger coupling apoptotic dephosphorylation to MAM localization not defined\", \"Stoichiometry within the NHERF1-IRBIT-NHE3 complex unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established that IRBIT family members multimerize and that N-terminal splicing dictates stability and target selectivity, providing a combinatorial code for IRBIT function.\",\n      \"evidence\": \"Co-IP for multimerization, protein stability and target-binding selectivity assays across family constructs, expression analysis\",\n      \"pmids\": [\"28348216\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological abundance of specific multimer combinations unknown\", \"Single-lab characterization without in vivo validation\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved the full phospho-regulatory circuit by which IRBIT controls NBCe1-B conformation and Cl- sensitivity through recruited kinases and phosphatases at defined sites.\",\n      \"evidence\": \"Phosphoproteomics, mutagenesis, electrophysiology, co-IP of recruited SPAK/CaMKII/PP1/calcineurin\",\n      \"pmids\": [\"30377224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamic switching between recruited enzymes in vivo not directly observed\", \"Generalization of the multi-enzyme recruitment model to other transporters untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined the structural mechanism of NBCe1-B activation as competitive electrostatic displacement of the autoinhibitory domain by IRBIT's bipartite binding arms, and linked IRBIT-RNR control to tissue homeostasis in vivo.\",\n      \"evidence\": \"Systematic NBCe1-B/IRBIT mutagenesis with voltage-clamp electrophysiology and co-IP; Drosophila midgut genetics with RNR epistasis\",\n      \"pmids\": [\"33237573\", \"32179478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Drosophila finding is Medium-confidence model-organism genetics\", \"Direct structural data on the IRBIT-NBCe1-B interface lacking\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified two new ligand-sensing and adaptor functions: SAH-sensing inhibition of PIK3C3/autophagy, and paralog-specific control of AE2 stability and activity.\",\n      \"evidence\": \"Co-IP, PIK3C3 activity and autophagy flux assays with domain dissection and in vivo validation; AE2 activity assays in IRBIT/L-IRBIT KO cells with bafilomycin rescue\",\n      \"pmids\": [\"33993848\", \"33727633\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological conditions raising SAH to engage IRBIT not defined\", \"Structural basis of opposing IRBIT vs L-IRBIT effects on AE2 unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linked IRBIT to neurodegeneration-relevant biology by showing tau interacts with IRBIT and alters its proximity to the IP3R.\",\n      \"evidence\": \"Protein microarray, co-IP from brain and cells, proximity ligation assay\",\n      \"pmids\": [\"35218773\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence of the tau-IRBIT interaction measured\", \"Disease relevance not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected IRBIT dosage to cardiac and redox physiology and to a cancer growth pathway, expanding its in vivo and disease-associated roles.\",\n      \"evidence\": \"AAV9 IRBIT overexpression/knockdown in mice with echocardiography and cardiomyocyte Ca2+ imaging; NAD salvage/redox electrophysiology on NBCe1-B; PREX2-AHCYL1 GEF assays, RAC1 activation, and NSCLC xenografts; IRBIT/LIMA1/SLC26A3 co-IP and activity assays\",\n      \"pmids\": [\"39929439\", \"39985648\", \"40365293\", \"40569378\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab in vivo models for cardiac and cancer roles\", \"Mechanism linking NAD redox cofactor binding to IRBIT activation not structurally defined\", \"Whether PREX2/RAC1 role intersects IRBIT's IP3R functions unknown\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Refined the pseudoligand mechanism by mapping the specific CK2 and PKA phosphosites that create the IP3-binding-core docking surface sufficient to inhibit Ca2+ release.\",\n      \"evidence\": \"In vitro PKA/CK2 kinase assays, IBC pulldown with defined phospho-mutants, computational binding estimation, Ca2+ imaging\",\n      \"pmids\": [\"42032162\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo kinase regulation of these sites not demonstrated\", \"How these sites coordinate with transporter-activation phospho-states unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How IRBIT's many parallel functions\\u2014IP3R suppression, transporter activation, RNR inhibition, CaMKII/PIK3C3/PREX2 regulation, and apoptosis\\u2014are partitioned spatially and temporally within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of full-length IRBIT in any complex\", \"Quantitative competition between competing binding partners untested\", \"Mechanism selecting which function dominates under a given stimulus unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 16, 22, 26]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [9, 17, 19, 27]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [1, 2, 28]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [22, 29]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [11, 16, 24]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 5, 6, 11]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7, 13]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [3, 6, 11, 13, 23, 27]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 11, 26]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [16]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [22]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"complexes\": [\n      \"CPSF (Fip1)\",\n      \"ribonucleotide reductase (RNR) complex\",\n      \"NHERF1-IRBIT-NHE3 macrocomplex\"\n    ],\n    \"partners\": [\n      \"ITPR1\",\n      \"NBCe1-B/SLC4A4 (pNBC1)\",\n      \"CFTR\",\n      \"SLC26A6\",\n      \"SLC26A3 (DRA)\",\n      \"NHE3/SLC9A3\",\n      \"CAMK2A\",\n      \"PIK3C3\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}