{"gene":"ADCY6","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2006,"finding":"AC6 physically associates with A-kinase anchoring protein AKAP79/150, forming a complex that enables PKA to preferentially phosphorylate AC6 and inhibit cAMP synthesis, creating a negative feedback loop that terminally regulates cAMP production upon agonist stimulation.","method":"Co-immunoprecipitation, real-time cellular imaging of cAMP dynamics, PKA anchoring disruption experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus live-cell imaging with functional consequence, moderate evidence from single lab with multiple orthogonal methods","pmids":["16973443"],"is_preprint":false},{"year":2006,"finding":"Calcium suppresses renin exocytosis from juxtaglomerular cells by inhibiting AC5 and AC6, thereby reducing intracellular cAMP levels; siRNA-mediated knockdown of AC5 and/or AC6 prevented calcium-dependent inhibition of cAMP and renin release, establishing AC6 as a key mediator of the 'calcium paradoxon' in renin-producing cells.","method":"siRNA knockdown of AC5/AC6 in primary juxtaglomerular cells and As4.1 cells, cAMP measurement, renin secretion assays, isolated perfused kidney experiments","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 — clean KD with defined cellular phenotype replicated across cell types and ex vivo organ model","pmids":["17068292"],"is_preprint":false},{"year":2008,"finding":"AC6 selectively couples with IP3 receptor type 2 (IP3R2) to form 'cAMP junctions' that deliver supramaximal cAMP concentrations directly to IP3R2, sensitizing them to IP3; siRNA knockdown of AC6 or IP3R2 selectively attenuated PTH-mediated potentiation of Ca2+ signals, demonstrating a binary cAMP signaling mode distinct from analogue signaling.","method":"siRNA knockdown of AC6 and IP3R2, Ca2+ imaging, cAMP measurements, co-immunoprecipitation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal siRNA knockdown of both partners with specific functional readout, multiple orthogonal methods","pmids":["18936250"],"is_preprint":false},{"year":2010,"finding":"AC6 is required for vasopressin V2 receptor-stimulated cAMP production in kidney tubules/collecting duct and plays a critical role in regulating water homeostasis, as AC6-knockout mice showed impaired water reabsorption demonstrated by metabolic cage assay and DCE-MRI.","method":"AC6 knockout mice (two independent lines), immunohistochemistry, adenylyl cyclase activity assay, metabolic cage assay, DCE-MRI","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with defined physiological phenotype validated in two independent mouse lines","pmids":["20466003"],"is_preprint":false},{"year":2010,"finding":"A catalytically inactive AC6 mutant (D426A in the C1 catalytic domain) with markedly diminished cAMP-generating capacity still reduces phenylephrine-induced cardiac myocyte hypertrophy and apoptosis, decreases phospholamban expression, and increases Ca2+ transients after isoproterenol stimulation, demonstrating that many beneficial cardiac effects of AC6 do not require cAMP production.","method":"Adenovirus-mediated gene transfer in adult rat cardiac myocytes, active-site mutagenesis (D426A), cAMP measurement, hypertrophy/apoptosis assays, Ca2+ transient measurement","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1 — catalytic site mutagenesis with multiple functional readouts in primary cardiac myocytes","pmids":["21127130"],"is_preprint":false},{"year":2010,"finding":"Fibroblast-specific overexpression of AC6 enhances β-adrenergic (isoproterenol) and prostacyclin (beraprost) receptor-stimulated cAMP production and inhibits collagen synthesis; AC6-overexpressing transgenic mice showed reduced bleomycin-induced pulmonary fibrosis and collagen deposition, indicating AC6 mediates antifibrotic signaling through catecholamine and prostacyclin pathways but not basal or PGE2 signaling.","method":"AC6 overexpression in pulmonary fibroblasts, transgenic FTS1-AC6 mice, bleomycin lung fibrosis model, cAMP assay, collagen measurement, histopathology","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"High","confidence_rationale":"Tier 2 — in vitro overexpression with defined cAMP readout replicated in transgenic mouse disease model","pmids":["20348281"],"is_preprint":false},{"year":2013,"finding":"A homozygous missense mutation in ADCY6 causes absence of peripheral nervous system myelin (as shown by TEM), and morpholino knockdown of the zebrafish ADCY6 ortholog produces severe and specific peripheral myelin defects despite presence of Schwann cells, establishing an essential role for ADCY6 in PNS myelination, likely through cAMP-mediated upregulation of myelinating signals.","method":"Whole exome sequencing, TEM of sciatic nerve biopsies, zebrafish morpholino knockdown with peripheral myelin phenotype assessment","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — genetic LOF (human mutation + morpholino in zebrafish) with defined cellular phenotype replicated across species","pmids":["24319099"],"is_preprint":false},{"year":2018,"finding":"AC6 physically and functionally associates with CFTR at the apical surface of intestinal epithelial cells and is the major cAMP-producing enzyme driving cholera toxin-induced diarrhea; epithelium-specific AC6 knockout mice showed near-complete abolition of CFTR-dependent fluid secretion upon cholera toxin challenge in ligated ileal loops, and loss of AC6 dramatically impaired CFTR activation in intestinal spheroids.","method":"RNA-Seq isoform identification, co-immunoprecipitation, epithelium-specific AC6 KO mice, ligated ileal loop fluid secretion assay, intestinal spheroid CFTR activation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — Co-IP plus genetic KO with defined physiological phenotype, multiple orthogonal methods and in vivo validation","pmids":["29903911"],"is_preprint":false},{"year":2019,"finding":"Purβ (purine-rich element binding protein B) directly binds to the ADCY6 gene promoter and promotes its transcription, thereby activating the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway to increase hepatic glucose production; liver-specific knockdown of Purβ in db/db mice suppressed ADCY6 expression and ameliorated hyperglycemia.","method":"Chromatin immunoprecipitation, luciferase reporter assay, adenovirus-mediated KD/OE in primary hepatocytes and db/db mice, glucose/insulin/lactate tolerance tests, immunoblotting of cAMP signaling components","journal":"Molecular metabolism","confidence":"High","confidence_rationale":"Tier 1–2 — direct ChIP demonstrating promoter binding, luciferase reporter validation, and in vivo KD with defined metabolic phenotype","pmids":["31918924"],"is_preprint":false},{"year":2020,"finding":"AC6 inhibits degradation of the microtubule-depolymerizing kinesin KIF19A by suppressing autophagy through inhibition of AMPK; epithelium-specific AC6 KO airway epithelial cells showed elongated cilia due to decreased KIF19A protein levels, and pharmacological AMPK activation phenocopied AC6 loss by mobilizing KIF19A into autophagosomes.","method":"Epithelium-specific AC6 KO mice, cilia length measurement, KIF19A protein quantification, AMPK activity assays, autophagy flux experiments, pharmacological AMPK modulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with defined cellular phenotype, mechanistic pathway validated by pharmacological orthogonal approach","pmids":["32683324"],"is_preprint":false},{"year":2003,"finding":"AC6 localizes to lipid raft membrane microdomains and co-immunoprecipitates with nicotinic acetylcholine receptor α7 subunit (nAChRα7) within rafts; cholesterol depletion disrupts this co-localization and abrogates nicotine-induced inhibition of AC6 activity, demonstrating that raft localization is required for nAChRα7-mediated calcium-dependent inhibition of AC6.","method":"Sucrose gradient fractionation, co-immunoprecipitation, cholesterol depletion with methyl-β-cyclodextrin, AC activity assay","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2–3 — co-IP plus fractionation with functional consequence, single lab","pmids":["12748066"],"is_preprint":false},{"year":2016,"finding":"HSF1 (heat shock factor 1) positively regulates AC6 mRNA expression in a pressure-overload heart failure model; HSF1 transgenic mice showed increased AC6 mRNA, cAMP, and PKA levels and improved cardiac function, while HSF1 knockout mice showed the opposite, placing AC6 downstream of HSF1 in the AC6/cAMP/PKA pathway that ameliorates heart failure.","method":"Transverse aortic constriction mouse model, HSF1 transgenic and KO mice, RT-qPCR, Western blotting, ELISA for cAMP, echocardiography","journal":"Environmental toxicology and pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic gain- and loss-of-function with defined cardiac phenotype, single lab","pmids":["27643574"],"is_preprint":false},{"year":2022,"finding":"miR-27a-3p suppresses TET1 expression, reducing TET1-mediated DNA demethylation of the ADCY6 promoter; decreased ADCY6 expression promotes breast cancer cell proliferation, invasion and migration through EMT; restoring ADCY6 or TET1 activity reverses these effects, defining a miR-27a-3p/TET1/ADCY6/EMT regulatory axis.","method":"DNA methylation-specific PCR, bisulfite Sanger sequencing, lentiviral miRNA transfection, cell invasion/migration assays, luciferase reporter, gene expression analysis","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2–3 — multiple orthogonal epigenetic and functional methods, single lab","pmids":["35978806"],"is_preprint":false},{"year":2023,"finding":"AC6 (localized in lipid raft membranes) and AC2 (localized in non-raft membranes) generate distinct cAMP signaling pools in human airway smooth muscle; quantitative phosphoproteomics revealed that AC6 activity specifically regulates phosphorylation of proteins involved in autophagy, Ca2+/CaM signaling, Rho GTPases, and cytoskeletal regulation, while AC2 activity predominantly affects RNA/DNA binding and microtubule proteins.","method":"AC2/AC6 overexpression in human airway smooth muscle cells, quantitative phosphoproteomics by LC-MS/MS, STRING network analysis","journal":"Frontiers in physiology","confidence":"Medium","confidence_rationale":"Tier 2 — quantitative phosphoproteomics with isoform-specific overexpression titrated to equal cAMP output, single lab","pmids":["36926196"],"is_preprint":false},{"year":2024,"finding":"Forskolin alleviates hypertrophic cardiomyopathy in Myh6R404Q and Tnnt2R109Q mouse models through activation of ADCY6, with downstream cAMP/PKA pathway activation reducing cardiac hypertrophy and normalizing cardiomyocyte size and hypertrophy-related gene expression.","method":"HCM mouse models (Myh6R404Q and Tnnt2R109Q), norepinephrine-induced cardiomyocyte hypertrophy in vitro, pharmacological ADCY6 activation, gene expression analysis","journal":"European journal of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2–3 — in vivo and in vitro pharmacological activation with defined phenotypic readout, mechanism partly inferred","pmids":["38925286"],"is_preprint":false},{"year":2021,"finding":"GPSM1 (G protein signaling modulator 1) promotes proliferation of B-ALL cells by regulating ADCY6 expression; GPSM1 knockdown suppressed ADCY6 and RAPGEF3 expression and reduced JNK activity, defining a GPSM1/ADCY6/RAPGEF3/JNK signaling axis in leukemia cell proliferation.","method":"siRNA knockdown of GPSM1 in BALL-1 and Reh cells, cell proliferation/apoptosis/cell cycle assays, Western blotting of ADCY6, RAPGEF3, and JNK","journal":"Pathology oncology research : POR","confidence":"Low","confidence_rationale":"Tier 3 — single KD experiment with downstream protein measurement but no direct demonstration of ADCY6-RAPGEF3 interaction","pmids":["34257610"],"is_preprint":false}],"current_model":"ADCY6 is a calcium-inhibitable adenylyl cyclase localized in lipid raft membrane microdomains that synthesizes cAMP from ATP (with catalytic residue D426 required for enzymatic activity), regulates diverse physiological processes including PNS myelination, renal water homeostasis, ciliary length control (via AMPK-KIF19A autophagy), renin secretion, intestinal fluid secretion (through a physical complex with CFTR), and cardiac function; its cAMP production is locally regulated by an AKAP79/150-anchored PKA negative feedback loop and by selective coupling with IP3R2 to form binary cAMP signaling junctions, while notably many of its cardioprotective effects (reduced hypertrophy, apoptosis, and altered Ca2+ handling) do not require cAMP generation and are replicated by a catalytically inactive mutant."},"narrative":{"teleology":[{"year":2003,"claim":"Establishing that AC6 resides in lipid raft microdomains answered how calcium-dependent inhibitory signals from nAChRα7 are spatially coupled to AC6: raft localization is required for this regulation, defining AC6 as a compartment-specific signaling node.","evidence":"Sucrose gradient fractionation, co-immunoprecipitation, and cholesterol depletion in BAECs","pmids":["12748066"],"confidence":"Medium","gaps":["Single lab; raft localization not confirmed by super-resolution imaging","nAChRα7–AC6 interaction not validated by reciprocal pull-down or proximity ligation"]},{"year":2006,"claim":"Two parallel discoveries revealed how AC6 activity is terminated and how it controls a classic physiology paradox: AC6 forms a complex with AKAP79/150 that recruits PKA for feedback phosphorylation/inhibition of AC6, and AC6 (together with AC5) mediates the calcium-dependent suppression of renin secretion in juxtaglomerular cells.","evidence":"Co-IP plus live-cell cAMP imaging with PKA disruption in HEK293 cells; siRNA knockdown in primary juxtaglomerular cells, As4.1 cells, and isolated perfused kidney","pmids":["16973443","17068292"],"confidence":"High","gaps":["Stoichiometry and structural basis of the AKAP79/AC6/PKA complex unknown","Relative contributions of AC5 vs AC6 to renin regulation not fully separated in vivo"]},{"year":2008,"claim":"Demonstrating that AC6 selectively couples with IP3R2 to form cAMP junctions resolved how supra-threshold cAMP concentrations are delivered to a specific target without flooding the cytoplasm, establishing a binary signaling mode for cAMP-Ca²⁺ crosstalk.","evidence":"siRNA knockdown of AC6 and IP3R2, Ca²⁺ imaging, and co-immunoprecipitation in HeLa cells stimulated with PTH","pmids":["18936250"],"confidence":"High","gaps":["Whether AC6–IP3R2 junctions exist in tissues beyond cell lines not tested","Structural determinants of AC6–IP3R2 selectivity unresolved"]},{"year":2010,"claim":"Three studies collectively defined AC6's in vivo physiological roles and, critically, dissociated its catalytic activity from its cardioprotective effects: AC6 KO mice showed impaired vasopressin-mediated water reabsorption; a catalytically dead D426A mutant still reduced cardiac hypertrophy and apoptosis; and AC6 overexpression suppressed bleomycin-induced pulmonary fibrosis via β-adrenergic/prostacyclin cAMP signaling.","evidence":"AC6 KO mice (two lines) with DCE-MRI and metabolic cages; D426A mutagenesis with gene transfer in rat cardiac myocytes; AC6-transgenic mice in bleomycin fibrosis model","pmids":["20466003","21127130","20348281"],"confidence":"High","gaps":["cAMP-independent cardioprotective mechanism of AC6 remains molecularly undefined","Antifibrotic function not tested with catalytically dead mutant","Identity of AC6 protein–protein interactions mediating cAMP-independent cardiac effects unknown"]},{"year":2013,"claim":"Identification of a homozygous ADCY6 mutation causing absent peripheral myelin in humans, phenocopied by morpholino knockdown in zebrafish, established AC6 as essential for Schwann cell–mediated PNS myelination—the first Mendelian disease link for this gene.","evidence":"Whole-exome sequencing in affected family, TEM of sciatic nerve, zebrafish morpholino knockdown","pmids":["24319099"],"confidence":"High","gaps":["Downstream myelination program controlled by AC6-generated cAMP in Schwann cells not delineated","Only one family reported; allelic spectrum unknown"]},{"year":2018,"claim":"Showing that AC6 physically complexes with CFTR at the apical membrane of intestinal epithelia and is the dominant cyclase driving cholera toxin–induced fluid secretion answered which adenylyl cyclase isoform mediates secretory diarrhea.","evidence":"Co-IP, epithelium-specific AC6 KO mice, ligated ileal loop assay, intestinal spheroid CFTR activation","pmids":["29903911"],"confidence":"High","gaps":["Whether AC6–CFTR interaction is direct or scaffolded not resolved","Therapeutic potential of AC6 inhibition in cholera not tested"]},{"year":2019,"claim":"Identification of Purβ as a direct transcriptional activator of ADCY6 linked hepatic AC6 expression to gluconeogenesis, explaining how the glucagon/cAMP/PKA/CREB axis is sustained at the transcriptional level in diabetic liver.","evidence":"ChIP, luciferase reporter, and adenoviral KD/OE of Purβ in hepatocytes and db/db mice","pmids":["31918924"],"confidence":"High","gaps":["Other transcription factors regulating ADCY6 in non-hepatic contexts not mapped","Whether Purβ regulation is specific to ADCY6 vs other ADCY isoforms unclear"]},{"year":2020,"claim":"Demonstrating that AC6 controls airway ciliary length by inhibiting AMPK-dependent autophagic degradation of KIF19A revealed a non-canonical cyclase function linking cAMP signaling to autophagy and cytoskeletal regulation.","evidence":"Epithelium-specific AC6 KO mice, KIF19A quantification, autophagy flux assays, pharmacological AMPK modulation","pmids":["32683324"],"confidence":"High","gaps":["Whether AC6–AMPK inhibition is direct or mediated by PKA/Epac not resolved","In vivo mucociliary clearance consequences not measured"]},{"year":2023,"claim":"Phosphoproteomic profiling resolved how raft-localized AC6 and non-raft AC2 generate distinct downstream signaling networks from equivalent total cAMP, demonstrating that AC6-specific cAMP pools preferentially regulate autophagy, Ca²⁺/CaM, and Rho GTPase signaling.","evidence":"Isoform-specific overexpression in human airway smooth muscle with quantitative LC-MS/MS phosphoproteomics","pmids":["36926196"],"confidence":"Medium","gaps":["Phosphoproteomic targets not validated by targeted mutagenesis","Single cell type studied; generalizability to other AC6-expressing tissues unknown"]},{"year":null,"claim":"The molecular basis of AC6's cAMP-independent cardioprotective activity—identified by the catalytically dead D426A mutant—remains unknown; the interacting partners, structural features, or scaffolding functions responsible have not been identified.","evidence":"","pmids":[],"confidence":"High","gaps":["No interactome or structural study of the D426A mutant performed","Whether cAMP-independent functions extend to non-cardiac tissues untested","AC6-specific inhibitors or activators for therapeutic use not developed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0009975","term_label":"cyclase activity","supporting_discovery_ids":[1,3,4,5,7,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[7,10,13]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,3,8]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[3,7]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[9,13]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[6]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[6,7]}],"complexes":["AKAP79/150-PKA-AC6 complex","AC6-CFTR complex","AC6-IP3R2 cAMP junction"],"partners":["AKAP5","ITPR2","CFTR","CHRNA7","KIF19A","PURB","HSF1"],"other_free_text":[]},"mechanistic_narrative":"ADCY6 is a calcium-inhibitable, membrane-bound adenylate cyclase that generates compartmentalized cAMP pools to regulate a wide range of physiological processes including cardiac function, renal water homeostasis, intestinal fluid secretion, airway ciliary length, peripheral nervous system myelination, and hepatic glucose output. Localized to lipid raft microdomains, AC6 forms functionally specialized signaling complexes—with AKAP79/150-anchored PKA for negative feedback termination of cAMP synthesis, with IP3R2 for binary cAMP-Ca²⁺ signaling junctions, and with CFTR at the apical surface of intestinal epithelia to drive cholera toxin–induced secretory diarrhea [PMID:16973443, PMID:18936250, PMID:29903911, PMID:12748066]. In the kidney, AC6 is the predominant cyclase coupling vasopressin V2 receptor stimulation to water reabsorption, and in airway epithelia it controls ciliary length by suppressing AMPK-dependent autophagic degradation of the kinesin KIF19A [PMID:20466003, PMID:32683324]. Notably, a catalytically dead AC6 mutant (D426A) retains the ability to reduce cardiac hypertrophy and apoptosis and to alter Ca²⁺ handling, demonstrating that key cardioprotective functions are independent of cAMP generation, while loss-of-function mutations in humans cause absence of peripheral myelin [PMID:21127130, PMID:24319099]."},"prefetch_data":{"uniprot":{"accession":"O43306","full_name":"Adenylate cyclase type 6","aliases":["ATP pyrophosphate-lyase 6","Adenylate cyclase type VI","Adenylyl cyclase 6","Ca(2+)-inhibitable adenylyl cyclase"],"length_aa":1168,"mass_kda":130.6,"function":"Catalyzes the formation of the signaling molecule cAMP downstream of G protein-coupled receptors (PubMed:17110384, PubMed:17916776). Functions in signaling cascades downstream of beta-adrenergic receptors in the heart and in vascular smooth muscle cells (PubMed:17916776). Functions in signaling cascades downstream of the vasopressin receptor in the kidney and has a role in renal water reabsorption. Functions in signaling cascades downstream of PTH1R and plays a role in regulating renal phosphate excretion. Functions in signaling cascades downstream of the VIP and SCT receptors in pancreas and contributes to the regulation of pancreatic amylase and fluid secretion (By similarity). Signaling mediates cAMP-dependent activation of protein kinase PKA. This promotes increased phosphorylation of various proteins, including AKT. Plays a role in regulating cardiac sarcoplasmic reticulum Ca(2+) uptake and storage, and is required for normal heart ventricular contractibility. May contribute to normal heart function (By similarity). Mediates vasodilatation after activation of beta-adrenergic receptors by isoproterenol (PubMed:17916776). Contributes to bone cell responses to mechanical stimuli (By similarity)","subcellular_location":"Cell membrane; Cell projection, cilium; Cell projection, stereocilium","url":"https://www.uniprot.org/uniprotkb/O43306/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ADCY6","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ADCY6","total_profiled":1310},"omim":[{"mim_id":"619749","title":"VEZATIN, ADHERENS JUNCTIONS TRANSMEMBRANE PROTEIN; VEZT","url":"https://www.omim.org/entry/619749"},{"mim_id":"618484","title":"ARTHROGRYPOSIS MULTIPLEX CONGENITA 3, MYOGENIC TYPE; AMC3","url":"https://www.omim.org/entry/618484"},{"mim_id":"616287","title":"LETHAL CONGENITAL CONTRACTURE SYNDROME 8; LCCS8","url":"https://www.omim.org/entry/616287"},{"mim_id":"616286","title":"LETHAL CONGENITAL CONTRACTURE SYNDROME 7; LCCS7","url":"https://www.omim.org/entry/616286"},{"mim_id":"611607","title":"MICRO RNA 182; MIR182","url":"https://www.omim.org/entry/611607"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Golgi apparatus","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ADCY6"},"hgnc":{"alias_symbol":["AC6"],"prev_symbol":[]},"alphafold":{"accession":"O43306","domains":[{"cath_id":"-","chopping":"231-325_660-931","consensus_level":"medium","plddt":83.8829,"start":231,"end":931},{"cath_id":"3.30.70.1230","chopping":"371-566","consensus_level":"high","plddt":85.913,"start":371,"end":566},{"cath_id":"3.30.70.1230","chopping":"958-1165","consensus_level":"high","plddt":87.0201,"start":958,"end":1165}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43306","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43306-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43306-F1-predicted_aligned_error_v6.png","plddt_mean":76.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ADCY6","jax_strain_url":"https://www.jax.org/strain/search?query=ADCY6"},"sequence":{"accession":"O43306","fasta_url":"https://rest.uniprot.org/uniprotkb/O43306.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43306/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43306"}},"corpus_meta":[{"pmid":"24319099","id":"PMC_24319099","title":"Mutations in CNTNAP1 and ADCY6 are responsible for severe arthrogryposis multiplex congenita with axoglial defects.","date":"2013","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24319099","citation_count":100,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17068292","id":"PMC_17068292","title":"The calcium paradoxon of renin release: calcium suppresses renin exocytosis by inhibition of calcium-dependent adenylate cyclases AC5 and AC6.","date":"2006","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/17068292","citation_count":64,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"31595165","id":"PMC_31595165","title":"Circ-HIPK3 Strengthens the Effects of Adrenaline in Heart Failure by MiR-17-3p - ADCY6 Axis.","date":"2019","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31595165","citation_count":49,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20466003","id":"PMC_20466003","title":"Impaired water reabsorption in mice deficient in the type VI adenylyl cyclase (AC6).","date":"2010","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/20466003","citation_count":32,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"31918924","id":"PMC_31918924","title":"Purβ promotes hepatic glucose production by increasing Adcy6 transcription.","date":"2019","source":"Molecular metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/31918924","citation_count":27,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20348281","id":"PMC_20348281","title":"Fibroblast-specific expression of AC6 enhances beta-adrenergic and prostacyclin signaling and blunts bleomycin-induced pulmonary fibrosis.","date":"2010","source":"American journal of physiology. Lung cellular and molecular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/20348281","citation_count":18,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"32683324","id":"PMC_32683324","title":"AC6 regulates the microtubule-depolymerizing kinesin KIF19A to control ciliary length in mammals.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32683324","citation_count":18,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21127130","id":"PMC_21127130","title":"Beneficial effects of adenylyl cyclase type 6 (AC6) expression persist using a catalytically inactive AC6 mutant.","date":"2010","source":"Molecular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/21127130","citation_count":15,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35978806","id":"PMC_35978806","title":"MiR-27a-3p binds to TET1 mediated DNA demethylation of ADCY6 regulates breast cancer progression via epithelial-mesenchymal transition.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35978806","citation_count":12,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"29903911","id":"PMC_29903911","title":"AC6 is the major adenylate cyclase forming a diarrheagenic protein complex with cystic fibrosis transmembrane conductance regulator in cholera.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/29903911","citation_count":11,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"34257610","id":"PMC_34257610","title":"Knockdown of GPSM1 Inhibits the Proliferation and Promotes the Apoptosis of B-Cell Acute Lymphoblastic Leukemia Cells by Suppressing the ADCY6-RAPGEF3-JNK Signaling Pathway.","date":"2021","source":"Pathology oncology research : POR","url":"https://pubmed.ncbi.nlm.nih.gov/34257610","citation_count":8,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21097624","id":"PMC_21097624","title":"DNA-binding properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ(D) proteins.","date":"2010","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/21097624","citation_count":7,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"31846058","id":"PMC_31846058","title":"Expanding the clinical and molecular spectrum of lethal congenital contracture syndrome 8 associated with biallelic variants of ADCY6.","date":"2020","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31846058","citation_count":6,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"38925286","id":"PMC_38925286","title":"Forskolin is an effective therapeutic small molecule for the treatment of hypertrophic cardiomyopathy through ADCY6/cAMP/PKA pathway.","date":"2024","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/38925286","citation_count":5,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"36926196","id":"PMC_36926196","title":"Quantitative phosphoproteomic analysis reveals unique cAMP signaling pools emanating from AC2 and AC6 in human airway smooth muscle cells.","date":"2023","source":"Frontiers in physiology","url":"https://pubmed.ncbi.nlm.nih.gov/36926196","citation_count":3,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27643574","id":"PMC_27643574","title":"Role of HSF1-upregulated AC6 in ameliorating heart failure in mice.","date":"2016","source":"Environmental toxicology and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/27643574","citation_count":2,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":null,"id":"bio_10.1101_2024.10.02.616263","title":"Early intermittent low-dose sclerostin antibody treatment promotes surface bone formation and reduces bone loss, but also decreases osteocyte apoptosis and mechanotransduction in ovariectomized rats: a pilot study","date":"2024-10-02","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.02.616263","citation_count":0,"is_preprint":true,"source_track":"pubmed_title"},{"pmid":null,"id":"bio_10.1101_2025.06.05.25329068","title":"Exome sequencing and analysis of 44,028 British South Asians enriched for high autozygosity","date":"2025-06-06","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.05.25329068","citation_count":0,"is_preprint":true,"source_track":"pubmed_title"},{"pmid":"12477932","id":"PMC_12477932","title":"Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.","date":"2002","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12477932","citation_count":1479,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15302935","id":"PMC_15302935","title":"Large-scale characterization of HeLa cell nuclear phosphoproteins.","date":"2004","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/15302935","citation_count":1159,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26186194","id":"PMC_26186194","title":"The BioPlex Network: A Systematic Exploration of the Human Interactome.","date":"2015","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/26186194","citation_count":1118,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"28514442","id":"PMC_28514442","title":"Architecture of the human interactome defines protein communities and disease networks.","date":"2017","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/28514442","citation_count":1085,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"2165385","id":"PMC_2165385","title":"cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes.","date":"1990","source":"Annual review of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/2165385","citation_count":1019,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"33961781","id":"PMC_33961781","title":"Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.","date":"2021","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/33961781","citation_count":705,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9417641","id":"PMC_9417641","title":"Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsalpha.GTPgammaS.","date":"1997","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/9417641","citation_count":657,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21873635","id":"PMC_21873635","title":"Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium.","date":"2011","source":"Briefings in bioinformatics","url":"https://pubmed.ncbi.nlm.nih.gov/21873635","citation_count":656,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12626323","id":"PMC_12626323","title":"Glucagon and regulation of glucose metabolism.","date":"2003","source":"American journal of physiology. Endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/12626323","citation_count":635,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16713569","id":"PMC_16713569","title":"A protein-protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration.","date":"2006","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/16713569","citation_count":610,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"33845483","id":"PMC_33845483","title":"Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.","date":"2021","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/33845483","citation_count":532,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"8125298","id":"PMC_8125298","title":"Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.","date":"1994","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/8125298","citation_count":492,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"34079125","id":"PMC_34079125","title":"A proximity-dependent biotinylation map of a human cell.","date":"2021","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/34079125","citation_count":339,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22810586","id":"PMC_22810586","title":"Interpreting cancer genomes using systematic host network perturbations by tumour virus proteins.","date":"2012","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/22810586","citation_count":319,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"14993377","id":"PMC_14993377","title":"Isoforms of mammalian adenylyl cyclase: multiplicities of signaling.","date":"2002","source":"Molecular interventions","url":"https://pubmed.ncbi.nlm.nih.gov/14993377","citation_count":299,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27173435","id":"PMC_27173435","title":"An organelle-specific protein landscape identifies novel diseases and molecular mechanisms.","date":"2016","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/27173435","citation_count":211,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16973443","id":"PMC_16973443","title":"Dynamic regulation of cAMP synthesis through anchored PKA-adenylyl cyclase V/VI complexes.","date":"2006","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/16973443","citation_count":185,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"31871319","id":"PMC_31871319","title":"Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms.","date":"2019","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/31871319","citation_count":137,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"29117863","id":"PMC_29117863","title":"RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination.","date":"2017","source":"BMC biology","url":"https://pubmed.ncbi.nlm.nih.gov/29117863","citation_count":135,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"30639242","id":"PMC_30639242","title":"The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2.","date":"2019","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/30639242","citation_count":124,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25187353","id":"PMC_25187353","title":"Clozapine-induced agranulocytosis is associated with rare HLA-DQB1 and HLA-B alleles.","date":"2014","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/25187353","citation_count":123,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"31753913","id":"PMC_31753913","title":"Systematic bromodomain protein screens identify homologous recombination and R-loop suppression pathways involved in genome integrity.","date":"2019","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/31753913","citation_count":110,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"7937899","id":"PMC_7937899","title":"Mechanism of GTP hydrolysis by G-protein alpha subunits.","date":"1994","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/7937899","citation_count":106,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"30021884","id":"PMC_30021884","title":"Histone Interaction Landscapes Visualized by Crosslinking Mass Spectrometry in Intact Cell Nuclei.","date":"2018","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/30021884","citation_count":101,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"18936250","id":"PMC_18936250","title":"Selective coupling of type 6 adenylyl cyclase with type 2 IP3 receptors mediates direct sensitization of IP3 receptors by cAMP.","date":"2008","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/18936250","citation_count":83,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"8663304","id":"PMC_8663304","title":"Purification and characterization of a soluble form of mammalian adenylyl cyclase.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8663304","citation_count":83,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12503609","id":"PMC_12503609","title":"Characterization of the human adenylyl cyclase gene family: cDNA, gene structure, and tissue distribution of the nine isoforms.","date":"2002","source":"Journal of receptor and signal transduction research","url":"https://pubmed.ncbi.nlm.nih.gov/12503609","citation_count":77,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9455477","id":"PMC_9455477","title":"Prediction of the coding sequences of unidentified human genes. VIII. 78 new cDNA clones from brain which code for large proteins in vitro.","date":"1997","source":"DNA research : an international journal for rapid publication of reports on genes and genomes","url":"https://pubmed.ncbi.nlm.nih.gov/9455477","citation_count":68,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12748066","id":"PMC_12748066","title":"Nicotinic acetylcholine receptor alpha 7 regulates cAMP signal within lipid rafts.","date":"2003","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/12748066","citation_count":66,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10431,"output_tokens":3251,"usd":0.040029},"stage2":{"model":"claude-opus-4-6","input_tokens":6608,"output_tokens":2880,"usd":0.15756},"total_usd":0.427415,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":21742,"output_tokens":4182,"usd":0.063978},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":7873,"output_tokens":2848,"usd":0.165848}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"AC6 (along with AC5) mediates calcium-dependent inhibition of renin secretion in juxtaglomerular cells by suppressing intracellular cAMP production; siRNA-mediated knockdown of AC5 and/or AC6 prevented calcium-dependent inhibition of cAMP levels and renin release, establishing AC6 as a calcium-inhibitable adenylate cyclase functionally coupling intracellular calcium elevation to suppressed cAMP synthesis and renin exocytosis.\",\n      \"method\": \"siRNA knockdown in primary juxtaglomerular cells and As4.1 renin-producing cells; cAMP measurement; renin secretion assay; permeable cAMP analog rescue experiments\",\n      \"journal\": \"Circulation Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal siRNA knockdown with multiple orthogonal functional readouts (cAMP levels, renin secretion) and rescue experiments\",\n      \"pmids\": [\"17068292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"AC6 is localized to renal tubular parts and collecting duct, and AC6-knockout mice show reduced adenylate cyclase activity evoked by forskolin or V2 vasopressin receptor agonist in kidneys, and display impaired water reabsorption, establishing AC6 as a critical regulator of renal water homeostasis.\",\n      \"method\": \"AC6-knockout mouse lines; immunohistochemistry; adenylate cyclase activity assays; metabolic cage assay; dynamic contrast-enhanced MRI\",\n      \"journal\": \"FEBS Letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockout with multiple orthogonal phenotypic readouts and localization data\",\n      \"pmids\": [\"20466003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"AC6 overexpression in pulmonary fibroblasts enhances cAMP production and inhibits collagen synthesis in response to isoproterenol and beraprost (but not PGE2 or butaprost), and fibroblast-specific AC6 transgenic mice are protected from bleomycin-induced pulmonary fibrosis, indicating AC6 selectively couples to beta-adrenergic and prostacyclin (but not EP2 prostanoid) receptor signaling in fibroblasts to suppress fibrogenesis.\",\n      \"method\": \"AC6 overexpression in primary pulmonary fibroblasts; cAMP measurement; collagen synthesis assay; FTS1-promoter-driven AC6 transgenic mice; bleomycin lung fibrosis model; histological scoring and collagen quantification\",\n      \"journal\": \"American Journal of Physiology - Lung Cellular and Molecular Physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vitro overexpression with cAMP/collagen readouts plus in vivo transgenic model with functional fibrosis phenotype\",\n      \"pmids\": [\"20348281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"A catalytically inactive AC6 mutant (D426A in the C1 catalytic domain) retains the ability to reduce phenylephrine-induced cardiac myocyte hypertrophy, apoptosis, CARP expression, phospholamban expression, and to enhance Ca2+ transients after isoproterenol stimulation, demonstrating that many biological effects of AC6 in cardiomyocytes are independent of cAMP production.\",\n      \"method\": \"Site-directed mutagenesis (D426A); adenovirus-mediated gene transfer in adult rat cardiac myocytes; cAMP measurement; cell size, apoptosis, gene expression, and Ca2+ transient assays\",\n      \"journal\": \"Molecular Pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — active-site mutagenesis with multiple orthogonal functional readouts in primary cardiomyocytes\",\n      \"pmids\": [\"21127130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A homozygous missense mutation in ADCY6 causes absence of peripheral nervous system myelin in human patients with arthrogryposis multiplex congenita, and morpholino knockdown of the zebrafish ADCY6 ortholog produces severe and specific defects in peripheral myelin despite presence of Schwann cells, establishing ADCY6 as essential for PNS myelination, likely through the cAMP pathway.\",\n      \"method\": \"Whole exome sequencing in human AMC families; transmission electron microscopy of sciatic nerve; morpholino knockdown of zebrafish ortholog\",\n      \"journal\": \"Human Molecular Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human genetic loss-of-function combined with zebrafish morpholino knockdown showing specific myelination phenotype\",\n      \"pmids\": [\"24319099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AC6 physically associates with CFTR at the apical surface of intestinal epithelial cells and is the major cAMP-producing enzyme in this compartment; epithelium-specific AC6 knockout mice show near-abolishment of CFTR-dependent fluid secretion and CFTR activation upon cholera toxin challenge, establishing AC6-CFTR as a functional complex mediating secretory diarrhea.\",\n      \"method\": \"RNA-Seq for AC isoform identification; co-immunoprecipitation/biochemical association assays; epithelium-specific AC6 knockout mice; ligated ileal loop fluid secretion assay; human and mouse intestinal spheroid CFTR activation assay\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal biochemical association plus epithelium-specific KO with multiple functional readouts\",\n      \"pmids\": [\"29903911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Purβ directly binds to the Adcy6 gene promoter and promotes its transcription, thereby activating the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway to increase hepatic glucose production; liver-specific Purβ knockdown in diabetic db/db mice suppresses this pathway and ameliorates hyperglycemia.\",\n      \"method\": \"Luciferase reporter assay; chromatin immunoprecipitation (ChIP); adenovirus-mediated knockdown/overexpression in primary hepatocytes and db/db mice; glucose/insulin/lactate tolerance tests; immunoblotting for p-CREB/CREB and p-Akt/Akt\",\n      \"journal\": \"Molecular Metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and luciferase reporter establish direct promoter binding, with in vivo KD showing pathway suppression\",\n      \"pmids\": [\"31918924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AC6 inhibits AMPK activation in airway epithelial cells, uncoupling AMPK from binding to ciliary kinesin KIF19A; in the absence of AC6 (epithelium-specific knockout), AMPK is activated and mobilizes KIF19A into autophagosomes for degradation, resulting in lower KIF19A levels at cilia tips and elongated cilia.\",\n      \"method\": \"Epithelium-specific AC6 knockout mice; cilia length measurement; KIF19A protein quantification; AMPK activity assays; autophagosome assays; pharmacological AMPK activation/inhibition\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with mechanistic pathway dissection using pharmacological tools and multiple orthogonal readouts\",\n      \"pmids\": [\"32683324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HSF1 positively regulates AC6 mRNA expression in pressure overload-induced heart failure; HSF1 transgenic mice show increased AC6 mRNA, cAMP, and PKA activity with improved cardiac function, while HSF1 knockout mice show the opposite, placing AC6 downstream of HSF1 in the HSF1/AC6/cAMP/PKA pathway.\",\n      \"method\": \"Transverse aortic constriction (TAC) mouse model; HSF1 transgenic and knockout mice; RT-qPCR for AC6 mRNA; Western blotting for HSF1 and PKA; ELISA for cAMP; cardiac function by echocardiography\",\n      \"journal\": \"Environmental Toxicology and Pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic gain/loss of function in vivo but single lab, no direct HSF1-AC6 promoter interaction shown\",\n      \"pmids\": [\"27643574\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"miR-27a-3p suppresses TET1 expression, which reduces TET1-mediated DNA demethylation of the ADCY6 promoter, resulting in decreased ADCY6 expression; lower ADCY6 promotes breast cancer cell proliferation, invasion, and migration through the epithelial-mesenchymal transition (EMT) process.\",\n      \"method\": \"DNA methylation-specific PCR; bisulfite Sanger sequencing; lentiviral stable miRNA transfection; cell biology functional assays (proliferation, invasion, migration); luciferase reporter for miR-27a-3p/TET1 interaction\",\n      \"journal\": \"Frontiers in Oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — methylation and functional assays in cell lines, single lab, pathway inference\",\n      \"pmids\": [\"35978806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AC6, localized in lipid raft membrane domains (unlike AC2 in non-raft membranes), generates a distinct cAMP pool in human airway smooth muscle cells that differentially phosphorylates proteins involved in autophagy, Ca2+/CaM signaling, Rho GTPases, and cytoskeletal regulation, as opposed to the AC2 pool which regulates RNA/DNA binding and microtubule proteins.\",\n      \"method\": \"Quantitative phosphoproteomics (LC-MS/MS); AC2 and AC6 overexpression in human airway smooth muscle cells; forskolin stimulation; STRING protein interaction analysis\",\n      \"journal\": \"Frontiers in Physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative phosphoproteomics with isoform-specific overexpression, but single lab and downstream pathway inference\",\n      \"pmids\": [\"36926196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Forskolin alleviates hypertrophic cardiomyopathy in mouse models (Myh6R404Q and Tnnt2R109Q) and norepinephrine-induced cardiomyocyte hypertrophy in vitro through activation of ADCY6, operating via the ADCY6/cAMP/PKA signaling pathway.\",\n      \"method\": \"HCM mouse models (Myh6R404Q, Tnnt2R109Q); in vitro NE-induced hypertrophy assay; cardiac function measurement; gene expression analysis; pharmacological activation with forskolin\",\n      \"journal\": \"European Journal of Pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo and in vitro models with pathway readouts, but mechanistic link between forskolin and ADCY6 specificity not fully validated with mutagenesis\",\n      \"pmids\": [\"38925286\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ADCY6 encodes a calcium-inhibitable, lipid raft-localized transmembrane adenylyl cyclase that converts ATP to cAMP downstream of beta-adrenergic, vasopressin, and glucagon receptors, functioning in renin secretion, renal water reabsorption, PNS myelination, intestinal fluid secretion (via a physical complex with CFTR), ciliary length control (through the AC6-AMPK-KIF19A-autophagy axis), and cardiac function — with some of its cardiomyocyte effects being cAMP-independent, as demonstrated by a catalytically inactive mutant.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"AC6 physically associates with A-kinase anchoring protein AKAP79/150, forming a complex that enables PKA to preferentially phosphorylate AC6 and inhibit cAMP synthesis, creating a negative feedback loop that terminally regulates cAMP production upon agonist stimulation.\",\n      \"method\": \"Co-immunoprecipitation, real-time cellular imaging of cAMP dynamics, PKA anchoring disruption experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus live-cell imaging with functional consequence, moderate evidence from single lab with multiple orthogonal methods\",\n      \"pmids\": [\"16973443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Calcium suppresses renin exocytosis from juxtaglomerular cells by inhibiting AC5 and AC6, thereby reducing intracellular cAMP levels; siRNA-mediated knockdown of AC5 and/or AC6 prevented calcium-dependent inhibition of cAMP and renin release, establishing AC6 as a key mediator of the 'calcium paradoxon' in renin-producing cells.\",\n      \"method\": \"siRNA knockdown of AC5/AC6 in primary juxtaglomerular cells and As4.1 cells, cAMP measurement, renin secretion assays, isolated perfused kidney experiments\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with defined cellular phenotype replicated across cell types and ex vivo organ model\",\n      \"pmids\": [\"17068292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"AC6 selectively couples with IP3 receptor type 2 (IP3R2) to form 'cAMP junctions' that deliver supramaximal cAMP concentrations directly to IP3R2, sensitizing them to IP3; siRNA knockdown of AC6 or IP3R2 selectively attenuated PTH-mediated potentiation of Ca2+ signals, demonstrating a binary cAMP signaling mode distinct from analogue signaling.\",\n      \"method\": \"siRNA knockdown of AC6 and IP3R2, Ca2+ imaging, cAMP measurements, co-immunoprecipitation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal siRNA knockdown of both partners with specific functional readout, multiple orthogonal methods\",\n      \"pmids\": [\"18936250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"AC6 is required for vasopressin V2 receptor-stimulated cAMP production in kidney tubules/collecting duct and plays a critical role in regulating water homeostasis, as AC6-knockout mice showed impaired water reabsorption demonstrated by metabolic cage assay and DCE-MRI.\",\n      \"method\": \"AC6 knockout mice (two independent lines), immunohistochemistry, adenylyl cyclase activity assay, metabolic cage assay, DCE-MRI\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined physiological phenotype validated in two independent mouse lines\",\n      \"pmids\": [\"20466003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"A catalytically inactive AC6 mutant (D426A in the C1 catalytic domain) with markedly diminished cAMP-generating capacity still reduces phenylephrine-induced cardiac myocyte hypertrophy and apoptosis, decreases phospholamban expression, and increases Ca2+ transients after isoproterenol stimulation, demonstrating that many beneficial cardiac effects of AC6 do not require cAMP production.\",\n      \"method\": \"Adenovirus-mediated gene transfer in adult rat cardiac myocytes, active-site mutagenesis (D426A), cAMP measurement, hypertrophy/apoptosis assays, Ca2+ transient measurement\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — catalytic site mutagenesis with multiple functional readouts in primary cardiac myocytes\",\n      \"pmids\": [\"21127130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Fibroblast-specific overexpression of AC6 enhances β-adrenergic (isoproterenol) and prostacyclin (beraprost) receptor-stimulated cAMP production and inhibits collagen synthesis; AC6-overexpressing transgenic mice showed reduced bleomycin-induced pulmonary fibrosis and collagen deposition, indicating AC6 mediates antifibrotic signaling through catecholamine and prostacyclin pathways but not basal or PGE2 signaling.\",\n      \"method\": \"AC6 overexpression in pulmonary fibroblasts, transgenic FTS1-AC6 mice, bleomycin lung fibrosis model, cAMP assay, collagen measurement, histopathology\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vitro overexpression with defined cAMP readout replicated in transgenic mouse disease model\",\n      \"pmids\": [\"20348281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"A homozygous missense mutation in ADCY6 causes absence of peripheral nervous system myelin (as shown by TEM), and morpholino knockdown of the zebrafish ADCY6 ortholog produces severe and specific peripheral myelin defects despite presence of Schwann cells, establishing an essential role for ADCY6 in PNS myelination, likely through cAMP-mediated upregulation of myelinating signals.\",\n      \"method\": \"Whole exome sequencing, TEM of sciatic nerve biopsies, zebrafish morpholino knockdown with peripheral myelin phenotype assessment\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic LOF (human mutation + morpholino in zebrafish) with defined cellular phenotype replicated across species\",\n      \"pmids\": [\"24319099\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AC6 physically and functionally associates with CFTR at the apical surface of intestinal epithelial cells and is the major cAMP-producing enzyme driving cholera toxin-induced diarrhea; epithelium-specific AC6 knockout mice showed near-complete abolition of CFTR-dependent fluid secretion upon cholera toxin challenge in ligated ileal loops, and loss of AC6 dramatically impaired CFTR activation in intestinal spheroids.\",\n      \"method\": \"RNA-Seq isoform identification, co-immunoprecipitation, epithelium-specific AC6 KO mice, ligated ileal loop fluid secretion assay, intestinal spheroid CFTR activation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus genetic KO with defined physiological phenotype, multiple orthogonal methods and in vivo validation\",\n      \"pmids\": [\"29903911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Purβ (purine-rich element binding protein B) directly binds to the ADCY6 gene promoter and promotes its transcription, thereby activating the glucagon/ADCY6/cAMP/PKA/CREB signaling pathway to increase hepatic glucose production; liver-specific knockdown of Purβ in db/db mice suppressed ADCY6 expression and ameliorated hyperglycemia.\",\n      \"method\": \"Chromatin immunoprecipitation, luciferase reporter assay, adenovirus-mediated KD/OE in primary hepatocytes and db/db mice, glucose/insulin/lactate tolerance tests, immunoblotting of cAMP signaling components\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct ChIP demonstrating promoter binding, luciferase reporter validation, and in vivo KD with defined metabolic phenotype\",\n      \"pmids\": [\"31918924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AC6 inhibits degradation of the microtubule-depolymerizing kinesin KIF19A by suppressing autophagy through inhibition of AMPK; epithelium-specific AC6 KO airway epithelial cells showed elongated cilia due to decreased KIF19A protein levels, and pharmacological AMPK activation phenocopied AC6 loss by mobilizing KIF19A into autophagosomes.\",\n      \"method\": \"Epithelium-specific AC6 KO mice, cilia length measurement, KIF19A protein quantification, AMPK activity assays, autophagy flux experiments, pharmacological AMPK modulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined cellular phenotype, mechanistic pathway validated by pharmacological orthogonal approach\",\n      \"pmids\": [\"32683324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"AC6 localizes to lipid raft membrane microdomains and co-immunoprecipitates with nicotinic acetylcholine receptor α7 subunit (nAChRα7) within rafts; cholesterol depletion disrupts this co-localization and abrogates nicotine-induced inhibition of AC6 activity, demonstrating that raft localization is required for nAChRα7-mediated calcium-dependent inhibition of AC6.\",\n      \"method\": \"Sucrose gradient fractionation, co-immunoprecipitation, cholesterol depletion with methyl-β-cyclodextrin, AC activity assay\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — co-IP plus fractionation with functional consequence, single lab\",\n      \"pmids\": [\"12748066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HSF1 (heat shock factor 1) positively regulates AC6 mRNA expression in a pressure-overload heart failure model; HSF1 transgenic mice showed increased AC6 mRNA, cAMP, and PKA levels and improved cardiac function, while HSF1 knockout mice showed the opposite, placing AC6 downstream of HSF1 in the AC6/cAMP/PKA pathway that ameliorates heart failure.\",\n      \"method\": \"Transverse aortic constriction mouse model, HSF1 transgenic and KO mice, RT-qPCR, Western blotting, ELISA for cAMP, echocardiography\",\n      \"journal\": \"Environmental toxicology and pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic gain- and loss-of-function with defined cardiac phenotype, single lab\",\n      \"pmids\": [\"27643574\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"miR-27a-3p suppresses TET1 expression, reducing TET1-mediated DNA demethylation of the ADCY6 promoter; decreased ADCY6 expression promotes breast cancer cell proliferation, invasion and migration through EMT; restoring ADCY6 or TET1 activity reverses these effects, defining a miR-27a-3p/TET1/ADCY6/EMT regulatory axis.\",\n      \"method\": \"DNA methylation-specific PCR, bisulfite Sanger sequencing, lentiviral miRNA transfection, cell invasion/migration assays, luciferase reporter, gene expression analysis\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — multiple orthogonal epigenetic and functional methods, single lab\",\n      \"pmids\": [\"35978806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AC6 (localized in lipid raft membranes) and AC2 (localized in non-raft membranes) generate distinct cAMP signaling pools in human airway smooth muscle; quantitative phosphoproteomics revealed that AC6 activity specifically regulates phosphorylation of proteins involved in autophagy, Ca2+/CaM signaling, Rho GTPases, and cytoskeletal regulation, while AC2 activity predominantly affects RNA/DNA binding and microtubule proteins.\",\n      \"method\": \"AC2/AC6 overexpression in human airway smooth muscle cells, quantitative phosphoproteomics by LC-MS/MS, STRING network analysis\",\n      \"journal\": \"Frontiers in physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative phosphoproteomics with isoform-specific overexpression titrated to equal cAMP output, single lab\",\n      \"pmids\": [\"36926196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Forskolin alleviates hypertrophic cardiomyopathy in Myh6R404Q and Tnnt2R109Q mouse models through activation of ADCY6, with downstream cAMP/PKA pathway activation reducing cardiac hypertrophy and normalizing cardiomyocyte size and hypertrophy-related gene expression.\",\n      \"method\": \"HCM mouse models (Myh6R404Q and Tnnt2R109Q), norepinephrine-induced cardiomyocyte hypertrophy in vitro, pharmacological ADCY6 activation, gene expression analysis\",\n      \"journal\": \"European journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — in vivo and in vitro pharmacological activation with defined phenotypic readout, mechanism partly inferred\",\n      \"pmids\": [\"38925286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GPSM1 (G protein signaling modulator 1) promotes proliferation of B-ALL cells by regulating ADCY6 expression; GPSM1 knockdown suppressed ADCY6 and RAPGEF3 expression and reduced JNK activity, defining a GPSM1/ADCY6/RAPGEF3/JNK signaling axis in leukemia cell proliferation.\",\n      \"method\": \"siRNA knockdown of GPSM1 in BALL-1 and Reh cells, cell proliferation/apoptosis/cell cycle assays, Western blotting of ADCY6, RAPGEF3, and JNK\",\n      \"journal\": \"Pathology oncology research : POR\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single KD experiment with downstream protein measurement but no direct demonstration of ADCY6-RAPGEF3 interaction\",\n      \"pmids\": [\"34257610\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ADCY6 is a calcium-inhibitable adenylyl cyclase localized in lipid raft membrane microdomains that synthesizes cAMP from ATP (with catalytic residue D426 required for enzymatic activity), regulates diverse physiological processes including PNS myelination, renal water homeostasis, ciliary length control (via AMPK-KIF19A autophagy), renin secretion, intestinal fluid secretion (through a physical complex with CFTR), and cardiac function; its cAMP production is locally regulated by an AKAP79/150-anchored PKA negative feedback loop and by selective coupling with IP3R2 to form binary cAMP signaling junctions, while notably many of its cardioprotective effects (reduced hypertrophy, apoptosis, and altered Ca2+ handling) do not require cAMP generation and are replicated by a catalytically inactive mutant.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ADCY6 encodes a calcium-inhibitable transmembrane adenylyl cyclase that generates compartmentalized cAMP pools to transduce signals from beta-adrenergic, vasopressin, and glucagon receptors into diverse physiological outputs. AC6 couples intracellular calcium elevation to suppression of cAMP and renin secretion in juxtaglomerular cells [PMID:17068292], mediates vasopressin-stimulated water reabsorption in the renal collecting duct [PMID:20466003], physically complexes with CFTR to drive intestinal chloride/fluid secretion [PMID:29903911], and inhibits AMPK to control ciliary length via KIF19A-targeted autophagy in airway epithelia [PMID:32683324]. In cardiomyocytes, a catalytically inactive AC6 mutant retains anti-hypertrophic and calcium-handling functions, demonstrating cAMP-independent scaffolding roles [PMID:21127130]. Homozygous loss-of-function mutations in ADCY6 cause lethal arthrogryposis multiplex congenita with absence of peripheral nervous system myelin [PMID:24319099].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing that AC6 is a calcium-inhibitable cyclase linking intracellular Ca²⁺ to cAMP suppression and renin release answered how juxtaglomerular cells sense calcium to regulate renin secretion.\",\n      \"evidence\": \"siRNA knockdown of AC5/AC6 in primary juxtaglomerular and As4.1 cells with cAMP and renin secretion readouts plus cAMP analog rescue\",\n      \"pmids\": [\"17068292\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AC5 and AC6 are functionally redundant or have distinct roles in JG cells\", \"Structural basis for calcium-mediated inhibition of AC6 catalysis\", \"In vivo confirmation in AC6-knockout kidney\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that AC6 knockout impairs renal water reabsorption and vasopressin-stimulated cyclase activity defined AC6 as the principal adenylyl cyclase mediating the antidiuretic response in the collecting duct.\",\n      \"evidence\": \"AC6-knockout mice; immunohistochemistry localizing AC6 to collecting duct; forskolin/AVP-stimulated cyclase assays; metabolic cage studies\",\n      \"pmids\": [\"20466003\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AC6 acts cell-autonomously in principal cells versus intercalated cells\", \"Compensatory changes in other AC isoforms in the knockout\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showing that a catalytically dead AC6 mutant (D426A) retained anti-hypertrophic, anti-apoptotic, and Ca²⁺-handling effects in cardiomyocytes revealed a cAMP-independent scaffolding function, fundamentally expanding the mechanistic repertoire of adenylyl cyclases.\",\n      \"evidence\": \"Site-directed mutagenesis of the C1 catalytic aspartate; adenoviral expression in adult rat cardiomyocytes; cell size, apoptosis, gene expression, and Ca²⁺ transient assays\",\n      \"pmids\": [\"21127130\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the cAMP-independent effector or binding partner mediating these effects\", \"Whether the catalytically inactive mutant retains normal membrane localization and protein interactions\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"AC6 overexpression selectively enhanced cAMP downstream of beta-adrenergic and prostacyclin—but not EP2—receptors in pulmonary fibroblasts, revealing receptor-selective coupling specificity that protects against fibrosis in vivo.\",\n      \"evidence\": \"AC6 overexpression in primary fibroblasts with agonist panels; fibroblast-specific AC6 transgenic mice in bleomycin fibrosis model\",\n      \"pmids\": [\"20348281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for receptor-selective coupling (scaffold proteins, lipid raft compartmentalization)\", \"Whether endogenous AC6 levels are limiting in human pulmonary fibrosis\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying a homozygous ADCY6 loss-of-function mutation as the cause of lethal arthrogryposis multiplex congenita with absent PNS myelin—confirmed by zebrafish morpholino phenocopy—established AC6 as essential for Schwann cell myelination.\",\n      \"evidence\": \"Whole-exome sequencing in consanguineous AMC families; TEM of sciatic nerve; zebrafish adcy6 morpholino knockdown\",\n      \"pmids\": [\"24319099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AC6 acts cell-autonomously in Schwann cells or through axonal signals\", \"Downstream cAMP effectors (PKA, EPAC) mediating myelination\", \"Whether heterozygous carriers have subclinical neuropathy\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placing AC6 downstream of HSF1 transcriptional control in pressure-overloaded hearts suggested a stress-responsive regulatory axis for cardiac AC6/cAMP/PKA signaling.\",\n      \"evidence\": \"HSF1 transgenic and knockout mice under TAC; AC6 mRNA quantification; cAMP/PKA activity measurement; echocardiography\",\n      \"pmids\": [\"27643574\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct HSF1 binding to the ADCY6 promoter demonstrated (ChIP or reporter)\", \"Single lab; not independently replicated\", \"Whether HSF1 regulation of AC6 occurs in non-cardiac tissues\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating that AC6 physically complexes with CFTR at the apical membrane and is the dominant cAMP source for CFTR activation in intestinal epithelium identified the AC6–CFTR signaling module as the critical mediator of cholera toxin-induced secretory diarrhea.\",\n      \"evidence\": \"Co-immunoprecipitation of AC6–CFTR; epithelium-specific AC6 knockout mice; ligated ileal loop secretion assay; intestinal organoid CFTR activation\",\n      \"pmids\": [\"29903911\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AC6–CFTR interaction is direct or scaffolded (e.g., by PDZ-domain proteins)\", \"Relevance to CFTR-dependent secretion in airway epithelium\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showing that AC6 normally restrains AMPK, which otherwise targets the ciliary kinesin KIF19A for autophagic degradation, defined a non-canonical AC6→AMPK→KIF19A→autophagy axis controlling ciliary length in airway epithelia.\",\n      \"evidence\": \"Epithelium-specific AC6 knockout mice; cilia length measurement; KIF19A quantification; AMPK activity assays; pharmacological AMPK modulation\",\n      \"pmids\": [\"32683324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the AMPK inhibition is cAMP/PKA-dependent or represents another cAMP-independent AC6 function\", \"Whether ciliary length changes alter mucociliary clearance in vivo\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identifying Purβ as a direct transcriptional activator of ADCY6 in hepatocytes linked glucagon-stimulated glucose production to the Purβ/ADCY6/cAMP/PKA/CREB axis, revealing an upstream regulatory node for hepatic gluconeogenesis.\",\n      \"evidence\": \"ChIP and luciferase reporter confirming Purβ binding to Adcy6 promoter; adenoviral knockdown/overexpression in primary hepatocytes and db/db mouse liver\",\n      \"pmids\": [\"31918924\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Purβ regulation of ADCY6 occurs in other AC6-expressing tissues\", \"Contribution of other AC isoforms in hepatocytes\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Phosphoproteomic profiling revealed that lipid raft-localized AC6 generates a cAMP pool distinct from non-raft AC2, preferentially phosphorylating substrates in autophagy, Ca²⁺/CaM signaling, and Rho GTPase pathways in airway smooth muscle cells.\",\n      \"evidence\": \"Quantitative LC-MS/MS phosphoproteomics comparing AC6- versus AC2-overexpressing human airway smooth muscle cells after forskolin stimulation\",\n      \"pmids\": [\"36926196\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Validation of key phosphosubstrates by targeted assays\", \"Whether lipid raft localization is required for the distinct signaling outcomes\", \"Single overexpression system; endogenous isoform contributions not dissected\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular basis for AC6's cAMP-independent functions in cardiomyocytes remains unresolved: no binding partner or structural domain mediating the scaffolding role has been identified, and the relationship between catalytic and non-catalytic functions across different tissues is undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No interacting partner identified for cAMP-independent cardiac effects\", \"No structural model of AC6 explaining dual catalytic/scaffolding functions\", \"Whether cAMP-independent roles extend to non-cardiac tissues\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0009975\", \"supporting_discovery_ids\": [0, 1, 2, 5, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 10]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 3, 5, 6, 7, 11]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [7, 10]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\n      \"AC6-CFTR apical signaling complex\"\n    ],\n    \"partners\": [\n      \"CFTR\",\n      \"PURB\",\n      \"HSF1\",\n      \"KIF19A\",\n      \"PRKAA1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"ADCY6 is a calcium-inhibitable, membrane-bound adenylate cyclase that generates compartmentalized cAMP pools to regulate a wide range of physiological processes including cardiac function, renal water homeostasis, intestinal fluid secretion, airway ciliary length, peripheral nervous system myelination, and hepatic glucose output. Localized to lipid raft microdomains, AC6 forms functionally specialized signaling complexes—with AKAP79/150-anchored PKA for negative feedback termination of cAMP synthesis, with IP3R2 for binary cAMP-Ca²⁺ signaling junctions, and with CFTR at the apical surface of intestinal epithelia to drive cholera toxin–induced secretory diarrhea [PMID:16973443, PMID:18936250, PMID:29903911, PMID:12748066]. In the kidney, AC6 is the predominant cyclase coupling vasopressin V2 receptor stimulation to water reabsorption, and in airway epithelia it controls ciliary length by suppressing AMPK-dependent autophagic degradation of the kinesin KIF19A [PMID:20466003, PMID:32683324]. Notably, a catalytically dead AC6 mutant (D426A) retains the ability to reduce cardiac hypertrophy and apoptosis and to alter Ca²⁺ handling, demonstrating that key cardioprotective functions are independent of cAMP generation, while loss-of-function mutations in humans cause absence of peripheral myelin [PMID:21127130, PMID:24319099].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing that AC6 resides in lipid raft microdomains answered how calcium-dependent inhibitory signals from nAChRα7 are spatially coupled to AC6: raft localization is required for this regulation, defining AC6 as a compartment-specific signaling node.\",\n      \"evidence\": \"Sucrose gradient fractionation, co-immunoprecipitation, and cholesterol depletion in BAECs\",\n      \"pmids\": [\"12748066\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; raft localization not confirmed by super-resolution imaging\", \"nAChRα7–AC6 interaction not validated by reciprocal pull-down or proximity ligation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Two parallel discoveries revealed how AC6 activity is terminated and how it controls a classic physiology paradox: AC6 forms a complex with AKAP79/150 that recruits PKA for feedback phosphorylation/inhibition of AC6, and AC6 (together with AC5) mediates the calcium-dependent suppression of renin secretion in juxtaglomerular cells.\",\n      \"evidence\": \"Co-IP plus live-cell cAMP imaging with PKA disruption in HEK293 cells; siRNA knockdown in primary juxtaglomerular cells, As4.1 cells, and isolated perfused kidney\",\n      \"pmids\": [\"16973443\", \"17068292\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and structural basis of the AKAP79/AC6/PKA complex unknown\", \"Relative contributions of AC5 vs AC6 to renin regulation not fully separated in vivo\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrating that AC6 selectively couples with IP3R2 to form cAMP junctions resolved how supra-threshold cAMP concentrations are delivered to a specific target without flooding the cytoplasm, establishing a binary signaling mode for cAMP-Ca²⁺ crosstalk.\",\n      \"evidence\": \"siRNA knockdown of AC6 and IP3R2, Ca²⁺ imaging, and co-immunoprecipitation in HeLa cells stimulated with PTH\",\n      \"pmids\": [\"18936250\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AC6–IP3R2 junctions exist in tissues beyond cell lines not tested\", \"Structural determinants of AC6–IP3R2 selectivity unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Three studies collectively defined AC6's in vivo physiological roles and, critically, dissociated its catalytic activity from its cardioprotective effects: AC6 KO mice showed impaired vasopressin-mediated water reabsorption; a catalytically dead D426A mutant still reduced cardiac hypertrophy and apoptosis; and AC6 overexpression suppressed bleomycin-induced pulmonary fibrosis via β-adrenergic/prostacyclin cAMP signaling.\",\n      \"evidence\": \"AC6 KO mice (two lines) with DCE-MRI and metabolic cages; D426A mutagenesis with gene transfer in rat cardiac myocytes; AC6-transgenic mice in bleomycin fibrosis model\",\n      \"pmids\": [\"20466003\", \"21127130\", \"20348281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"cAMP-independent cardioprotective mechanism of AC6 remains molecularly undefined\", \"Antifibrotic function not tested with catalytically dead mutant\", \"Identity of AC6 protein–protein interactions mediating cAMP-independent cardiac effects unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identification of a homozygous ADCY6 mutation causing absent peripheral myelin in humans, phenocopied by morpholino knockdown in zebrafish, established AC6 as essential for Schwann cell–mediated PNS myelination—the first Mendelian disease link for this gene.\",\n      \"evidence\": \"Whole-exome sequencing in affected family, TEM of sciatic nerve, zebrafish morpholino knockdown\",\n      \"pmids\": [\"24319099\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream myelination program controlled by AC6-generated cAMP in Schwann cells not delineated\", \"Only one family reported; allelic spectrum unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showing that AC6 physically complexes with CFTR at the apical membrane of intestinal epithelia and is the dominant cyclase driving cholera toxin–induced fluid secretion answered which adenylyl cyclase isoform mediates secretory diarrhea.\",\n      \"evidence\": \"Co-IP, epithelium-specific AC6 KO mice, ligated ileal loop assay, intestinal spheroid CFTR activation\",\n      \"pmids\": [\"29903911\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AC6–CFTR interaction is direct or scaffolded not resolved\", \"Therapeutic potential of AC6 inhibition in cholera not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identification of Purβ as a direct transcriptional activator of ADCY6 linked hepatic AC6 expression to gluconeogenesis, explaining how the glucagon/cAMP/PKA/CREB axis is sustained at the transcriptional level in diabetic liver.\",\n      \"evidence\": \"ChIP, luciferase reporter, and adenoviral KD/OE of Purβ in hepatocytes and db/db mice\",\n      \"pmids\": [\"31918924\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Other transcription factors regulating ADCY6 in non-hepatic contexts not mapped\", \"Whether Purβ regulation is specific to ADCY6 vs other ADCY isoforms unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrating that AC6 controls airway ciliary length by inhibiting AMPK-dependent autophagic degradation of KIF19A revealed a non-canonical cyclase function linking cAMP signaling to autophagy and cytoskeletal regulation.\",\n      \"evidence\": \"Epithelium-specific AC6 KO mice, KIF19A quantification, autophagy flux assays, pharmacological AMPK modulation\",\n      \"pmids\": [\"32683324\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AC6–AMPK inhibition is direct or mediated by PKA/Epac not resolved\", \"In vivo mucociliary clearance consequences not measured\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Phosphoproteomic profiling resolved how raft-localized AC6 and non-raft AC2 generate distinct downstream signaling networks from equivalent total cAMP, demonstrating that AC6-specific cAMP pools preferentially regulate autophagy, Ca²⁺/CaM, and Rho GTPase signaling.\",\n      \"evidence\": \"Isoform-specific overexpression in human airway smooth muscle with quantitative LC-MS/MS phosphoproteomics\",\n      \"pmids\": [\"36926196\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphoproteomic targets not validated by targeted mutagenesis\", \"Single cell type studied; generalizability to other AC6-expressing tissues unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular basis of AC6's cAMP-independent cardioprotective activity—identified by the catalytically dead D426A mutant—remains unknown; the interacting partners, structural features, or scaffolding functions responsible have not been identified.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No interactome or structural study of the D426A mutant performed\", \"Whether cAMP-independent functions extend to non-cardiac tissues untested\", \"AC6-specific inhibitors or activators for therapeutic use not developed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0009975\", \"supporting_discovery_ids\": [1, 3, 4, 5, 7, 9]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [7, 10, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0009975\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 3, 8]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [3, 7]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [9, 13]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [6, 7]}\n    ],\n    \"complexes\": [\n      \"AKAP79/150-PKA-AC6 complex\",\n      \"AC6-CFTR complex\",\n      \"AC6-IP3R2 cAMP junction\"\n    ],\n    \"partners\": [\n      \"AKAP5\",\n      \"ITPR2\",\n      \"CFTR\",\n      \"CHRNA7\",\n      \"KIF19A\",\n      \"PURB\",\n      \"HSF1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}