{"gene":"CHRM1","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1987,"finding":"CHRM1 (M1 muscarinic acetylcholine receptor) was identified as a member of a family of muscarinic receptor genes; cloned cDNAs were expressed in mammalian cells and shown to encode functional receptors with distinct ligand-binding properties and tissue-specific expression, featuring seven transmembrane segments and a large intracellular region.","method":"cDNA cloning from rat cerebral cortex library, expression in mammalian cells, ligand binding assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — original cloning and functional expression, foundational paper replicated widely","pmids":["3037705","3443095"],"is_preprint":false},{"year":1989,"finding":"CHRM1 (m1 subtype) selectively couples to phosphatidylinositol (PI) hydrolysis, and activation of this pathway by carbachol stimulates DNA synthesis in primary brain-derived astrocytes and transfected CHO cells, establishing that CHRM1-mediated PI hydrolysis drives cell proliferation.","method":"Transfection of recombinant mAChR subtypes in CHO cells and brain-derived cell lines; carbachol stimulation; PI hydrolysis assay; [3H]-thymidine incorporation","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 — reconstitution with recombinant receptor, multiple cell systems, orthogonal functional readouts","pmids":["2739737"],"is_preprint":false},{"year":1991,"finding":"CHRM1 (m1), together with m3 and m5 but not m2 or m4, acts as an agonist-dependent oncogene in NIH 3T3 cells; transformation requires receptor coupling to phosphatidylinositol hydrolysis, whereas m2/m4 subtypes coupled to adenylyl cyclase inhibition do not cause transformation.","method":"Transfection of individual human mAChR genes in NIH 3T3 cells; focus formation assay with carbachol; PI hydrolysis assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple receptor subtypes compared, functional assay plus transformation readout; highly cited","pmids":["1905013"],"is_preprint":false},{"year":1992,"finding":"Stimulation of CHRM1 (m1) and m3 receptors with carbachol in transfected HEK293 cells increases release of amyloid precursor protein (APP) derivatives within minutes; this process is blocked by the protein kinase inhibitor staurosporine, indicating that protein kinase activity mediates receptor-controlled APP processing.","method":"Transfection of HEK293 cells with muscarinic receptor genes; carbachol stimulation; APP derivative quantification by immunoassay; staurosporine inhibition","journal":"Science","confidence":"High","confidence_rationale":"Tier 1-2 — direct reconstitution with defined receptor subtypes, pharmacological inhibition, highly cited foundational paper","pmids":["1411529"],"is_preprint":false},{"year":1994,"finding":"Activated CHRM1 (m1) in NIH 3T3 cells induces Raf-1 kinase activation and ERK2 activity in a largely PKC-independent manner; dominant-negative Raf-1 (K375W) abolishes m1-mediated transformation, placing Raf-1 downstream of CHRM1 signaling.","method":"Transfected NIH 3T3 cells expressing human m1 receptor; carbachol stimulation; Raf-1 kinase activity assay using MEK substrate; phosphoamino acid analysis; dominant-negative Raf-1 co-transfection; PKC inhibition (GF 109203X, phorbol ester down-regulation)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — epistasis via dominant-negative mutant plus kinase assay plus pharmacological dissection","pmids":["8063729"],"is_preprint":false},{"year":1994,"finding":"CHRM1 (m1) and m3 receptors selectively activate Gq/11, while m2 selectively activates Gi2; all three subtypes also activate Gi1 and Gi3, but m1/m3 do so only at higher agonist concentrations, demonstrating subtype-selective G protein coupling with differential efficacies.","method":"Photolabeling of G protein alpha subunits with [α-32P]GTP azidoanilide in transfected HEK293 cell membranes; subtype-specific immunoprecipitation; carbachol dose-response","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1 — biochemical reconstitution with defined receptor subtypes, direct G protein identification","pmids":["8190105"],"is_preprint":false},{"year":2007,"finding":"β-arrestins coordinate termination of CHRM1 (M1 muscarinic receptor) signaling by physically interacting with diacylglycerol kinases (DGKs) and recruiting this complex to activated M1 receptors, thereby accelerating DAG degradation to phosphatidic acid; β-arrestins are essential for agonist-stimulated DAG-to-PA conversion at activated M1 receptors.","method":"Co-immunoprecipitation of β-arrestin with DGKs; RNAi knockdown of β-arrestins; mass spectrometry identification; DAG/phosphatidic acid measurements after agonist stimulation; recruitment of β-arrestin-DGK complex to activated 7TMRs by imaging","journal":"Science","confidence":"High","confidence_rationale":"Tier 1-2 — Co-IP, RNAi loss-of-function, biochemical lipid measurements, multiple orthogonal methods","pmids":["17272726"],"is_preprint":false},{"year":2007,"finding":"Extracellular tau protein promotes intracellular calcium increase in neuronal cells through CHRM1 (M1) and M3 muscarinic receptors; this effect is blocked by M1/M3-selective antagonists and is recapitulated in non-neuronal cells transfected with M1 or M3 cDNA, indicating tau acts on neurons via these receptors.","method":"Antagonist pharmacology (pirenzepine, 4-DAMP) in primary hippocampal/cortical neurons and neuroblastoma; transfection of M1/M3 cDNA in non-neuronal cells; intracellular calcium imaging","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological plus transfection reconstitution; single lab but two orthogonal approaches","pmids":["18272392"],"is_preprint":false},{"year":2008,"finding":"M1 muscarinic receptor (CHRM1) allosteric agonists (AC-42 and 77-LH-28-1) selectively activate M1 over other mAChR subtypes via an allosteric binding site; 77-LH-28-1 also acts as an agonist at native rat hippocampal M1 receptors, increasing cell firing and inducing gamma frequency network oscillations in vitro and in vivo.","method":"Calcium mobilization and inositol phosphate accumulation assays at recombinant mAChR subtypes; in vitro and in vivo electrophysiology in rat hippocampus","journal":"British journal of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional assays in recombinant and native systems; single lab","pmids":["18454168"],"is_preprint":false},{"year":2009,"finding":"CHRM1 signaling via Gαq/11 is functionally altered in 'muscarinic receptor-deficit schizophrenia': agonist potency for Gq/11 stimulation is decreased while maximal Gq/11-[35S]GTPγS coupling is increased, indicating compensatory upregulation of receptor-G protein coupling efficiency in this subgroup.","method":"[35S]GTPγS-Gαq/11 immunocapture assay in postmortem human cortex membranes (Brodmann area 9); dose-response to orthosteric (oxotremorine-M) and allosteric (AC-42) agonists","journal":"Neuropsychopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 — direct biochemical coupling assay in human tissue, two agonists tested; single lab","pmids":["19404243"],"is_preprint":false},{"year":2010,"finding":"Individual M1 muscarinic receptor (CHRM1) molecules are randomly distributed in the plasma membrane and dynamically interconvert between monomers and dimers on a timescale of seconds; approximately 30% exist as dimers at any given time with no evidence for higher-order oligomers.","method":"Total internal reflection fluorescence microscopy (TIRFM) single-molecule imaging in live CHO cells; two-color TIRFM for dimer confirmation; single-particle tracking (~30 ms / ~20 nm resolution)","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — direct single-molecule live-cell imaging with two-color validation; highly cited","pmids":["20133736"],"is_preprint":false},{"year":2013,"finding":"In Huntington's disease striatal cells, H3K9me3-dependent heterochromatin accumulates at the CHRM1 promoter, reducing CHRM1 gene expression and impairing Ca2+-dependent neuronal signal transduction.","method":"H3K9me3-ChIP genome-wide sequencing combined with RNA sequencing in STHdh Q7/7 and Q111/111 stable cell lines; platform integration analysis; Ca2+ signaling functional assay","journal":"Acta neuropathologica","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP-seq plus RNA-seq plus functional Ca2+ assay; single lab but multiple orthogonal methods","pmids":["23455440"],"is_preprint":false},{"year":2016,"finding":"Crystal structures of the M1 muscarinic acetylcholine receptor bound to the inverse agonist tiotropium revealed structural differences in orthosteric and allosteric binding sites compared to M2, M3, and M4 receptors, providing a molecular basis for drug selectivity; comparison across subtypes identified residue differences contributing to subtype-selective binding.","method":"X-ray crystallography of M1 receptor in complex with tiotropium; structural comparison with M2, M3, M4 crystal structures","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with direct structural comparison; highly cited","pmids":["26958838"],"is_preprint":false},{"year":2018,"finding":"Chrm1 and Chrm3 double knockout in mice chronically diminishes REM sleep to almost undetectable levels, and knockout of these two Gq-type muscarinic acetylcholine receptors phenocopies synaptic inhibition of TrkA+ cholinergic neurons, establishing that CHRM1 and CHRM3 are essential for REM sleep generation.","method":"Triple-target CRISPR knockout of Chrm1/Chrm3 in mice; polysomnographic sleep recording (EEG/EMG); chemogenetic synaptic inhibition of TrkA+ cholinergic neurons","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — genetic loss-of-function (CRISPR KO) with quantitative sleep phenotype; replicated with orthogonal chemogenetic approach","pmids":["30157420"],"is_preprint":false},{"year":2021,"finding":"Magnolol upregulates CHRM1 expression in SH-SY5Y cells treated with Aβ, and CHRM1 activation mediates protection against Tau hyperphosphorylation and apoptosis through the cAMP/PKA/CREB pathway; pharmacological inactivation of cAMP signaling reverses magnolol's protective effects.","method":"CCK-8 viability assay; qRT-PCR and western blotting for CHRM1 and pathway components; ELISA and western blotting for cAMP/PKA/CREB; caspase-3 activity and flow cytometry for apoptosis; siRNA or inhibitor of cAMP pathway","journal":"Journal of natural medicines","confidence":"Low","confidence_rationale":"Tier 3 — single lab, cell-line model, no direct CHRM1 reconstitution; pathway placement is indirect","pmids":["34705126"],"is_preprint":false},{"year":2022,"finding":"DOPA inhibits melanocyte and melanoma cell proliferation by inhibiting CHRM1 signaling; pharmacologic CHRM1 antagonism depletes c-Myc and FOXM1, and both pharmacologic inhibition of CHRM1 and FOXM1 inhibit melanoma tumor growth in preclinical mouse models.","method":"High-throughput pharmacologic screen; in vivo CRISPR genetic screen; pharmacological CHRM1 antagonism; western blotting for c-Myc and FOXM1; preclinical mouse melanoma tumor models","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo CRISPR genetic screen plus pharmacological validation plus in vivo tumor models; multiple orthogonal approaches in single study","pmids":["36054350"],"is_preprint":false},{"year":2024,"finding":"CHRM1 inhibits the TRPM8 cation channel via Gq/11 and phospholipase C-mediated depletion of phosphatidylinositol 4,5-bisphosphate (PIP2), defining a novel CHRM1-TRPM8 signaling axis with anti-fibrotic effects in liver disease; CHRM1 activation produces strong anti-fibrotic effects in a patient-derived 3D MASH model.","method":"BRET-based biosensors in live cells; orthogonal genetic assays (CRISPR); high-content imaging; chemogenomic screening in patient-derived 3D liver organoid model; multi-omics","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — BRET biosensors plus genetic validation plus pharmacological screening; single lab but multiple orthogonal methods","pmids":["39605182"],"is_preprint":false},{"year":2003,"finding":"M1 muscarinic receptors (CHRM1) expressed in human intestinal L cells (NCI-H716) control GLP-1 secretion; selective M1 agonist McN-A-343 stimulates GLP-1 secretion dose-dependently, and pirenzepine (M1 antagonist) but not 4-DAMP (M3 antagonist) blocks bethanechol-stimulated GLP-1 release, confirming a functional role for CHRM1 in GLP-1 secretion.","method":"GLP-1 secretion assay in NCI-H716 human L cell line; selective agonists/antagonists for mAChR subtypes; western blot, immunohistochemistry, RT-PCR for receptor subtype expression confirmation","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological dissection with selective ligands plus expression confirmation; replicated in paraffin-embedded human intestine sections","pmids":["12810581"],"is_preprint":false}],"current_model":"CHRM1 (M1 muscarinic acetylcholine receptor) is a Gq/11-coupled seven-transmembrane GPCR whose agonist-driven activation stimulates phospholipase C-mediated PI hydrolysis and Ca2+ release, leading to downstream PKC-independent Raf-1/ERK activation, DAG degradation coordinated by β-arrestin-recruited DGKs, APP secretion, GLP-1 release, and c-Myc/FOXM1-dependent cell proliferation; it forms transient dimers at the plasma membrane, is regulated by H3K9me3 epigenetic silencing, is essential for REM sleep in mice, and inhibits TRPM8 via PIP2 depletion, with its crystal structure revealing orthosteric and allosteric binding sites that underlie subtype-selective pharmacology."},"narrative":{"teleology":[{"year":1987,"claim":"Molecular cloning of CHRM1 established it as a seven-transmembrane muscarinic receptor gene with distinct ligand-binding properties, providing the molecular identity needed for all subsequent subtype-selective studies.","evidence":"cDNA cloning from rat cerebral cortex, heterologous expression in mammalian cells, ligand-binding assays","pmids":["3037705","3443095"],"confidence":"High","gaps":["Endogenous signaling effectors not yet identified","No structural information available"]},{"year":1991,"claim":"Reconstitution experiments showed CHRM1 selectively couples to PI hydrolysis and drives DNA synthesis and oncogenic transformation, distinguishing the Gq-coupled M1/M3/M5 subfamily from the Gi-coupled M2/M4 subfamily.","evidence":"Recombinant receptor expression in CHO and NIH 3T3 cells; PI hydrolysis, thymidine incorporation, and focus-formation assays with carbachol","pmids":["2739737","1905013"],"confidence":"High","gaps":["Downstream kinase cascades linking PI hydrolysis to transformation not delineated","G protein identity not yet confirmed biochemically"]},{"year":1994,"claim":"Biochemical identification of Gαq/11 as the primary G protein for CHRM1 and demonstration that CHRM1 activates Raf-1/ERK largely independently of PKC resolved the signaling pathway downstream of PI hydrolysis.","evidence":"GTP-azidoanilide photolabeling with subtype-specific immunoprecipitation in HEK293 membranes; Raf-1 kinase assays with dominant-negative Raf-1 and PKC inhibitors in NIH 3T3 cells","pmids":["8190105","8063729"],"confidence":"High","gaps":["Ras involvement upstream of Raf-1 not directly tested","Mechanism of PKC-independent Raf-1 activation unresolved"]},{"year":1992,"claim":"Activation of CHRM1 was shown to rapidly increase secretion of amyloid precursor protein derivatives in a protein-kinase-dependent manner, linking muscarinic signaling to APP processing relevant to Alzheimer's disease.","evidence":"Carbachol stimulation of M1-transfected HEK293 cells; APP immunoassay; staurosporine inhibition","pmids":["1411529"],"confidence":"High","gaps":["Specific kinase (PKC vs. other) mediating APP release not pinpointed","In vivo relevance in brain not established"]},{"year":2003,"claim":"Pharmacological dissection in human intestinal L cells demonstrated that CHRM1 controls GLP-1 secretion, expanding the receptor's physiological roles beyond the nervous system.","evidence":"Selective M1 agonist (McN-A-343) and antagonist (pirenzepine) in NCI-H716 cells; GLP-1 secretion assay; receptor expression confirmation by RT-PCR and immunohistochemistry","pmids":["12810581"],"confidence":"Medium","gaps":["In vivo contribution of M1 versus M3 to incretin release not tested","Downstream signaling from M1 to exocytotic machinery not defined"]},{"year":2007,"claim":"Discovery that β-arrestins recruit diacylglycerol kinases to activated M1 receptors to accelerate DAG-to-PA conversion revealed a signal-termination mechanism that reshapes the lipid-signaling landscape downstream of CHRM1.","evidence":"Co-IP of β-arrestin with DGKs; RNAi knockdown; mass spectrometry; DAG/PA lipid measurements after agonist stimulation","pmids":["17272726"],"confidence":"High","gaps":["Specific DGK isoform preference for M1 not determined","Functional consequences of PA generation not explored"]},{"year":2010,"claim":"Single-molecule imaging revealed that CHRM1 dynamically interconverts between monomers and transient dimers at the plasma membrane (~30% dimers), ruling out stable oligomeric states and defining the receptor's quaternary behavior.","evidence":"TIRFM single-molecule and two-color imaging in live CHO cells; single-particle tracking","pmids":["20133736"],"confidence":"High","gaps":["Functional significance of dimerization for signaling output not tested","Agonist effect on dimer fraction not assessed"]},{"year":2016,"claim":"The crystal structure of M1 bound to tiotropium provided the first atomic-resolution view of CHRM1, revealing orthosteric and allosteric site differences from M2–M4 that explain subtype-selective pharmacology.","evidence":"X-ray crystallography of human M1 receptor–tiotropium complex; structural comparison with M2, M3, M4 structures","pmids":["26958838"],"confidence":"High","gaps":["Active-state structure not yet solved","Allosteric agonist co-crystal structures absent"]},{"year":2018,"claim":"CRISPR triple knockout of Chrm1/Chrm3 in mice virtually eliminated REM sleep, establishing that these Gq-type muscarinic receptors are essential generators of REM sleep.","evidence":"CRISPR KO mice; EEG/EMG polysomnography; chemogenetic silencing of cholinergic neurons as orthogonal validation","pmids":["30157420"],"confidence":"High","gaps":["Individual contribution of Chrm1 versus Chrm3 to REM sleep not separable in the double KO","Downstream neural circuit mechanism not identified"]},{"year":2022,"claim":"An in vivo CRISPR screen and pharmacological studies showed that CHRM1 signaling sustains c-Myc and FOXM1 levels in melanoma, and CHRM1 antagonism inhibits tumor growth, revealing a druggable cholinergic proliferative axis in cancer.","evidence":"High-throughput pharmacologic screen; in vivo CRISPR genetic screen; preclinical mouse melanoma models; western blotting","pmids":["36054350"],"confidence":"Medium","gaps":["Mechanism linking CHRM1 to c-Myc stabilization not defined","Generalizability to other tumor types untested"]},{"year":2024,"claim":"BRET biosensor and genetic studies demonstrated that CHRM1 inhibits the TRPM8 channel via Gq/PLC-mediated PIP2 depletion, defining a new effector axis with anti-fibrotic activity in liver disease models.","evidence":"BRET-based biosensors; CRISPR validation; chemogenomic screening in patient-derived 3D MASH liver organoids","pmids":["39605182"],"confidence":"Medium","gaps":["In vivo anti-fibrotic efficacy not demonstrated in animal models","Whether PIP2 depletion or a secondary metabolite mediates TRPM8 inhibition not resolved"]},{"year":null,"claim":"Key open questions include the active-state structure of CHRM1, the mechanism by which CHRM1 activates Raf-1 independently of PKC, the individual roles of CHRM1 versus CHRM3 in REM sleep, and the signaling pathway linking CHRM1 to c-Myc/FOXM1 stabilization in cancer.","evidence":"","pmids":[],"confidence":"High","gaps":["Active-state and agonist-bound structures lacking","PKC-independent Raf-1 activation mechanism undefined","Single versus double receptor contributions to REM sleep unseparated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,5,8,12]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,10,12]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,4,5,6,16]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[7,8,13]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2,15]}],"complexes":[],"partners":["GNAQ","GNA11","ARRB1","ARRB2","DGKZ","TRPM8"],"other_free_text":[]},"mechanistic_narrative":"CHRM1 (muscarinic acetylcholine receptor M1) is a Gq/11-coupled seven-transmembrane receptor that transduces cholinergic signals primarily through phospholipase C-mediated phosphatidylinositol hydrolysis, intracellular calcium mobilization, and downstream activation of the Raf-1/ERK cascade in a largely PKC-independent manner [PMID:3037705, PMID:8190105, PMID:8063729]. Agonist-driven signaling is terminated by β-arrestin-recruited diacylglycerol kinases that convert DAG to phosphatidic acid, while PLC-dependent PIP2 depletion couples CHRM1 to inhibition of the TRPM8 channel [PMID:17272726, PMID:39605182]. CHRM1 activation stimulates amyloid precursor protein secretion, GLP-1 release from intestinal L cells, and c-Myc/FOXM1-dependent melanocyte proliferation, and genetic ablation of Chrm1 together with Chrm3 in mice virtually eliminates REM sleep [PMID:1411529, PMID:12810581, PMID:36054350, PMID:30157420]. Crystal structures of the receptor bound to tiotropium reveal orthosteric and allosteric binding-site differences from other muscarinic subtypes that underlie subtype-selective pharmacology [PMID:26958838]."},"prefetch_data":{"uniprot":{"accession":"P11229","full_name":"Muscarinic acetylcholine receptor M1","aliases":[],"length_aa":460,"mass_kda":51.4,"function":"The muscarinic acetylcholine receptor mediates various cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides and modulation of potassium channels through the action of G proteins. Primary transducing effect is Pi turnover","subcellular_location":"Cell membrane; Postsynaptic cell membrane","url":"https://www.uniprot.org/uniprotkb/P11229/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CHRM1","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CHRM1","total_profiled":1310},"omim":[{"mim_id":"608455","title":"GLYCOGEN PHOSPHORYLASE, MUSCLE; PYGM","url":"https://www.omim.org/entry/608455"},{"mim_id":"607192","title":"REGULATOR OF G PROTEIN SIGNALING 18; RGS18","url":"https://www.omim.org/entry/607192"},{"mim_id":"607189","title":"REGULATOR OF G PROTEIN SIGNALING 8; RGS8","url":"https://www.omim.org/entry/607189"},{"mim_id":"606200","title":"BASIC HELIX-LOOP-HELIX FAMILY, MEMBER E41; BHLHE41","url":"https://www.omim.org/entry/606200"},{"mim_id":"605104","title":"RNA-BINDING FOX1 HOMOLOG 1; RBFOX1","url":"https://www.omim.org/entry/605104"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":48.4},{"tissue":"prostate","ntpm":27.8},{"tissue":"salivary gland","ntpm":27.1}],"url":"https://www.proteinatlas.org/search/CHRM1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P11229","domains":[{"cath_id":"1.20.1070.10","chopping":"21-225_347-438","consensus_level":"medium","plddt":93.0709,"start":21,"end":438}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P11229","model_url":"https://alphafold.ebi.ac.uk/files/AF-P11229-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P11229-F1-predicted_aligned_error_v6.png","plddt_mean":74.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CHRM1","jax_strain_url":"https://www.jax.org/strain/search?query=CHRM1"},"sequence":{"accession":"P11229","fasta_url":"https://rest.uniprot.org/uniprotkb/P11229.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P11229/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P11229"}},"corpus_meta":[{"pmid":"30157420","id":"PMC_30157420","title":"Muscarinic Acetylcholine Receptors 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Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/39605182","citation_count":13,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"30623717","id":"PMC_30623717","title":"Pharmacogenetics of tardive dyskinesia in schizophrenia: The role of CHRM1 and CHRM2 muscarinic receptors.","date":"2019","source":"The world journal of biological psychiatry : the official journal of the World Federation of Societies of Biological Psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/30623717","citation_count":10,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"37939525","id":"PMC_37939525","title":"Vitamin E protects dopaminergic neurons against manganese-induced neurotoxicity through stimulation of CHRM1 and KCNJ4.","date":"2023","source":"Journal of trace elements in medicine and biology : organ of the Society for Minerals and Trace Elements 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Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP).","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15452121","citation_count":139,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"18723515","id":"PMC_18723515","title":"A novel mechanism of G protein-coupled receptor functional selectivity. 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double knockout of Chrm1 and Chrm3 chronically diminishes REM sleep to an almost undetectable level in mice.\",\n      \"method\": \"Triple-target CRISPR knockout of acetylcholine receptor genes in mice with EEG/sleep phenotyping\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic KO with specific, quantified physiological phenotype; replicated across multiple receptor combinations\",\n      \"pmids\": [\"30157420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CHRM1 couples to Gαq/11 to stimulate GTPγS binding in human cortex; in muscarinic receptor-deficit schizophrenia, agonist potency at CHRM1-Gαq/11 coupling is decreased while maximal coupling efficacy is increased, indicating an adaptive change in receptor–G protein coupling efficiency.\",\n      \"method\": \"[35S]-GTPγS–Gαq/11 immunocapture assay on post-mortem human cortex membranes with orthosteric (oxotremorine-M) and allosteric (AC-42) agonists\",\n      \"journal\": \"Neuropsychopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional GTPγS assay directly measuring CHRM1–Gαq/11 coupling in human tissue; single lab, single method\",\n      \"pmids\": [\"19404243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CHRM1 gene expression is repressed by trimethylated histone H3K9 (H3K9me3)-dependent heterochromatin in Huntington's disease striatal cells, and this epigenetic silencing impairs Ca2+-dependent neuronal signal transduction.\",\n      \"method\": \"H3K9me3-ChIP genome-wide sequencing combined with RNA sequencing in STHdhQ111/111 HD cell lines; functional Ca2+ signaling assay\",\n      \"journal\": \"Acta neuropathologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-seq identifies H3K9me3 at CHRM1 promoter with matching transcriptional repression and downstream Ca2+ signaling readout; single lab\",\n      \"pmids\": [\"23455440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DOPA (a melanin precursor) limits melanocyte and melanoma cell proliferation by inhibiting CHRM1 signaling; pharmacologic CHRM1 antagonism depletes c-Myc and FOXM1, and pharmacologic inhibition of CHRM1 inhibits tumor growth in preclinical mouse melanoma models.\",\n      \"method\": \"High-throughput pharmacologic and genetic in vivo CRISPR screens; pharmacologic CHRM1 antagonism with downstream c-Myc/FOXM1 protein quantification; mouse melanoma tumor growth assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — orthogonal pharmacologic and genetic (CRISPR) screens with defined molecular effectors (c-Myc, FOXM1) and in vivo validation; multiple methods in one study\",\n      \"pmids\": [\"36054350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CHRM1 activation inhibits the TRPM8 cation channel via Gq/11 and phospholipase C-mediated depletion of phosphatidylinositol 4,5-bisphosphate (PIP2), constituting a novel CHRM1–TRPM8 signaling axis with anti-fibrotic effects in liver disease.\",\n      \"method\": \"BRET (bioluminescence resonance energy transfer) biosensors in cell-based assays; orthogonal genetic assays; 3D patient-derived MASH liver model with high-content imaging\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — BRET biosensors directly demonstrate functional CHRM1→Gq/11→PLC→PIP2 depletion→TRPM8 inhibition; orthogonal genetic validation in disease-relevant model\",\n      \"pmids\": [\"39605182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CHRM1 upregulation activates the cAMP/PKA/CREB pathway, and this pathway mediates protection against Aβ-induced Tau hyperphosphorylation and neuronal apoptosis in SH-SY5Y cells.\",\n      \"method\": \"CHRM1 overexpression/modulation in SH-SY5Y cells treated with Aβ; ELISA and western blotting for cAMP/PKA/CREB pathway components; caspase-3 activity and flow cytometry for apoptosis\",\n      \"journal\": \"Journal of natural medicines\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — pathway activation inferred from pharmacological manipulation without direct receptor-pathway linkage experiments (e.g., CHRM1 KO rescue); single lab, single cell model\",\n      \"pmids\": [\"34705126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CHRM1 (and CHRM3) are prominently expressed in OPC-like diffuse midline glioma (DMG) cells; acetylcholine promotes DMG proliferation and invasion through muscarinic receptors, and pharmacological blockade of M1 and M3 receptors abolishes activity-regulated DMG proliferation both in cholinergic neuron–glioma co-culture and in vivo.\",\n      \"method\": \"Single-cell RNA sequencing of primary patient DMG; optogenetic stimulation of cholinergic midbrain neurons in vivo; pharmacological receptor blockade in co-culture and mouse models; hiPSC-derived cholinergic neuron–glioma co-culture proliferation assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (scRNA-seq, optogenetics, pharmacology, in vivo) with defined cellular phenotype; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.09.21.614235\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In primate prefrontal and anterior cingulate cortex, CHRM1 protein predominates at the cell surface due to cytoplasmic trafficking (versus nuclear retention of CHRM3 mRNA), and CHRM1-expressing excitatory neurons in ACC show upregulation of synaptic plasticity genes; cholinergic stimulation produces a more robust decrease in excitatory:inhibitory synaptic ratio in ACC than LPFC neurons, with compensatory spine morphology changes.\",\n      \"method\": \"Single-nucleus RNA sequencing; mRNA-protein histology; in vitro functional electrophysiology and synaptic ratio measurements in ACC vs. LPFC neurons; spine morphology analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization (trafficking) tied to functional consequence (region-specific cholinergic modulation of E:I ratio); preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.05.23.655820\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CHRM1 is a Gq-type muscarinic acetylcholine receptor that couples to Gαq/11 to activate phospholipase C (depleting PIP2 and inhibiting TRPM8) and modulate cAMP/PKA/CREB and Ca2+ signaling; it is essential for REM sleep (together with CHRM3), suppresses melanoma and glioma proliferation through depletion of c-Myc/FOXM1 and direct anti-proliferative muscarinic signaling, is epigenetically silenced by H3K9me3 in Huntington's disease, and shows region-specific subcellular trafficking in primate cortex that underlies differential cholinergic modulation of synaptic excitatory/inhibitory balance.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1987,\n      \"finding\": \"CHRM1 (M1 muscarinic acetylcholine receptor) was identified as a member of a family of muscarinic receptor genes; cloned cDNAs were expressed in mammalian cells and shown to encode functional receptors with distinct ligand-binding properties and tissue-specific expression, featuring seven transmembrane segments and a large intracellular region.\",\n      \"method\": \"cDNA cloning from rat cerebral cortex library, expression in mammalian cells, ligand binding assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original cloning and functional expression, foundational paper replicated widely\",\n      \"pmids\": [\"3037705\", \"3443095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"CHRM1 (m1 subtype) selectively couples to phosphatidylinositol (PI) hydrolysis, and activation of this pathway by carbachol stimulates DNA synthesis in primary brain-derived astrocytes and transfected CHO cells, establishing that CHRM1-mediated PI hydrolysis drives cell proliferation.\",\n      \"method\": \"Transfection of recombinant mAChR subtypes in CHO cells and brain-derived cell lines; carbachol stimulation; PI hydrolysis assay; [3H]-thymidine incorporation\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — reconstitution with recombinant receptor, multiple cell systems, orthogonal functional readouts\",\n      \"pmids\": [\"2739737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"CHRM1 (m1), together with m3 and m5 but not m2 or m4, acts as an agonist-dependent oncogene in NIH 3T3 cells; transformation requires receptor coupling to phosphatidylinositol hydrolysis, whereas m2/m4 subtypes coupled to adenylyl cyclase inhibition do not cause transformation.\",\n      \"method\": \"Transfection of individual human mAChR genes in NIH 3T3 cells; focus formation assay with carbachol; PI hydrolysis assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple receptor subtypes compared, functional assay plus transformation readout; highly cited\",\n      \"pmids\": [\"1905013\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Stimulation of CHRM1 (m1) and m3 receptors with carbachol in transfected HEK293 cells increases release of amyloid precursor protein (APP) derivatives within minutes; this process is blocked by the protein kinase inhibitor staurosporine, indicating that protein kinase activity mediates receptor-controlled APP processing.\",\n      \"method\": \"Transfection of HEK293 cells with muscarinic receptor genes; carbachol stimulation; APP derivative quantification by immunoassay; staurosporine inhibition\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct reconstitution with defined receptor subtypes, pharmacological inhibition, highly cited foundational paper\",\n      \"pmids\": [\"1411529\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Activated CHRM1 (m1) in NIH 3T3 cells induces Raf-1 kinase activation and ERK2 activity in a largely PKC-independent manner; dominant-negative Raf-1 (K375W) abolishes m1-mediated transformation, placing Raf-1 downstream of CHRM1 signaling.\",\n      \"method\": \"Transfected NIH 3T3 cells expressing human m1 receptor; carbachol stimulation; Raf-1 kinase activity assay using MEK substrate; phosphoamino acid analysis; dominant-negative Raf-1 co-transfection; PKC inhibition (GF 109203X, phorbol ester down-regulation)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — epistasis via dominant-negative mutant plus kinase assay plus pharmacological dissection\",\n      \"pmids\": [\"8063729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"CHRM1 (m1) and m3 receptors selectively activate Gq/11, while m2 selectively activates Gi2; all three subtypes also activate Gi1 and Gi3, but m1/m3 do so only at higher agonist concentrations, demonstrating subtype-selective G protein coupling with differential efficacies.\",\n      \"method\": \"Photolabeling of G protein alpha subunits with [α-32P]GTP azidoanilide in transfected HEK293 cell membranes; subtype-specific immunoprecipitation; carbachol dose-response\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — biochemical reconstitution with defined receptor subtypes, direct G protein identification\",\n      \"pmids\": [\"8190105\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"β-arrestins coordinate termination of CHRM1 (M1 muscarinic receptor) signaling by physically interacting with diacylglycerol kinases (DGKs) and recruiting this complex to activated M1 receptors, thereby accelerating DAG degradation to phosphatidic acid; β-arrestins are essential for agonist-stimulated DAG-to-PA conversion at activated M1 receptors.\",\n      \"method\": \"Co-immunoprecipitation of β-arrestin with DGKs; RNAi knockdown of β-arrestins; mass spectrometry identification; DAG/phosphatidic acid measurements after agonist stimulation; recruitment of β-arrestin-DGK complex to activated 7TMRs by imaging\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — Co-IP, RNAi loss-of-function, biochemical lipid measurements, multiple orthogonal methods\",\n      \"pmids\": [\"17272726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Extracellular tau protein promotes intracellular calcium increase in neuronal cells through CHRM1 (M1) and M3 muscarinic receptors; this effect is blocked by M1/M3-selective antagonists and is recapitulated in non-neuronal cells transfected with M1 or M3 cDNA, indicating tau acts on neurons via these receptors.\",\n      \"method\": \"Antagonist pharmacology (pirenzepine, 4-DAMP) in primary hippocampal/cortical neurons and neuroblastoma; transfection of M1/M3 cDNA in non-neuronal cells; intracellular calcium imaging\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological plus transfection reconstitution; single lab but two orthogonal approaches\",\n      \"pmids\": [\"18272392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"M1 muscarinic receptor (CHRM1) allosteric agonists (AC-42 and 77-LH-28-1) selectively activate M1 over other mAChR subtypes via an allosteric binding site; 77-LH-28-1 also acts as an agonist at native rat hippocampal M1 receptors, increasing cell firing and inducing gamma frequency network oscillations in vitro and in vivo.\",\n      \"method\": \"Calcium mobilization and inositol phosphate accumulation assays at recombinant mAChR subtypes; in vitro and in vivo electrophysiology in rat hippocampus\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional assays in recombinant and native systems; single lab\",\n      \"pmids\": [\"18454168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CHRM1 signaling via Gαq/11 is functionally altered in 'muscarinic receptor-deficit schizophrenia': agonist potency for Gq/11 stimulation is decreased while maximal Gq/11-[35S]GTPγS coupling is increased, indicating compensatory upregulation of receptor-G protein coupling efficiency in this subgroup.\",\n      \"method\": \"[35S]GTPγS-Gαq/11 immunocapture assay in postmortem human cortex membranes (Brodmann area 9); dose-response to orthosteric (oxotremorine-M) and allosteric (AC-42) agonists\",\n      \"journal\": \"Neuropsychopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical coupling assay in human tissue, two agonists tested; single lab\",\n      \"pmids\": [\"19404243\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Individual M1 muscarinic receptor (CHRM1) molecules are randomly distributed in the plasma membrane and dynamically interconvert between monomers and dimers on a timescale of seconds; approximately 30% exist as dimers at any given time with no evidence for higher-order oligomers.\",\n      \"method\": \"Total internal reflection fluorescence microscopy (TIRFM) single-molecule imaging in live CHO cells; two-color TIRFM for dimer confirmation; single-particle tracking (~30 ms / ~20 nm resolution)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct single-molecule live-cell imaging with two-color validation; highly cited\",\n      \"pmids\": [\"20133736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In Huntington's disease striatal cells, H3K9me3-dependent heterochromatin accumulates at the CHRM1 promoter, reducing CHRM1 gene expression and impairing Ca2+-dependent neuronal signal transduction.\",\n      \"method\": \"H3K9me3-ChIP genome-wide sequencing combined with RNA sequencing in STHdh Q7/7 and Q111/111 stable cell lines; platform integration analysis; Ca2+ signaling functional assay\",\n      \"journal\": \"Acta neuropathologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP-seq plus RNA-seq plus functional Ca2+ assay; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"23455440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structures of the M1 muscarinic acetylcholine receptor bound to the inverse agonist tiotropium revealed structural differences in orthosteric and allosteric binding sites compared to M2, M3, and M4 receptors, providing a molecular basis for drug selectivity; comparison across subtypes identified residue differences contributing to subtype-selective binding.\",\n      \"method\": \"X-ray crystallography of M1 receptor in complex with tiotropium; structural comparison with M2, M3, M4 crystal structures\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with direct structural comparison; highly cited\",\n      \"pmids\": [\"26958838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Chrm1 and Chrm3 double knockout in mice chronically diminishes REM sleep to almost undetectable levels, and knockout of these two Gq-type muscarinic acetylcholine receptors phenocopies synaptic inhibition of TrkA+ cholinergic neurons, establishing that CHRM1 and CHRM3 are essential for REM sleep generation.\",\n      \"method\": \"Triple-target CRISPR knockout of Chrm1/Chrm3 in mice; polysomnographic sleep recording (EEG/EMG); chemogenetic synaptic inhibition of TrkA+ cholinergic neurons\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic loss-of-function (CRISPR KO) with quantitative sleep phenotype; replicated with orthogonal chemogenetic approach\",\n      \"pmids\": [\"30157420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Magnolol upregulates CHRM1 expression in SH-SY5Y cells treated with Aβ, and CHRM1 activation mediates protection against Tau hyperphosphorylation and apoptosis through the cAMP/PKA/CREB pathway; pharmacological inactivation of cAMP signaling reverses magnolol's protective effects.\",\n      \"method\": \"CCK-8 viability assay; qRT-PCR and western blotting for CHRM1 and pathway components; ELISA and western blotting for cAMP/PKA/CREB; caspase-3 activity and flow cytometry for apoptosis; siRNA or inhibitor of cAMP pathway\",\n      \"journal\": \"Journal of natural medicines\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, cell-line model, no direct CHRM1 reconstitution; pathway placement is indirect\",\n      \"pmids\": [\"34705126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DOPA inhibits melanocyte and melanoma cell proliferation by inhibiting CHRM1 signaling; pharmacologic CHRM1 antagonism depletes c-Myc and FOXM1, and both pharmacologic inhibition of CHRM1 and FOXM1 inhibit melanoma tumor growth in preclinical mouse models.\",\n      \"method\": \"High-throughput pharmacologic screen; in vivo CRISPR genetic screen; pharmacological CHRM1 antagonism; western blotting for c-Myc and FOXM1; preclinical mouse melanoma tumor models\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo CRISPR genetic screen plus pharmacological validation plus in vivo tumor models; multiple orthogonal approaches in single study\",\n      \"pmids\": [\"36054350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CHRM1 inhibits the TRPM8 cation channel via Gq/11 and phospholipase C-mediated depletion of phosphatidylinositol 4,5-bisphosphate (PIP2), defining a novel CHRM1-TRPM8 signaling axis with anti-fibrotic effects in liver disease; CHRM1 activation produces strong anti-fibrotic effects in a patient-derived 3D MASH model.\",\n      \"method\": \"BRET-based biosensors in live cells; orthogonal genetic assays (CRISPR); high-content imaging; chemogenomic screening in patient-derived 3D liver organoid model; multi-omics\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — BRET biosensors plus genetic validation plus pharmacological screening; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"39605182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"M1 muscarinic receptors (CHRM1) expressed in human intestinal L cells (NCI-H716) control GLP-1 secretion; selective M1 agonist McN-A-343 stimulates GLP-1 secretion dose-dependently, and pirenzepine (M1 antagonist) but not 4-DAMP (M3 antagonist) blocks bethanechol-stimulated GLP-1 release, confirming a functional role for CHRM1 in GLP-1 secretion.\",\n      \"method\": \"GLP-1 secretion assay in NCI-H716 human L cell line; selective agonists/antagonists for mAChR subtypes; western blot, immunohistochemistry, RT-PCR for receptor subtype expression confirmation\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological dissection with selective ligands plus expression confirmation; replicated in paraffin-embedded human intestine sections\",\n      \"pmids\": [\"12810581\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CHRM1 (M1 muscarinic acetylcholine receptor) is a Gq/11-coupled seven-transmembrane GPCR whose agonist-driven activation stimulates phospholipase C-mediated PI hydrolysis and Ca2+ release, leading to downstream PKC-independent Raf-1/ERK activation, DAG degradation coordinated by β-arrestin-recruited DGKs, APP secretion, GLP-1 release, and c-Myc/FOXM1-dependent cell proliferation; it forms transient dimers at the plasma membrane, is regulated by H3K9me3 epigenetic silencing, is essential for REM sleep in mice, and inhibits TRPM8 via PIP2 depletion, with its crystal structure revealing orthosteric and allosteric binding sites that underlie subtype-selective pharmacology.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CHRM1 is a Gq/11-coupled muscarinic acetylcholine receptor that activates phospholipase C to deplete PIP2 and mobilize intracellular calcium, thereby modulating ion channel activity, synaptic signaling, and cell proliferation. CHRM1 signals through Gαq/11 to inhibit TRPM8 via PIP2 depletion [PMID:39605182] and couples to cAMP/PKA/CREB signaling in neuronal contexts [PMID:34705126]; genetic ablation of Chrm1 together with Chrm3 nearly eliminates REM sleep in mice, establishing these Gq-type receptors as essential regulators of sleep architecture [PMID:30157420]. In cancer, CHRM1 signaling promotes melanocyte and melanoma proliferation through c-Myc and FOXM1, and pharmacological CHRM1 antagonism suppresses tumor growth in vivo [PMID:36054350]. CHRM1 expression is epigenetically silenced by H3K9me3-mediated heterochromatin in Huntington's disease striatal cells, impairing downstream Ca²⁺-dependent neuronal signaling [PMID:23455440].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrating that CHRM1 directly couples to Gαq/11 in human cortex established the primary G-protein signaling partner and revealed that this coupling is functionally altered in muscarinic receptor-deficit schizophrenia.\",\n      \"evidence\": \"[35S]-GTPγS–Gαq/11 immunocapture assay on post-mortem human cortical membranes with orthosteric and allosteric agonists\",\n      \"pmids\": [\"19404243\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single lab and single method; no independent replication of the coupling efficiency change in schizophrenia\",\n        \"Downstream effectors of altered coupling efficiency not characterized\",\n        \"Whether coupling changes are cause or consequence of disease is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying H3K9me3-mediated epigenetic silencing of CHRM1 in Huntington's disease striatal cells linked chromatin pathology to impaired muscarinic Ca²⁺ signaling, providing a mechanism for cholinergic signaling deficits in HD.\",\n      \"evidence\": \"H3K9me3-ChIP-seq with matched RNA-seq in STHdhQ111/111 HD cell lines; functional Ca²⁺ signaling assay\",\n      \"pmids\": [\"23455440\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single cell-line model; not validated in primary human HD striatal tissue\",\n        \"Whether restoring CHRM1 expression rescues Ca²⁺ signaling in vivo is untested\",\n        \"Identity of the H3K9 methyltransferase responsible for silencing at the CHRM1 locus is unknown\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Genetic elimination of Chrm1 and Chrm3 revealed that these two Gq-type muscarinic receptors are the essential, non-redundant receptors for REM sleep, resolving decades of pharmacological ambiguity about which receptor subtypes control this sleep stage.\",\n      \"evidence\": \"Triple-target CRISPR knockout of muscarinic receptor genes in mice with EEG/sleep phenotyping across multiple receptor combinations\",\n      \"pmids\": [\"30157420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Downstream neural circuits through which Chrm1 specifically (versus Chrm3) contributes to REM sleep are undefined\",\n        \"Whether the REM sleep phenotype reflects developmental compensation in constitutive knockouts is not addressed\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linking CHRM1 upregulation to cAMP/PKA/CREB activation and protection against Aβ-induced Tau phosphorylation expanded the known signaling repertoire of CHRM1 beyond canonical Gq/PLC to include a neuroprotective cAMP pathway.\",\n      \"evidence\": \"CHRM1 overexpression and pharmacological modulation in Aβ-treated SH-SY5Y cells with ELISA/western blotting for pathway components\",\n      \"pmids\": [\"34705126\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No CHRM1 knockout or rescue experiment to confirm receptor-pathway specificity; pathway activation inferred indirectly\",\n        \"Single immortalized cell line; not validated in primary neurons or in vivo\",\n        \"Mechanism linking a Gq-coupled receptor to cAMP elevation not delineated\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying CHRM1 as a proliferative driver in melanocytes and melanoma — with antagonism depleting c-Myc and FOXM1 and suppressing tumor growth — established a direct oncogenic signaling role and a candidate therapeutic vulnerability.\",\n      \"evidence\": \"High-throughput pharmacologic and CRISPR screens; CHRM1 antagonism with c-Myc/FOXM1 protein quantification; in vivo mouse melanoma tumor growth assays\",\n      \"pmids\": [\"36054350\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The intracellular signaling cascade connecting CHRM1 activation to c-Myc/FOXM1 stabilization or transcription is not mapped\",\n        \"Whether CHRM1 drives proliferation in non-melanoma cancers through the same effectors is unknown\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Delineating a CHRM1→Gq/11→PLC→PIP2 depletion→TRPM8 inhibition axis defined the complete signaling chain by which CHRM1 gates a specific ion channel and linked this mechanism to anti-fibrotic effects in liver disease.\",\n      \"evidence\": \"BRET biosensors for each signaling step in cell-based assays; orthogonal genetic validation; 3D patient-derived MASH liver model\",\n      \"pmids\": [\"39605182\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether CHRM1–TRPM8 coupling occurs in neurons or other tissues beyond hepatic stellate cells is untested\",\n        \"Structural basis of PIP2-dependent TRPM8 gating by CHRM1 signaling is not resolved\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showing that acetylcholine promotes diffuse midline glioma proliferation through CHRM1/CHRM3 on OPC-like tumor cells identified muscarinic receptors as mediators of activity-dependent glioma growth. (preprint)\",\n      \"evidence\": \"(preprint) scRNA-seq of patient DMG; optogenetic cholinergic neuron stimulation in vivo; pharmacological blockade in co-culture and mouse models\",\n      \"pmids\": [\"bio_10.1101_2024.09.21.614235\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Preprint; not yet peer-reviewed\",\n        \"Relative contributions of CHRM1 versus CHRM3 to glioma proliferation not disentangled\",\n        \"Downstream signaling effectors in DMG cells not identified\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating region-specific subcellular trafficking of CHRM1 protein (plasma membrane enrichment) in primate cortex, coupled with differential cholinergic modulation of excitatory:inhibitory balance, linked receptor trafficking to circuit-level function. (preprint)\",\n      \"evidence\": \"(preprint) Single-nucleus RNA-seq; mRNA-protein histology; in vitro electrophysiology and spine morphology in ACC vs. LPFC primate neurons\",\n      \"pmids\": [\"bio_10.1101_2025.05.23.655820\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Preprint; not yet peer-reviewed\",\n        \"Trafficking machinery responsible for differential CHRM1 surface expression across cortical regions is unidentified\",\n        \"Whether region-specific CHRM1 trafficking is conserved outside primates is unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The intracellular signaling cascades that connect CHRM1–Gq activation to transcriptional effectors such as c-Myc and FOXM1, the structural basis of CHRM1 coupling selectivity, and whether CHRM1 trafficking differences underlie region-specific cholinergic vulnerability in neurodegeneration remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No high-resolution structure of CHRM1 in complex with Gαq/11 and downstream effectors\",\n        \"Mechanism linking CHRM1 to c-Myc/FOXM1 regulation is unmapped\",\n        \"Whether CHRM1 epigenetic silencing extends beyond HD to other neurodegenerative diseases is untested\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 4, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 4, 5]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"GNAQ\",\n      \"GNA11\",\n      \"TRPM8\",\n      \"CHRM3\",\n      \"MYC\",\n      \"FOXM1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"CHRM1 (muscarinic acetylcholine receptor M1) is a Gq/11-coupled seven-transmembrane receptor that transduces cholinergic signals primarily through phospholipase C-mediated phosphatidylinositol hydrolysis, intracellular calcium mobilization, and downstream activation of the Raf-1/ERK cascade in a largely PKC-independent manner [PMID:3037705, PMID:8190105, PMID:8063729]. Agonist-driven signaling is terminated by β-arrestin-recruited diacylglycerol kinases that convert DAG to phosphatidic acid, while PLC-dependent PIP2 depletion couples CHRM1 to inhibition of the TRPM8 channel [PMID:17272726, PMID:39605182]. CHRM1 activation stimulates amyloid precursor protein secretion, GLP-1 release from intestinal L cells, and c-Myc/FOXM1-dependent melanocyte proliferation, and genetic ablation of Chrm1 together with Chrm3 in mice virtually eliminates REM sleep [PMID:1411529, PMID:12810581, PMID:36054350, PMID:30157420]. Crystal structures of the receptor bound to tiotropium reveal orthosteric and allosteric binding-site differences from other muscarinic subtypes that underlie subtype-selective pharmacology [PMID:26958838].\",\n  \"teleology\": [\n    {\n      \"year\": 1987,\n      \"claim\": \"Molecular cloning of CHRM1 established it as a seven-transmembrane muscarinic receptor gene with distinct ligand-binding properties, providing the molecular identity needed for all subsequent subtype-selective studies.\",\n      \"evidence\": \"cDNA cloning from rat cerebral cortex, heterologous expression in mammalian cells, ligand-binding assays\",\n      \"pmids\": [\"3037705\", \"3443095\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous signaling effectors not yet identified\", \"No structural information available\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Reconstitution experiments showed CHRM1 selectively couples to PI hydrolysis and drives DNA synthesis and oncogenic transformation, distinguishing the Gq-coupled M1/M3/M5 subfamily from the Gi-coupled M2/M4 subfamily.\",\n      \"evidence\": \"Recombinant receptor expression in CHO and NIH 3T3 cells; PI hydrolysis, thymidine incorporation, and focus-formation assays with carbachol\",\n      \"pmids\": [\"2739737\", \"1905013\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream kinase cascades linking PI hydrolysis to transformation not delineated\", \"G protein identity not yet confirmed biochemically\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Biochemical identification of Gαq/11 as the primary G protein for CHRM1 and demonstration that CHRM1 activates Raf-1/ERK largely independently of PKC resolved the signaling pathway downstream of PI hydrolysis.\",\n      \"evidence\": \"GTP-azidoanilide photolabeling with subtype-specific immunoprecipitation in HEK293 membranes; Raf-1 kinase assays with dominant-negative Raf-1 and PKC inhibitors in NIH 3T3 cells\",\n      \"pmids\": [\"8190105\", \"8063729\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ras involvement upstream of Raf-1 not directly tested\", \"Mechanism of PKC-independent Raf-1 activation unresolved\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Activation of CHRM1 was shown to rapidly increase secretion of amyloid precursor protein derivatives in a protein-kinase-dependent manner, linking muscarinic signaling to APP processing relevant to Alzheimer's disease.\",\n      \"evidence\": \"Carbachol stimulation of M1-transfected HEK293 cells; APP immunoassay; staurosporine inhibition\",\n      \"pmids\": [\"1411529\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific kinase (PKC vs. other) mediating APP release not pinpointed\", \"In vivo relevance in brain not established\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Pharmacological dissection in human intestinal L cells demonstrated that CHRM1 controls GLP-1 secretion, expanding the receptor's physiological roles beyond the nervous system.\",\n      \"evidence\": \"Selective M1 agonist (McN-A-343) and antagonist (pirenzepine) in NCI-H716 cells; GLP-1 secretion assay; receptor expression confirmation by RT-PCR and immunohistochemistry\",\n      \"pmids\": [\"12810581\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo contribution of M1 versus M3 to incretin release not tested\", \"Downstream signaling from M1 to exocytotic machinery not defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Discovery that β-arrestins recruit diacylglycerol kinases to activated M1 receptors to accelerate DAG-to-PA conversion revealed a signal-termination mechanism that reshapes the lipid-signaling landscape downstream of CHRM1.\",\n      \"evidence\": \"Co-IP of β-arrestin with DGKs; RNAi knockdown; mass spectrometry; DAG/PA lipid measurements after agonist stimulation\",\n      \"pmids\": [\"17272726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific DGK isoform preference for M1 not determined\", \"Functional consequences of PA generation not explored\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Single-molecule imaging revealed that CHRM1 dynamically interconverts between monomers and transient dimers at the plasma membrane (~30% dimers), ruling out stable oligomeric states and defining the receptor's quaternary behavior.\",\n      \"evidence\": \"TIRFM single-molecule and two-color imaging in live CHO cells; single-particle tracking\",\n      \"pmids\": [\"20133736\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional significance of dimerization for signaling output not tested\", \"Agonist effect on dimer fraction not assessed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The crystal structure of M1 bound to tiotropium provided the first atomic-resolution view of CHRM1, revealing orthosteric and allosteric site differences from M2–M4 that explain subtype-selective pharmacology.\",\n      \"evidence\": \"X-ray crystallography of human M1 receptor–tiotropium complex; structural comparison with M2, M3, M4 structures\",\n      \"pmids\": [\"26958838\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Active-state structure not yet solved\", \"Allosteric agonist co-crystal structures absent\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"CRISPR triple knockout of Chrm1/Chrm3 in mice virtually eliminated REM sleep, establishing that these Gq-type muscarinic receptors are essential generators of REM sleep.\",\n      \"evidence\": \"CRISPR KO mice; EEG/EMG polysomnography; chemogenetic silencing of cholinergic neurons as orthogonal validation\",\n      \"pmids\": [\"30157420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Individual contribution of Chrm1 versus Chrm3 to REM sleep not separable in the double KO\", \"Downstream neural circuit mechanism not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"An in vivo CRISPR screen and pharmacological studies showed that CHRM1 signaling sustains c-Myc and FOXM1 levels in melanoma, and CHRM1 antagonism inhibits tumor growth, revealing a druggable cholinergic proliferative axis in cancer.\",\n      \"evidence\": \"High-throughput pharmacologic screen; in vivo CRISPR genetic screen; preclinical mouse melanoma models; western blotting\",\n      \"pmids\": [\"36054350\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking CHRM1 to c-Myc stabilization not defined\", \"Generalizability to other tumor types untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"BRET biosensor and genetic studies demonstrated that CHRM1 inhibits the TRPM8 channel via Gq/PLC-mediated PIP2 depletion, defining a new effector axis with anti-fibrotic activity in liver disease models.\",\n      \"evidence\": \"BRET-based biosensors; CRISPR validation; chemogenomic screening in patient-derived 3D MASH liver organoids\",\n      \"pmids\": [\"39605182\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo anti-fibrotic efficacy not demonstrated in animal models\", \"Whether PIP2 depletion or a secondary metabolite mediates TRPM8 inhibition not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the active-state structure of CHRM1, the mechanism by which CHRM1 activates Raf-1 independently of PKC, the individual roles of CHRM1 versus CHRM3 in REM sleep, and the signaling pathway linking CHRM1 to c-Myc/FOXM1 stabilization in cancer.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Active-state and agonist-bound structures lacking\", \"PKC-independent Raf-1 activation mechanism undefined\", \"Single versus double receptor contributions to REM sleep unseparated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 5, 8, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 10, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 4, 5, 6, 16]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [7, 8, 13]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"GNAQ\",\n      \"GNA11\",\n      \"ARRB1\",\n      \"ARRB2\",\n      \"DGKZ\",\n      \"TRPM8\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}