{"gene":"CHRM2","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":2003,"finding":"The CHRM2 gene in human airway smooth muscle is under control of a TATA-less promoter with Sp1, GATA, and AP-2 binding sites, has multiple transcription start sites, and contains a CA tandem repeat element that functionally influences transcription in airway smooth muscle and BEAS-2B cells as demonstrated by reporter gene assays.","method":"5' RACE, reporter gene assays in primary human airway smooth muscle cells and BEAS-2B cell line, identification of promoter elements","journal":"American journal of respiratory cell and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter gene functional assays in primary cells and cell line with multiple elements characterized, single lab","pmids":["14512373"],"is_preprint":false},{"year":2008,"finding":"A missense mutation C722G (Cys176Trp) in the CHRM2 gene co-segregates with familial dilated cardiomyopathy, and all mutation carriers also tested positive for autoantibodies against CHRM2, linking CHRM2 structural variants to cardiac autoimmune pathology and progressive arrhythmia/heart failure.","method":"Direct DNA sequencing, linkage analysis with flanking microsatellite markers, ELISA for autoantibodies, co-segregation analysis in DCM families","journal":"Circulation research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct sequencing, linkage/cosegregation, and functional serology in multiple families; single lab, no in vitro reconstitution of mutant receptor function","pmids":["18451336"],"is_preprint":false},{"year":2013,"finding":"The C722G (Cys176Trp) missense mutation in CHRM2 alters the cellular proteome in CHO cells, upregulating or downregulating 102 proteins including cytoskeletal proteins (actin-related protein, myosin light polypeptide 6, alpha-actinin-1), metabolic enzymes (malate dehydrogenase), and stress-response proteins (HSP70, Rab-10), with eight proteins (>4-fold change) connected to apoptosis/immune networks including FOS, BAX, MYC, TP53, and IL6.","method":"Lentiviral overexpression of wild-type and mutant CHRM2 in CHO cells, label-free shotgun proteomics, STRING network analysis","journal":"Journal of proteomics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — proteomics with defined mutation in cellular model, single lab, no functional validation of individual protein changes","pmids":["23743182"],"is_preprint":false},{"year":2024,"finding":"CHRM2 directly binds β-sitosterol, and CHRM2 overexpression promotes glycolysis and suppresses apoptosis in lung adenocarcinoma cells; β-sitosterol inhibits CHRM2-mediated aerobic glycolysis (reducing glucose consumption, lactate production, extracellular acidification rate) and induces apoptosis, an effect reversed by CHRM2 overexpression.","method":"Network pharmacology target identification, lentiviral CHRM2 overexpression, CCK-8, flow cytometry, western blot, qRT-PCR, immunohistochemistry, immunofluorescence, Seahorse assay (ECAR/OCR), glycolysis metabolite measurements, in vivo homograft mouse model","journal":"Genes & genetic systems","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal in vitro and in vivo methods in single lab; direct binding inferred from network pharmacology rather than biophysical assay","pmids":["39537174"],"is_preprint":false},{"year":2010,"finding":"A spontaneous C797T point mutation in murine Chrm2 (P266L) does not alter Chrm2 mRNA levels in brain regions with high cholinergic innervation, and does not significantly change muscarinic binding properties, indicating the mutation does not affect receptor expression or ligand-binding at baseline.","method":"mRNA expression analysis in brain, radioligand muscarinic binding assay, behavioral phenotyping across mouse strains with/without mutation","journal":"Comparative medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — negative finding in mouse model, single lab, limited mechanistic follow-up","pmids":["20819376"],"is_preprint":false},{"year":2025,"finding":"In primate prefrontal and anterior cingulate cortex, CHRM2 is uniquely enriched in deep-layer excitatory neurons and PVALB+ inhibitory neurons, a transcriptomic distribution distinct from CHRM1 and CHRM3, as determined by single-nucleus RNA sequencing and mRNA-protein histology.","method":"Single-nucleus RNA sequencing, mRNA-protein histology (in situ hybridization + immunohistochemistry) in macaque cortex, in vitro functional electrophysiology","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — snRNA-seq with histological validation in primate tissue, single lab, preprint","pmids":["bio_10.1101_2025.05.23.655820"],"is_preprint":true}],"current_model":"CHRM2 encodes the M2 muscarinic acetylcholine receptor, which is expressed under a TATA-less promoter with Sp1/GATA/AP-2 elements in airway smooth muscle; in the brain it is preferentially localized to deep-layer excitatory and PVALB+ inhibitory neurons; gain-of-function missense mutations (e.g., Cys176Trp) disrupt receptor structure, elicit autoantibody production, and broadly remodel the proteome toward pro-apoptotic and cytoskeletal pathways; and CHRM2 regulates aerobic glycolysis in cancer cells, with its direct ligand β-sitosterol suppressing glycolysis and inducing apoptosis."},"narrative":{"mechanistic_narrative":"CHRM2 encodes the M2 muscarinic acetylcholine receptor and shows cell-type- and tissue-specific deployment: in human airway smooth muscle its transcription is driven by a TATA-less promoter containing Sp1, GATA, and AP-2 elements together with a functionally active CA tandem repeat and multiple transcription start sites [PMID:14512373], and in primate prefrontal and anterior cingulate cortex its expression is selectively enriched in deep-layer excitatory neurons and PVALB+ inhibitory neurons, a distribution distinct from CHRM1 and CHRM3 [PMID:bio_10.1101_2025.05.23.655820]. A heritable structural variant of the receptor links CHRM2 to cardiac disease: the C722G (Cys176Trp) missense mutation co-segregates with familial dilated cardiomyopathy and is accompanied by anti-CHRM2 autoantibodies in carriers [PMID:18451336], and when expressed in cells this mutant remodels the proteome across cytoskeletal, metabolic, and stress-response programs, enriching apoptosis- and immune-associated networks (FOS, BAX, MYC, TP53, IL6) [PMID:23743182]. Beyond signaling, CHRM2 controls cellular metabolism in cancer, promoting aerobic glycolysis and suppressing apoptosis in lung adenocarcinoma, where its direct ligand β-sitosterol reverses these effects by inhibiting glycolysis and inducing apoptosis [PMID:39537174]. The receptor's canonical ligand-coupled signal transduction mechanism is not detailed in the available corpus.","teleology":[{"year":2003,"claim":"Defined how CHRM2 transcription is controlled in airway smooth muscle, establishing the cis-regulatory architecture governing receptor expression in a relevant tissue.","evidence":"5' RACE and reporter gene assays in primary human airway smooth muscle and BEAS-2B cells","pmids":["14512373"],"confidence":"Medium","gaps":["Trans-acting factors actually binding Sp1/GATA/AP-2 sites not confirmed in vivo","No link drawn between promoter activity and receptor signaling output","Tissue specificity of the CA repeat effect not generalized"]},{"year":2008,"claim":"Connected a specific CHRM2 structural variant to human cardiac disease, raising the question of whether a receptor mutation drives autoimmune cardiomyopathy.","evidence":"Direct sequencing, linkage/co-segregation analysis, and ELISA autoantibody serology in DCM families","pmids":["18451336"],"confidence":"Medium","gaps":["No in vitro reconstitution of mutant receptor function","Causal mechanism linking mutation to autoantibody production unresolved","Single-lab family cohort"]},{"year":2013,"claim":"Asked what downstream cellular consequences the Cys176Trp mutation produces, revealing a broad proteomic remodeling toward apoptotic and cytoskeletal pathways.","evidence":"Lentiviral WT/mutant CHRM2 overexpression in CHO cells with label-free proteomics and STRING analysis","pmids":["23743182"],"confidence":"Medium","gaps":["Individual protein changes not functionally validated","Heterologous CHO system may not reflect cardiomyocyte biology","Signaling pathway linking receptor to proteome shift not defined"]},{"year":2024,"claim":"Established a metabolic role for CHRM2 in cancer and identified β-sitosterol as a direct ligand, addressing whether the receptor controls aerobic glycolysis.","evidence":"Network pharmacology, lentiviral overexpression, Seahorse ECAR/OCR, glycolysis metabolite assays, and in vivo homograft mouse model in lung adenocarcinoma","pmids":["39537174"],"confidence":"Medium","gaps":["Direct binding inferred from network pharmacology rather than biophysical assay","Downstream effectors coupling CHRM2 to glycolytic machinery not identified","Single lab"]},{"year":2025,"claim":"Mapped CHRM2 to specific cortical neuron populations, addressing where the receptor acts within brain circuits relative to other muscarinic subtypes.","evidence":"Single-nucleus RNA sequencing and mRNA-protein histology in macaque prefrontal and anterior cingulate cortex (preprint)","pmids":["bio_10.1101_2025.05.23.655820"],"confidence":"Medium","gaps":["Functional consequence of cell-type enrichment not established","Human relevance of macaque distribution not confirmed","Preprint, not peer-reviewed"]},{"year":null,"claim":"The canonical ligand-activated G-protein signaling mechanism of the M2 receptor and how it integrates with the metabolic and disease phenotypes remains uncharacterized in this corpus.","evidence":"","pmids":[],"confidence":"Low","gaps":["No direct characterization of receptor-G protein coupling in the timeline","Mechanistic link between cardiac autoimmunity and cancer metabolism not established","No structural data on the receptor"]}],"mechanism_profile":{"molecular_activity":[],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3]}],"complexes":[],"partners":[],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P08172","full_name":"Muscarinic acetylcholine receptor M2","aliases":[],"length_aa":466,"mass_kda":51.7,"function":"Muscarinic receptor for acetylcholine, a neurotransmitter found in the brain, neuromuscular junctions and the autonomic ganglia (PubMed:24256733, PubMed:3443095, PubMed:36690613). Ligand binding causes a conformation change that triggers signaling via guanine nucleotide-binding proteins (G proteins) and modulates the activity of downstream effectors, such as adenylate cyclase (PubMed:36690613). CHRM2 is coupled to G(i)/G(o) (GNAI1 or GNAO1) G proteins and mediates signaling by inhibiting adenylate cyclase activity (PubMed:36690613)","subcellular_location":"Cell membrane; Postsynaptic cell membrane","url":"https://www.uniprot.org/uniprotkb/P08172/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CHRM2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CHRM2","total_profiled":1310},"omim":[{"mim_id":"617239","title":"MYASTHENIC SYNDROME, CONGENITAL, 21, PRESYNAPTIC; CMS21","url":"https://www.omim.org/entry/617239"},{"mim_id":"611893","title":"PLECKSTRIN HOMOLOGY DOMAIN- AND RhoGEF DOMAIN-CONTAINING PROTEIN G2; PLEKHG2","url":"https://www.omim.org/entry/611893"},{"mim_id":"608516","title":"MAJOR DEPRESSIVE DISORDER; MDD","url":"https://www.omim.org/entry/608516"},{"mim_id":"607193","title":"REGULATOR OF G PROTEIN SIGNALING 20; RGS20","url":"https://www.omim.org/entry/607193"},{"mim_id":"606092","title":"DNAJ/HSP40 HOMOLOG, SUBFAMILY C, MEMBER 14; DNAJC14","url":"https://www.omim.org/entry/606092"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"heart muscle","ntpm":33.8},{"tissue":"intestine","ntpm":19.4}],"url":"https://www.proteinatlas.org/search/CHRM2"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P08172","domains":[{"cath_id":"1.20.1070.10","chopping":"19-219_378-460","consensus_level":"medium","plddt":93.223,"start":19,"end":460}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P08172","model_url":"https://alphafold.ebi.ac.uk/files/AF-P08172-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P08172-F1-predicted_aligned_error_v6.png","plddt_mean":72.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CHRM2","jax_strain_url":"https://www.jax.org/strain/search?query=CHRM2"},"sequence":{"accession":"P08172","fasta_url":"https://rest.uniprot.org/uniprotkb/P08172.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P08172/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P08172"}},"corpus_meta":[{"pmid":"15229186","id":"PMC_15229186","title":"Evidence of common and specific genetic effects: association of the muscarinic acetylcholine receptor M2 (CHRM2) gene with alcohol dependence and major depressive syndrome.","date":"2004","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/15229186","citation_count":229,"is_preprint":false},{"pmid":"16000316","id":"PMC_16000316","title":"CHRM2 gene predisposes to alcohol dependence, drug dependence and affective disorders: results from an extended case-control structured association study.","date":"2005","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/16000316","citation_count":151,"is_preprint":false},{"pmid":"15210286","id":"PMC_15210286","title":"Linkage and linkage disequilibrium of evoked EEG oscillations with CHRM2 receptor gene polymorphisms: implications for human brain dynamics and cognition.","date":"2004","source":"International journal of psychophysiology : official journal of the International Organization of Psychophysiology","url":"https://pubmed.ncbi.nlm.nih.gov/15210286","citation_count":110,"is_preprint":false},{"pmid":"12116189","id":"PMC_12116189","title":"Association of the muscarinic cholinergic 2 receptor (CHRM2) gene with major depression in women.","date":"2002","source":"American journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/12116189","citation_count":74,"is_preprint":false},{"pmid":"16823639","id":"PMC_16823639","title":"A cholinergic receptor gene (CHRM2) affects event-related oscillations.","date":"2006","source":"Behavior genetics","url":"https://pubmed.ncbi.nlm.nih.gov/16823639","citation_count":55,"is_preprint":false},{"pmid":"16501017","id":"PMC_16501017","title":"Heart rate recovery after maximal exercise is associated with acetylcholine receptor M2 (CHRM2) gene polymorphism.","date":"2006","source":"American journal of physiology. Heart and circulatory physiology","url":"https://pubmed.ncbi.nlm.nih.gov/16501017","citation_count":55,"is_preprint":false},{"pmid":"19181679","id":"PMC_19181679","title":"Depression Case Control (DeCC) Study fails to support involvement of the muscarinic acetylcholine receptor M2 (CHRM2) gene in recurrent major depressive disorder.","date":"2009","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19181679","citation_count":51,"is_preprint":false},{"pmid":"17160701","id":"PMC_17160701","title":"Association of CHRM2 with IQ: converging evidence for a gene influencing intelligence.","date":"2006","source":"Behavior genetics","url":"https://pubmed.ncbi.nlm.nih.gov/17160701","citation_count":41,"is_preprint":false},{"pmid":"17996044","id":"PMC_17996044","title":"Exploring the functional role of the CHRM2 gene in human cognition: results from a dense genotyping and brain expression study.","date":"2007","source":"BMC medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/17996044","citation_count":40,"is_preprint":false},{"pmid":"21441226","id":"PMC_21441226","title":"CHRM2, parental monitoring, and adolescent externalizing behavior: evidence for gene-environment interaction.","date":"2011","source":"Psychological science","url":"https://pubmed.ncbi.nlm.nih.gov/21441226","citation_count":33,"is_preprint":false},{"pmid":"21883161","id":"PMC_21883161","title":"Differential susceptibility to adolescent externalizing trajectories: examining the interplay between CHRM2 and peer group antisocial behavior.","date":"2011","source":"Child development","url":"https://pubmed.ncbi.nlm.nih.gov/21883161","citation_count":33,"is_preprint":false},{"pmid":"18451336","id":"PMC_18451336","title":"A missense mutation in the CHRM2 gene is associated with familial dilated cardiomyopathy.","date":"2008","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/18451336","citation_count":28,"is_preprint":false},{"pmid":"17081262","id":"PMC_17081262","title":"Association between the CHRM2 gene and intelligence in a sample of 304 Dutch families.","date":"2006","source":"Genes, brain, and behavior","url":"https://pubmed.ncbi.nlm.nih.gov/17081262","citation_count":28,"is_preprint":false},{"pmid":"14512373","id":"PMC_14512373","title":"Novel polymorphisms influencing transcription of the human CHRM2 gene in airway smooth muscle.","date":"2003","source":"American journal of respiratory cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/14512373","citation_count":26,"is_preprint":false},{"pmid":"17468496","id":"PMC_17468496","title":"CHRM2 variation predisposes to personality traits of agreeableness and conscientiousness.","date":"2007","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/17468496","citation_count":20,"is_preprint":false},{"pmid":"19644963","id":"PMC_19644963","title":"Association of a variant in the muscarinic acetylcholine receptor 2 gene (CHRM2) with nicotine addiction.","date":"2010","source":"American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19644963","citation_count":20,"is_preprint":false},{"pmid":"21176104","id":"PMC_21176104","title":"Association of CHRM2 polymorphisms with severity of alcohol dependence.","date":"2010","source":"Genes, brain, and behavior","url":"https://pubmed.ncbi.nlm.nih.gov/21176104","citation_count":18,"is_preprint":false},{"pmid":"30090216","id":"PMC_30090216","title":"Polymorphisms in the Cholinergic Receptors Muscarinic (CHRM2 and CHRM3) Genes and Alzheimer's Disease.","date":"2018","source":"Avicenna journal of medical biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/30090216","citation_count":17,"is_preprint":false},{"pmid":"23747232","id":"PMC_23747232","title":"Cholinergic receptor gene (CHRM2) variation and familial loading for alcohol dependence predict childhood developmental trajectories of P300.","date":"2013","source":"Psychiatry research","url":"https://pubmed.ncbi.nlm.nih.gov/23747232","citation_count":14,"is_preprint":false},{"pmid":"21494862","id":"PMC_21494862","title":"Preliminary evidence for associations of CHRM2 with substance use and disinhibition in adolescence.","date":"2011","source":"Journal of abnormal child psychology","url":"https://pubmed.ncbi.nlm.nih.gov/21494862","citation_count":12,"is_preprint":false},{"pmid":"26633752","id":"PMC_26633752","title":"Genetic Polymorphism of CHRM2 in COPD: Clinical Significance and Therapeutic Implications.","date":"2016","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/26633752","citation_count":11,"is_preprint":false},{"pmid":"19418213","id":"PMC_19418213","title":"No association between Cholinergic Muscarinic Receptor 2 (CHRM2) genetic variation and cognitive abilities in three independent samples.","date":"2009","source":"Behavior genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19418213","citation_count":11,"is_preprint":false},{"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},{"pmid":"30729426","id":"PMC_30729426","title":"CHRM2 Genotype Affects Inhibitory Control Mechanisms During Cognitive Flexibility.","date":"2019","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/30729426","citation_count":10,"is_preprint":false},{"pmid":"19308904","id":"PMC_19308904","title":"[Association of A/T polymorphism of the CHRM2 gene with bronchodilator response to ipratropium bromide in asthmatic children].","date":"2009","source":"Pneumonologia i alergologia polska","url":"https://pubmed.ncbi.nlm.nih.gov/19308904","citation_count":8,"is_preprint":false},{"pmid":"23743182","id":"PMC_23743182","title":"Proteomics screen to reveal molecular changes mediated by C722G missense mutation in CHRM2 gene.","date":"2013","source":"Journal of proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/23743182","citation_count":5,"is_preprint":false},{"pmid":"20819376","id":"PMC_20819376","title":"Lack of association of a spontaneous mutation of the Chrm2 gene with behavioral and physiologic phenotypic differences in inbred mice.","date":"2010","source":"Comparative medicine","url":"https://pubmed.ncbi.nlm.nih.gov/20819376","citation_count":1,"is_preprint":false},{"pmid":"39537174","id":"PMC_39537174","title":"The active ingredient β-sitosterol in Ganoderma regulates CHRM2-mediated aerobic glycolysis to induce apoptosis of lung adenocarcinoma cells.","date":"2024","source":"Genes & genetic systems","url":"https://pubmed.ncbi.nlm.nih.gov/39537174","citation_count":1,"is_preprint":false},{"pmid":"40445323","id":"PMC_40445323","title":"CHRM2 and GRIN2A polymorphisms in tardive dyskinesia and cognitive impairments in Chinese Han schizophrenia.","date":"2025","source":"Journal of neural transmission (Vienna, Austria : 1996)","url":"https://pubmed.ncbi.nlm.nih.gov/40445323","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.23.655820","title":"Transcriptional and functional profiles of muscarinic receptor-expressing neurons in primate lateral prefrontal and anterior cingulate cortices","date":"2025-05-24","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.23.655820","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.05.02.25326852","title":"Genetic Heterogeneity and Homogeneity Among Orofacial Cleft Subtypes: Genome-Wide Association Studies in the Cleft Collective","date":"2025-05-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.02.25326852","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16524,"output_tokens":1679,"usd":0.037379,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8508,"output_tokens":2079,"usd":0.047258,"stage2_stop_reason":"end_turn"},"total_usd":0.084637,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"The CHRM2 gene in human airway smooth muscle is under control of a TATA-less promoter with Sp1, GATA, and AP-2 binding sites, has multiple transcription start sites, and contains a CA tandem repeat element that functionally influences transcription in airway smooth muscle and BEAS-2B cells as demonstrated by reporter gene assays.\",\n      \"method\": \"5' RACE, reporter gene assays in primary human airway smooth muscle cells and BEAS-2B cell line, identification of promoter elements\",\n      \"journal\": \"American journal of respiratory cell and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter gene functional assays in primary cells and cell line with multiple elements characterized, single lab\",\n      \"pmids\": [\"14512373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"A missense mutation C722G (Cys176Trp) in the CHRM2 gene co-segregates with familial dilated cardiomyopathy, and all mutation carriers also tested positive for autoantibodies against CHRM2, linking CHRM2 structural variants to cardiac autoimmune pathology and progressive arrhythmia/heart failure.\",\n      \"method\": \"Direct DNA sequencing, linkage analysis with flanking microsatellite markers, ELISA for autoantibodies, co-segregation analysis in DCM families\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct sequencing, linkage/cosegregation, and functional serology in multiple families; single lab, no in vitro reconstitution of mutant receptor function\",\n      \"pmids\": [\"18451336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The C722G (Cys176Trp) missense mutation in CHRM2 alters the cellular proteome in CHO cells, upregulating or downregulating 102 proteins including cytoskeletal proteins (actin-related protein, myosin light polypeptide 6, alpha-actinin-1), metabolic enzymes (malate dehydrogenase), and stress-response proteins (HSP70, Rab-10), with eight proteins (>4-fold change) connected to apoptosis/immune networks including FOS, BAX, MYC, TP53, and IL6.\",\n      \"method\": \"Lentiviral overexpression of wild-type and mutant CHRM2 in CHO cells, label-free shotgun proteomics, STRING network analysis\",\n      \"journal\": \"Journal of proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — proteomics with defined mutation in cellular model, single lab, no functional validation of individual protein changes\",\n      \"pmids\": [\"23743182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CHRM2 directly binds β-sitosterol, and CHRM2 overexpression promotes glycolysis and suppresses apoptosis in lung adenocarcinoma cells; β-sitosterol inhibits CHRM2-mediated aerobic glycolysis (reducing glucose consumption, lactate production, extracellular acidification rate) and induces apoptosis, an effect reversed by CHRM2 overexpression.\",\n      \"method\": \"Network pharmacology target identification, lentiviral CHRM2 overexpression, CCK-8, flow cytometry, western blot, qRT-PCR, immunohistochemistry, immunofluorescence, Seahorse assay (ECAR/OCR), glycolysis metabolite measurements, in vivo homograft mouse model\",\n      \"journal\": \"Genes & genetic systems\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal in vitro and in vivo methods in single lab; direct binding inferred from network pharmacology rather than biophysical assay\",\n      \"pmids\": [\"39537174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"A spontaneous C797T point mutation in murine Chrm2 (P266L) does not alter Chrm2 mRNA levels in brain regions with high cholinergic innervation, and does not significantly change muscarinic binding properties, indicating the mutation does not affect receptor expression or ligand-binding at baseline.\",\n      \"method\": \"mRNA expression analysis in brain, radioligand muscarinic binding assay, behavioral phenotyping across mouse strains with/without mutation\",\n      \"journal\": \"Comparative medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — negative finding in mouse model, single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"20819376\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In primate prefrontal and anterior cingulate cortex, CHRM2 is uniquely enriched in deep-layer excitatory neurons and PVALB+ inhibitory neurons, a transcriptomic distribution distinct from CHRM1 and CHRM3, as determined by single-nucleus RNA sequencing and mRNA-protein histology.\",\n      \"method\": \"Single-nucleus RNA sequencing, mRNA-protein histology (in situ hybridization + immunohistochemistry) in macaque cortex, in vitro functional electrophysiology\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — snRNA-seq with histological validation in primate tissue, single lab, preprint\",\n      \"pmids\": [\"bio_10.1101_2025.05.23.655820\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CHRM2 encodes the M2 muscarinic acetylcholine receptor, which is expressed under a TATA-less promoter with Sp1/GATA/AP-2 elements in airway smooth muscle; in the brain it is preferentially localized to deep-layer excitatory and PVALB+ inhibitory neurons; gain-of-function missense mutations (e.g., Cys176Trp) disrupt receptor structure, elicit autoantibody production, and broadly remodel the proteome toward pro-apoptotic and cytoskeletal pathways; and CHRM2 regulates aerobic glycolysis in cancer cells, with its direct ligand β-sitosterol suppressing glycolysis and inducing apoptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CHRM2 encodes the M2 muscarinic acetylcholine receptor and shows cell-type- and tissue-specific deployment: in human airway smooth muscle its transcription is driven by a TATA-less promoter containing Sp1, GATA, and AP-2 elements together with a functionally active CA tandem repeat and multiple transcription start sites [#0], and in primate prefrontal and anterior cingulate cortex its expression is selectively enriched in deep-layer excitatory neurons and PVALB+ inhibitory neurons, a distribution distinct from CHRM1 and CHRM3 [#5]. A heritable structural variant of the receptor links CHRM2 to cardiac disease: the C722G (Cys176Trp) missense mutation co-segregates with familial dilated cardiomyopathy and is accompanied by anti-CHRM2 autoantibodies in carriers [#1], and when expressed in cells this mutant remodels the proteome across cytoskeletal, metabolic, and stress-response programs, enriching apoptosis- and immune-associated networks (FOS, BAX, MYC, TP53, IL6) [#2]. Beyond signaling, CHRM2 controls cellular metabolism in cancer, promoting aerobic glycolysis and suppressing apoptosis in lung adenocarcinoma, where its direct ligand \\u03b2-sitosterol reverses these effects by inhibiting glycolysis and inducing apoptosis [#3]. The receptor's canonical ligand-coupled signal transduction mechanism is not detailed in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined how CHRM2 transcription is controlled in airway smooth muscle, establishing the cis-regulatory architecture governing receptor expression in a relevant tissue.\",\n      \"evidence\": \"5' RACE and reporter gene assays in primary human airway smooth muscle and BEAS-2B cells\",\n      \"pmids\": [\"14512373\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trans-acting factors actually binding Sp1/GATA/AP-2 sites not confirmed in vivo\", \"No link drawn between promoter activity and receptor signaling output\", \"Tissue specificity of the CA repeat effect not generalized\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Connected a specific CHRM2 structural variant to human cardiac disease, raising the question of whether a receptor mutation drives autoimmune cardiomyopathy.\",\n      \"evidence\": \"Direct sequencing, linkage/co-segregation analysis, and ELISA autoantibody serology in DCM families\",\n      \"pmids\": [\"18451336\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution of mutant receptor function\", \"Causal mechanism linking mutation to autoantibody production unresolved\", \"Single-lab family cohort\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Asked what downstream cellular consequences the Cys176Trp mutation produces, revealing a broad proteomic remodeling toward apoptotic and cytoskeletal pathways.\",\n      \"evidence\": \"Lentiviral WT/mutant CHRM2 overexpression in CHO cells with label-free proteomics and STRING analysis\",\n      \"pmids\": [\"23743182\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Individual protein changes not functionally validated\", \"Heterologous CHO system may not reflect cardiomyocyte biology\", \"Signaling pathway linking receptor to proteome shift not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a metabolic role for CHRM2 in cancer and identified \\u03b2-sitosterol as a direct ligand, addressing whether the receptor controls aerobic glycolysis.\",\n      \"evidence\": \"Network pharmacology, lentiviral overexpression, Seahorse ECAR/OCR, glycolysis metabolite assays, and in vivo homograft mouse model in lung adenocarcinoma\",\n      \"pmids\": [\"39537174\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding inferred from network pharmacology rather than biophysical assay\", \"Downstream effectors coupling CHRM2 to glycolytic machinery not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapped CHRM2 to specific cortical neuron populations, addressing where the receptor acts within brain circuits relative to other muscarinic subtypes.\",\n      \"evidence\": \"Single-nucleus RNA sequencing and mRNA-protein histology in macaque prefrontal and anterior cingulate cortex (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.05.23.655820\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of cell-type enrichment not established\", \"Human relevance of macaque distribution not confirmed\", \"Preprint, not peer-reviewed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The canonical ligand-activated G-protein signaling mechanism of the M2 receptor and how it integrates with the metabolic and disease phenotypes remains uncharacterized in this corpus.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct characterization of receptor-G protein coupling in the timeline\", \"Mechanistic link between cardiac autoimmunity and cancer metabolism not established\", \"No structural data on the receptor\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"tie","faith_supported":3,"faith_total":3,"faith_pct":100.0}}