{"gene":"TPRA1","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2015,"finding":"GPR175/TPRA1 (also known as Tpra40) localizes to primary cilia upon Hedgehog (Hh) stimulation and positively regulates Hh signaling by coupling to Gαi1 to inhibit adenylyl cyclase, thereby reducing cAMP levels, suppressing PKA activity, and reducing Gli3 processing into its repressor form. Interaction experiments placed Gpr175 at the level of PKA and upstream of Gαi1; depletion of either Gpr175 or Gαi1 led to increased cAMP and increased Gli3 repressor formation.","method":"Knockdown/depletion experiments, cAMP measurement, Gli3 processing assays, interaction/co-localization experiments in primary cilia, epistasis with PKA and Gαi1","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, multiple orthogonal methods (KD, cAMP measurement, Gli3 processing, localization, interaction experiments)","pmids":["26451044"],"is_preprint":false},{"year":2008,"finding":"TPRA40/GPR175 regulates cell division of early mouse embryos. The C-terminal region of TPRA40 is essential for this regulation. NA14 (nuclear antigen of 14 kDa, a Sjögren's syndrome autoantigen) binds directly to the C-terminal region of TPRA40, as confirmed by GST pull-down and co-immunoprecipitation. NA14 mediates functional transport of TPRA40 from cytosol to the plasma membrane via microtubules; an N-terminal deletion mutant of NA14 (NA14ΔN) that cannot bind microtubules but retains TPRA40 binding inhibits this transport. TPRA40ΔC, lacking the NA14-binding C-terminus, also shows impaired plasma membrane transport.","method":"shRNA knockdown in mouse embryos, yeast two-hybrid screening, GST pull-down, co-immunoprecipitation, live imaging of FLAG-TPRA40 subcellular localization, dominant-negative NA14 mutant experiments","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, multiple orthogonal methods (Y2H, GST pulldown, Co-IP, live imaging, mutagenesis, in vivo embryo KD)","pmids":["18459117"],"is_preprint":false},{"year":1999,"finding":"TPRA40 (TPRA1) encodes a novel 298-amino acid, seven-transmembrane domain orphan receptor identified in 3T3-L1 adipocytes. Its mRNA and protein levels are higher in differentiated 3T3-L1 adipocytes than in fibroblasts, and TPRA40 mRNA is significantly upregulated (2–3 fold) in epididymal white adipose tissue of aged mice and in db/db and ob/ob diabetic mice, with no change in brown fat or other tissues.","method":"PCR cloning from 3T3-L1 adipocyte cDNA library, Northern blot/PCR expression analysis across tissues and disease models","journal":"Endocrinology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, cloning and expression characterization only, no functional mechanistic experiment","pmids":["10342878"],"is_preprint":false},{"year":2001,"finding":"Rat TPRA40 mRNA levels decrease significantly during ischemic hypoxia and reoxygenation in H9c2 cardiomyoblast cells, and the rat ortholog has a longer C-terminal region (369 amino acids) than the mouse protein.","method":"RT-PCR cloning, mRNA expression analysis during ischemic hypoxia/reoxygenation in H9c2 cells","journal":"Biochimica et biophysica acta","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method (RT-PCR expression), no functional mechanistic experiment","pmids":["11267675"],"is_preprint":false},{"year":2014,"finding":"GPR175 (TPRA1) was identified as a binding partner of Neuroligin 3 (NLGN3) intracellular fragment via yeast two-hybrid screening of a human fetal cDNA library, and the interaction was validated by co-immunoprecipitation in human neuroblastoma cells or brain tissues.","method":"Yeast two-hybrid screening, co-immunoprecipitation in neuroblastoma cells and brain tissue","journal":"Journal of molecular neuroscience : MN","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Y2H plus confirmatory Co-IP, no downstream functional characterization of TPRA1 in this context","pmids":["25464930"],"is_preprint":false},{"year":2025,"finding":"TPRA1 was identified as a receptor for oncolytic virus M1 (OVM) through a membrane-protein targeted CRISPR-Cas9 screen. Mechanistically, the extracellular region of TPRA1 binds OVM particles directly via glycosylation, while the cytoplasmic tail mediates viral endocytosis, collectively enabling viral entry and cancer cell lysis. TPRA1 also promotes entry of Semliki Forest Virus, suggesting a conserved role in alphavirus infection.","method":"Membrane-protein targeted CRISPR-Cas9 screen, direct binding assays (extracellular region to OVM particles), glycosylation characterization, cytoplasmic tail deletion/mutagenesis functional assays, viral infection/lysis assays in cell lines and mouse models","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — CRISPR screen plus domain-level mechanistic dissection (extracellular binding, endocytosis via cytoplasmic tail), glycosylation characterization, validated in cell lines, mouse models, and patient samples; single lab but multiple orthogonal methods","pmids":["41053536"],"is_preprint":false},{"year":2025,"finding":"TPRA1 can interact with the promoter region of lncRNA XLOC-040580 and regulate its expression during zygotic genome activation (ZGA) in porcine embryos. Knockdown of TPRA1 by siRNA at the one-cell stage blocked porcine blastocyst development and affected ZGA-related gene expression, recapitulating the phenotype of XLOC-040580 knockdown. Dual-luciferase reporter assays confirmed XLOC-040580 as a downstream transcriptional target of TPRA1.","method":"siRNA microinjection at one-cell stage, blastocyst development rate quantification, qRT-PCR, dual-luciferase reporter assay, single-cell mRNA sequencing","journal":"Cell transplantation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, multiple methods (siRNA KD, luciferase reporter, scRNA-seq), functional embryonic phenotype established","pmids":["40245181"],"is_preprint":false}],"current_model":"TPRA1 (GPR175/TPRA40) is an orphan seven-transmembrane GPCR that localizes to primary cilia upon Hedgehog stimulation where it couples to Gαi1 to suppress cAMP production and PKA activity, thereby maximizing Hedgehog signaling; it also serves as a direct receptor for oncolytic alphavirus M1 through glycosylation-mediated extracellular binding and cytoplasmic tail-dependent endocytosis, regulates early embryonic cell division via NA14-mediated plasma membrane transport, acts as a transcriptional regulator of lncRNA XLOC-040580 during zygotic genome activation, and binds the intracellular domain of Neuroligin 3."},"narrative":{"mechanistic_narrative":"TPRA1 (GPR175/TPRA40) is an orphan seven-transmembrane receptor that functions as a positive regulator of Hedgehog signaling: upon Hedgehog stimulation it localizes to primary cilia and couples to Gαi1 to inhibit adenylyl cyclase, lowering cAMP, suppressing PKA activity, and reducing the formation of the Gli3 repressor [PMID:26451044]. Beyond this signaling role, TPRA1 controls early embryonic cell division through its C-terminal region, which binds NA14 to direct microtubule-dependent transport of the receptor from the cytosol to the plasma membrane [PMID:18459117], and it acts in zygotic genome activation by interacting with the promoter of lncRNA XLOC-040580 to drive its expression, an activity required for blastocyst development [PMID:40245181]. Independently, TPRA1 serves as an entry receptor for the oncolytic alphavirus M1, binding virus particles through glycosylation of its extracellular region while its cytoplasmic tail mediates viral endocytosis, a function extending to Semliki Forest Virus [PMID:41053536]. A direct interaction between TPRA1 and the intracellular fragment of Neuroligin 3 has also been documented [PMID:25464930], and its expression is modulated in adipocyte differentiation and metabolic disease models [PMID:10342878].","teleology":[{"year":1999,"claim":"Established the existence and tissue context of TPRA1 by cloning a novel seven-transmembrane orphan receptor whose expression tracks adipocyte differentiation and metabolic disease states.","evidence":"PCR cloning from 3T3-L1 adipocyte cDNA and Northern/PCR expression profiling across tissues and db/db, ob/ob mice","pmids":["10342878"],"confidence":"Low","gaps":["Expression correlation only; no functional or signaling mechanism tested","No ligand or coupling partner identified","Causal role in adipogenesis or diabetes not established"]},{"year":2001,"claim":"Extended expression regulation to cardiac stress, showing the receptor's transcript is responsive to ischemic hypoxia and noting species-level C-terminal divergence.","evidence":"RT-PCR cloning and mRNA expression analysis during hypoxia/reoxygenation in rat H9c2 cardiomyoblasts","pmids":["11267675"],"confidence":"Low","gaps":["Descriptive expression change only; no mechanism","Functional consequence of C-terminal length difference unexplored"]},{"year":2008,"claim":"Provided the first functional mechanism by linking TPRA1's C-terminus to NA14-dependent, microtubule-mediated trafficking required for embryonic cell division.","evidence":"Yeast two-hybrid, GST pull-down, Co-IP, live imaging of FLAG-TPRA40, dominant-negative NA14 mutants, and shRNA knockdown in mouse embryos","pmids":["18459117"],"confidence":"Medium","gaps":["Downstream signaling driving cell division not defined","Whether plasma membrane localization is required for a specific signaling output untested","Single lab"]},{"year":2014,"claim":"Identified a candidate protein partner by showing TPRA1 binds the intracellular domain of Neuroligin 3.","evidence":"Yeast two-hybrid screen of human fetal cDNA and Co-IP in neuroblastoma cells and brain tissue","pmids":["25464930"],"confidence":"Low","gaps":["No functional consequence of the interaction characterized","No reciprocal validation or mapping of the binding interface","Physiological relevance in neurons untested"]},{"year":2015,"claim":"Defined TPRA1's signaling mechanism, placing it in primary cilia as a Gαi1-coupled positive regulator of Hedgehog signaling acting through cAMP/PKA control of Gli3 processing.","evidence":"Knockdown/depletion, cAMP measurement, Gli3 processing assays, ciliary localization, and epistasis with PKA and Gαi1","pmids":["26451044"],"confidence":"Medium","gaps":["Endogenous ligand/activating stimulus for the receptor unidentified","Structural basis of Gαi1 coupling not resolved","Single lab"]},{"year":2025,"claim":"Revealed an unrelated functional role by identifying TPRA1 as an alphavirus entry receptor, dissecting glycosylation-dependent extracellular binding and cytoplasmic-tail-driven endocytosis.","evidence":"Membrane-protein CRISPR-Cas9 screen, direct virus-binding and glycosylation assays, cytoplasmic-tail mutagenesis, and infection/lysis assays in cells, mouse models, and patient samples","pmids":["41053536"],"confidence":"High","gaps":["Specific glycan moiety mediating binding not fully defined","Endocytic machinery recruited by the cytoplasmic tail unknown","Relationship to the receptor's signaling function unaddressed"]},{"year":2025,"claim":"Implicated TPRA1 in transcriptional control of early development by showing it binds and activates the lncRNA XLOC-040580 promoter to enable zygotic genome activation.","evidence":"siRNA microinjection at one-cell stage, blastocyst development quantification, dual-luciferase reporter assays, and single-cell RNA-seq in porcine embryos","pmids":["40245181"],"confidence":"Medium","gaps":["Mechanism by which a 7TM receptor reaches/acts on a promoter unexplained","Whether the effect is direct DNA binding or indirect not resolved","Single lab and single species"]},{"year":null,"claim":"The endogenous ligand of this orphan receptor and how its diverse roles — ciliary Hedgehog signaling, embryonic trafficking, transcriptional regulation, and viral entry — are mechanistically unified remain unknown.","evidence":"","pmids":[],"confidence":"Low","gaps":["No deorphanizing ligand identified","No structural model of the receptor or its complexes","Connection between membrane signaling and reported nuclear/transcriptional activity unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[5]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[0]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,5]}],"pathway":[],"complexes":[],"partners":["GNAI1","NA14","NLGN3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86W33","full_name":"Transmembrane protein adipocyte-associated 1","aliases":["Integral membrane protein GPR175","Transmembrane protein 227"],"length_aa":373,"mass_kda":41.1,"function":"","subcellular_location":"Membrane","url":"https://www.uniprot.org/uniprotkb/Q86W33/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TPRA1","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/TPRA1","total_profiled":1310},"omim":[{"mim_id":"612008","title":"CELIAC DISEASE, SUSCEPTIBILITY TO, 10; CELIAC10","url":"https://www.omim.org/entry/612008"},{"mim_id":"608336","title":"TRANSMEMBRANE PROTEIN, ADIPOCYTE-ASSOCIATED 1; TPRA1","url":"https://www.omim.org/entry/608336"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TPRA1"},"hgnc":{"alias_symbol":["TPRA40","FLJ32197","TMEM227"],"prev_symbol":["GPR175"]},"alphafold":{"accession":"Q86W33","domains":[{"cath_id":"1.20.1070,1.20.1070","chopping":"45-286","consensus_level":"medium","plddt":89.524,"start":45,"end":286}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86W33","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86W33-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86W33-F1-predicted_aligned_error_v6.png","plddt_mean":73.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TPRA1","jax_strain_url":"https://www.jax.org/strain/search?query=TPRA1"},"sequence":{"accession":"Q86W33","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86W33.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86W33/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86W33"}},"corpus_meta":[{"pmid":"18550530","id":"PMC_18550530","title":"TRPA1 mediates the noxious effects of natural sesquiterpene deterrents.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18550530","citation_count":78,"is_preprint":false},{"pmid":"19389958","id":"PMC_19389958","title":"Identification of internal control genes for quantitative polymerase chain reaction in mammary tissue of lactating cows receiving lipid supplements.","date":"2009","source":"Journal of dairy science","url":"https://pubmed.ncbi.nlm.nih.gov/19389958","citation_count":74,"is_preprint":false},{"pmid":"26451044","id":"PMC_26451044","title":"The Orphan G Protein-coupled Receptor Gpr175 (Tpra40) Enhances Hedgehog Signaling by Modulating cAMP Levels.","date":"2015","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26451044","citation_count":46,"is_preprint":false},{"pmid":"29852243","id":"PMC_29852243","title":"Acrolein Contributes to the Neuropathic Pain and Neuron Damage after Ischemic-Reperfusion Spinal Cord Injury.","date":"2018","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/29852243","citation_count":33,"is_preprint":false},{"pmid":"25464930","id":"PMC_25464930","title":"Novel interactive partners of neuroligin 3: new aspects for pathogenesis of autism.","date":"2014","source":"Journal of molecular neuroscience : MN","url":"https://pubmed.ncbi.nlm.nih.gov/25464930","citation_count":24,"is_preprint":false},{"pmid":"33810314","id":"PMC_33810314","title":"Role of TRPA1 in Tissue Damage and Kidney Disease.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33810314","citation_count":19,"is_preprint":false},{"pmid":"30669148","id":"PMC_30669148","title":"Genetic-Epigenetic Interactions in Asthma Revealed by a Genome-Wide Gene-Centric Search.","date":"2019","source":"Human heredity","url":"https://pubmed.ncbi.nlm.nih.gov/30669148","citation_count":17,"is_preprint":false},{"pmid":"35614079","id":"PMC_35614079","title":"Pan-cancer analyses reveal the genetic and pharmacogenomic landscape of transient receptor potential channels.","date":"2022","source":"NPJ genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35614079","citation_count":16,"is_preprint":false},{"pmid":"18459117","id":"PMC_18459117","title":"TPRA40/GPR175 regulates early mouse embryogenesis through functional membrane transport by Sjögren's syndrome-associated protein NA14.","date":"2008","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18459117","citation_count":15,"is_preprint":false},{"pmid":"34828272","id":"PMC_34828272","title":"Discovering of Genomic Variations Associated to Growth Traits by GWAS in Braunvieh Cattle.","date":"2021","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/34828272","citation_count":14,"is_preprint":false},{"pmid":"27413384","id":"PMC_27413384","title":"Synergistic Effect of Ferulic Acid and Z-Ligustilide, Major Components of A. sinensis, on Regulating Cold-Sensing Protein TRPM8 and TPRA1 In Vitro.","date":"2016","source":"Evidence-based complementary and alternative medicine : eCAM","url":"https://pubmed.ncbi.nlm.nih.gov/27413384","citation_count":9,"is_preprint":false},{"pmid":"10342878","id":"PMC_10342878","title":"Differential expression of a novel seven transmembrane domain protein in epididymal fat from aged and diabetic mice.","date":"1999","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/10342878","citation_count":8,"is_preprint":false},{"pmid":"11267675","id":"PMC_11267675","title":"Molecular cloning of rat transmembrane domain protein of 40 kDa regulated in adipocytes and its expression in H9c2 cells exposed to ischemic hypoxia and reoxygenation.","date":"2001","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/11267675","citation_count":8,"is_preprint":false},{"pmid":"39257693","id":"PMC_39257693","title":"Integrated Long Noncoding RNA and Messenger RNA Expression Analysis Identifies Molecules Specifically Associated With Resiliency and Susceptibility to Depression and Antidepressant Response.","date":"2024","source":"Biological psychiatry global open science","url":"https://pubmed.ncbi.nlm.nih.gov/39257693","citation_count":6,"is_preprint":false},{"pmid":"25519347","id":"PMC_25519347","title":"Mixed-effects models for joint modeling of sequence data in longitudinal studies.","date":"2014","source":"BMC proceedings","url":"https://pubmed.ncbi.nlm.nih.gov/25519347","citation_count":5,"is_preprint":false},{"pmid":"40245181","id":"PMC_40245181","title":"LncRNA XLOC-040580 targeted by TPRA1 coordinate zygotic genome activation during porcine embryonic development.","date":"2025","source":"Cell transplantation","url":"https://pubmed.ncbi.nlm.nih.gov/40245181","citation_count":2,"is_preprint":false},{"pmid":"39656803","id":"PMC_39656803","title":"Synovial transcriptome-wide association study implicates novel genes underlying rheumatoid arthritis risk.","date":"2025","source":"Rheumatology (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/39656803","citation_count":2,"is_preprint":false},{"pmid":"41053536","id":"PMC_41053536","title":"Identification of TPRA1 as a Novel Receptor and Predictive Biomarker for Oncolytic Virus M1.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/41053536","citation_count":1,"is_preprint":false},{"pmid":"39778364","id":"PMC_39778364","title":"Identification of quantitative trait loci and candidate genes associated with growth curve parameters in chinese wenshang barred chickens.","date":"2025","source":"Poultry science","url":"https://pubmed.ncbi.nlm.nih.gov/39778364","citation_count":1,"is_preprint":false},{"pmid":"28032561","id":"PMC_28032561","title":"Expression of transient receptor potential ankyrin 1 correlating to the recovery of colonic transit after pelvic nerve denervation in rats.","date":"2016","source":"The Journal of surgical research","url":"https://pubmed.ncbi.nlm.nih.gov/28032561","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12115,"output_tokens":2212,"usd":0.034763,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9236,"output_tokens":2598,"usd":0.055565,"stage2_stop_reason":"end_turn"},"total_usd":0.090328,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2015,\n      \"finding\": \"GPR175/TPRA1 (also known as Tpra40) localizes to primary cilia upon Hedgehog (Hh) stimulation and positively regulates Hh signaling by coupling to Gαi1 to inhibit adenylyl cyclase, thereby reducing cAMP levels, suppressing PKA activity, and reducing Gli3 processing into its repressor form. Interaction experiments placed Gpr175 at the level of PKA and upstream of Gαi1; depletion of either Gpr175 or Gαi1 led to increased cAMP and increased Gli3 repressor formation.\",\n      \"method\": \"Knockdown/depletion experiments, cAMP measurement, Gli3 processing assays, interaction/co-localization experiments in primary cilia, epistasis with PKA and Gαi1\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, multiple orthogonal methods (KD, cAMP measurement, Gli3 processing, localization, interaction experiments)\",\n      \"pmids\": [\"26451044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TPRA40/GPR175 regulates cell division of early mouse embryos. The C-terminal region of TPRA40 is essential for this regulation. NA14 (nuclear antigen of 14 kDa, a Sjögren's syndrome autoantigen) binds directly to the C-terminal region of TPRA40, as confirmed by GST pull-down and co-immunoprecipitation. NA14 mediates functional transport of TPRA40 from cytosol to the plasma membrane via microtubules; an N-terminal deletion mutant of NA14 (NA14ΔN) that cannot bind microtubules but retains TPRA40 binding inhibits this transport. TPRA40ΔC, lacking the NA14-binding C-terminus, also shows impaired plasma membrane transport.\",\n      \"method\": \"shRNA knockdown in mouse embryos, yeast two-hybrid screening, GST pull-down, co-immunoprecipitation, live imaging of FLAG-TPRA40 subcellular localization, dominant-negative NA14 mutant experiments\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, multiple orthogonal methods (Y2H, GST pulldown, Co-IP, live imaging, mutagenesis, in vivo embryo KD)\",\n      \"pmids\": [\"18459117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"TPRA40 (TPRA1) encodes a novel 298-amino acid, seven-transmembrane domain orphan receptor identified in 3T3-L1 adipocytes. Its mRNA and protein levels are higher in differentiated 3T3-L1 adipocytes than in fibroblasts, and TPRA40 mRNA is significantly upregulated (2–3 fold) in epididymal white adipose tissue of aged mice and in db/db and ob/ob diabetic mice, with no change in brown fat or other tissues.\",\n      \"method\": \"PCR cloning from 3T3-L1 adipocyte cDNA library, Northern blot/PCR expression analysis across tissues and disease models\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, cloning and expression characterization only, no functional mechanistic experiment\",\n      \"pmids\": [\"10342878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Rat TPRA40 mRNA levels decrease significantly during ischemic hypoxia and reoxygenation in H9c2 cardiomyoblast cells, and the rat ortholog has a longer C-terminal region (369 amino acids) than the mouse protein.\",\n      \"method\": \"RT-PCR cloning, mRNA expression analysis during ischemic hypoxia/reoxygenation in H9c2 cells\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method (RT-PCR expression), no functional mechanistic experiment\",\n      \"pmids\": [\"11267675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GPR175 (TPRA1) was identified as a binding partner of Neuroligin 3 (NLGN3) intracellular fragment via yeast two-hybrid screening of a human fetal cDNA library, and the interaction was validated by co-immunoprecipitation in human neuroblastoma cells or brain tissues.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation in neuroblastoma cells and brain tissue\",\n      \"journal\": \"Journal of molecular neuroscience : MN\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Y2H plus confirmatory Co-IP, no downstream functional characterization of TPRA1 in this context\",\n      \"pmids\": [\"25464930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TPRA1 was identified as a receptor for oncolytic virus M1 (OVM) through a membrane-protein targeted CRISPR-Cas9 screen. Mechanistically, the extracellular region of TPRA1 binds OVM particles directly via glycosylation, while the cytoplasmic tail mediates viral endocytosis, collectively enabling viral entry and cancer cell lysis. TPRA1 also promotes entry of Semliki Forest Virus, suggesting a conserved role in alphavirus infection.\",\n      \"method\": \"Membrane-protein targeted CRISPR-Cas9 screen, direct binding assays (extracellular region to OVM particles), glycosylation characterization, cytoplasmic tail deletion/mutagenesis functional assays, viral infection/lysis assays in cell lines and mouse models\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — CRISPR screen plus domain-level mechanistic dissection (extracellular binding, endocytosis via cytoplasmic tail), glycosylation characterization, validated in cell lines, mouse models, and patient samples; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"41053536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TPRA1 can interact with the promoter region of lncRNA XLOC-040580 and regulate its expression during zygotic genome activation (ZGA) in porcine embryos. Knockdown of TPRA1 by siRNA at the one-cell stage blocked porcine blastocyst development and affected ZGA-related gene expression, recapitulating the phenotype of XLOC-040580 knockdown. Dual-luciferase reporter assays confirmed XLOC-040580 as a downstream transcriptional target of TPRA1.\",\n      \"method\": \"siRNA microinjection at one-cell stage, blastocyst development rate quantification, qRT-PCR, dual-luciferase reporter assay, single-cell mRNA sequencing\",\n      \"journal\": \"Cell transplantation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, multiple methods (siRNA KD, luciferase reporter, scRNA-seq), functional embryonic phenotype established\",\n      \"pmids\": [\"40245181\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TPRA1 (GPR175/TPRA40) is an orphan seven-transmembrane GPCR that localizes to primary cilia upon Hedgehog stimulation where it couples to Gαi1 to suppress cAMP production and PKA activity, thereby maximizing Hedgehog signaling; it also serves as a direct receptor for oncolytic alphavirus M1 through glycosylation-mediated extracellular binding and cytoplasmic tail-dependent endocytosis, regulates early embryonic cell division via NA14-mediated plasma membrane transport, acts as a transcriptional regulator of lncRNA XLOC-040580 during zygotic genome activation, and binds the intracellular domain of Neuroligin 3.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TPRA1 (GPR175/TPRA40) is an orphan seven-transmembrane receptor that functions as a positive regulator of Hedgehog signaling: upon Hedgehog stimulation it localizes to primary cilia and couples to Gαi1 to inhibit adenylyl cyclase, lowering cAMP, suppressing PKA activity, and reducing the formation of the Gli3 repressor [#0]. Beyond this signaling role, TPRA1 controls early embryonic cell division through its C-terminal region, which binds NA14 to direct microtubule-dependent transport of the receptor from the cytosol to the plasma membrane [#1], and it acts in zygotic genome activation by interacting with the promoter of lncRNA XLOC-040580 to drive its expression, an activity required for blastocyst development [#6]. Independently, TPRA1 serves as an entry receptor for the oncolytic alphavirus M1, binding virus particles through glycosylation of its extracellular region while its cytoplasmic tail mediates viral endocytosis, a function extending to Semliki Forest Virus [#5]. A direct interaction between TPRA1 and the intracellular fragment of Neuroligin 3 has also been documented [#4], and its expression is modulated in adipocyte differentiation and metabolic disease models [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established the existence and tissue context of TPRA1 by cloning a novel seven-transmembrane orphan receptor whose expression tracks adipocyte differentiation and metabolic disease states.\",\n      \"evidence\": \"PCR cloning from 3T3-L1 adipocyte cDNA and Northern/PCR expression profiling across tissues and db/db, ob/ob mice\",\n      \"pmids\": [\"10342878\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Expression correlation only; no functional or signaling mechanism tested\", \"No ligand or coupling partner identified\", \"Causal role in adipogenesis or diabetes not established\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Extended expression regulation to cardiac stress, showing the receptor's transcript is responsive to ischemic hypoxia and noting species-level C-terminal divergence.\",\n      \"evidence\": \"RT-PCR cloning and mRNA expression analysis during hypoxia/reoxygenation in rat H9c2 cardiomyoblasts\",\n      \"pmids\": [\"11267675\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Descriptive expression change only; no mechanism\", \"Functional consequence of C-terminal length difference unexplored\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Provided the first functional mechanism by linking TPRA1's C-terminus to NA14-dependent, microtubule-mediated trafficking required for embryonic cell division.\",\n      \"evidence\": \"Yeast two-hybrid, GST pull-down, Co-IP, live imaging of FLAG-TPRA40, dominant-negative NA14 mutants, and shRNA knockdown in mouse embryos\",\n      \"pmids\": [\"18459117\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream signaling driving cell division not defined\", \"Whether plasma membrane localization is required for a specific signaling output untested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified a candidate protein partner by showing TPRA1 binds the intracellular domain of Neuroligin 3.\",\n      \"evidence\": \"Yeast two-hybrid screen of human fetal cDNA and Co-IP in neuroblastoma cells and brain tissue\",\n      \"pmids\": [\"25464930\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No functional consequence of the interaction characterized\", \"No reciprocal validation or mapping of the binding interface\", \"Physiological relevance in neurons untested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined TPRA1's signaling mechanism, placing it in primary cilia as a Gαi1-coupled positive regulator of Hedgehog signaling acting through cAMP/PKA control of Gli3 processing.\",\n      \"evidence\": \"Knockdown/depletion, cAMP measurement, Gli3 processing assays, ciliary localization, and epistasis with PKA and Gαi1\",\n      \"pmids\": [\"26451044\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous ligand/activating stimulus for the receptor unidentified\", \"Structural basis of Gαi1 coupling not resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed an unrelated functional role by identifying TPRA1 as an alphavirus entry receptor, dissecting glycosylation-dependent extracellular binding and cytoplasmic-tail-driven endocytosis.\",\n      \"evidence\": \"Membrane-protein CRISPR-Cas9 screen, direct virus-binding and glycosylation assays, cytoplasmic-tail mutagenesis, and infection/lysis assays in cells, mouse models, and patient samples\",\n      \"pmids\": [\"41053536\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific glycan moiety mediating binding not fully defined\", \"Endocytic machinery recruited by the cytoplasmic tail unknown\", \"Relationship to the receptor's signaling function unaddressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated TPRA1 in transcriptional control of early development by showing it binds and activates the lncRNA XLOC-040580 promoter to enable zygotic genome activation.\",\n      \"evidence\": \"siRNA microinjection at one-cell stage, blastocyst development quantification, dual-luciferase reporter assays, and single-cell RNA-seq in porcine embryos\",\n      \"pmids\": [\"40245181\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which a 7TM receptor reaches/acts on a promoter unexplained\", \"Whether the effect is direct DNA binding or indirect not resolved\", \"Single lab and single species\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The endogenous ligand of this orphan receptor and how its diverse roles — ciliary Hedgehog signaling, embryonic trafficking, transcriptional regulation, and viral entry — are mechanistically unified remain unknown.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No deorphanizing ligand identified\", \"No structural model of the receptor or its complexes\", \"Connection between membrane signaling and reported nuclear/transcriptional activity unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0007224\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"GNAI1\", \"NA14\", \"NLGN3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":4,"faith_total":4,"faith_pct":100.0}}