{"gene":"MRGPRF","run_date":"2026-06-10T02:59:51","timeline":{"discoveries":[{"year":2022,"finding":"MrgprF reduces PI3K complex formation between p101 and p110γ subunits, thereby inhibiting the critical step of PIP2-to-PIP3 conversion and suppressing downstream PI3K/Akt signaling in cutaneous melanoma cells. Forced expression inhibits tumor cell proliferation, migration, xenograft growth, and metastasis; knockdown promotes proliferation and transformation. The Akt-specific agonist SC79 reverses these effects.","method":"Forced expression and knockdown in melanoma cell lines; xenograft tumor models; co-immunoprecipitation/pulldown of PI3K subunits; rescue with Akt agonist SC79; in vitro and in vivo functional assays","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional assays (OE + KD), epistasis rescue with Akt agonist, in vivo xenograft, and PI3K subunit interaction data in one study; replicated directionally by a second independent study (PMID:38805406)","pmids":["35504869"],"is_preprint":false},{"year":2024,"finding":"GPR168 (MRGPRF) overexpression in mouse melanoma B16-F10 cells inhibits proliferation and migration through the Akt signaling pathway, evidenced by decreased p-Akt, p-GSK-3β, β-catenin, Myc, CyclinD1, and CDK4. A blocking anti-GPR168 polyclonal antibody restores proliferation and migration, confirming that signaling depends on GPR168 receptor functionality.","method":"GPR168 overexpression in B16-F10 cells; xenograft tumor model; western blot for pathway components; rescue with anti-GPR168 blocking antibody","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean OE with xenograft plus antibody rescue experiment, single lab, consistent with PMID:35504869","pmids":["38805406"],"is_preprint":false},{"year":2025,"finding":"USP45 directly binds the N-terminal domain of MRGPRF via its catalytic domain and stabilizes MRGPRF protein by removing K63-linked ubiquitination. USP45 knockdown destabilizes MRGPRF and promotes melanoma cell malignancy; these effects are rescued by MRGPRF overexpression, placing USP45 upstream of MRGPRF in a deubiquitination-dependent tumor suppressor axis.","method":"Screening of 40 USPs; Co-IP and pulldown of USP45-MRGPRF interaction; domain mapping (N-terminal MRGPRF); ubiquitination assays (K63-linkage); USP45 OE/KD functional assays; MRGPRF depletion/rescue epistasis; xenograft mouse model","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — biochemical deubiquitination assay with linkage specificity, domain mapping, reciprocal epistasis rescue, and in vivo xenograft in a single rigorous study","pmids":["40788071"],"is_preprint":false},{"year":2025,"finding":"In the mouse spinal cord, MrgprF (MRGPRF) protein levels are regulated by RNA m6A methylation of MrgprF mRNA. Gut microbiota depletion reduces expression of the m6A demethylase FTO (via inactivation of the transcription factor ETS1 at the Fto promoter), leading to increased m6A methylation on MrgprF mRNA and decreased MRGPRF protein in the dorsal horn, resulting in reduced itch sensation. Oral administration of acetyl-L-carnitine (ALC) from Bacteroides fragilis restores FTO and MRGPRF levels.","method":"Antibiotic-treated and germ-free mouse models; oral gavage with B. fragilis and ALC; m6A site mapping on MrgprF mRNA; FTO expression analysis; ETS1 chromatin binding at Fto promoter; behavioral itch assays","journal":"Gut microbes","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic pathway defined with multiple methods (m6A mapping, transcription factor binding, metabolite rescue) in a single lab study","pmids":["40289281"],"is_preprint":false},{"year":2025,"finding":"Global knockout of MrgF (MRGPRF) in mice reduces pain-related behavioral responses to thermal and chemical (formalin) nociceptive stimuli, without affecting motor coordination or cerebellar histology. In MrgF-/- DRG, expression of nociceptive modulation genes c-fos, Runx1, Nav1.7, Nav1.8, and Nav1.9 is downregulated, placing MrgF in a pathway that modulates pain sensitivity.","method":"MrgF global knockout mouse model; behavioral tests (hot plate, tail flick, formalin, rotarod, pole, balance beam, treadmill); qPCR for nociceptive genes in DRG; cerebellar histology","journal":"Hereditas","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO model with specific pain phenotype and molecular readout (nociceptive gene downregulation in DRG), single lab","pmids":["40033362"],"is_preprint":false},{"year":2011,"finding":"MrgF (MRGPRF) is expressed in distinct enteric neuronal subpopulations in the murine ileum, including sensory, secretomotor, and vasodilator neurons, and in nerve fibers of the tunica muscularis and lamina propria. The number of MrgF-expressing enteric neurons is significantly reduced during intestinal schistosomiasis and chemically induced ileitis, indicating that inflammation negatively regulates MrgF expression in the enteric nervous system.","method":"RT-PCR, quantitative PCR, and immunohistochemistry on murine ileum from control, schistosomiasis, and chemically induced ileitis models","journal":"Histochemistry and cell biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization by IHC with quantified loss-of-function in two independent inflammation models, single lab","pmids":["21912971"],"is_preprint":false},{"year":2008,"finding":"Deletion of MrgE in mice results in upregulation of MrgF (MRGPRF) expression in DRG as the sole significantly affected Mrg family member, suggesting a regulatory relationship between MrgE and MrgF expression.","method":"MrgE knockout mice; gene expression analysis of Mrg family members and 84 sensory neuron genes in DRG","journal":"Molecular pain","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single gene expression observation from a KO study focused on MrgE, single lab, no mechanistic follow-up on MRGPRF","pmids":["18197975"],"is_preprint":false},{"year":2023,"finding":"MRGPRF is essential for adipocyte viability and regulates intracellular cAMP levels in a (pre)adipocyte cell model, as determined by functional knockdown/overexpression experiments in the context of a GPCRome screen of adipose tissue.","method":"RNA-seq database (FATTLAS); functional assays in (pre)adipocyte cell model with MRGPRF manipulation; cAMP level measurement","journal":"iScience","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, limited methodological detail in abstract, no mutagenesis or reconstitution; cAMP regulation is a novel functional finding but minimally characterized","pmids":["37766984"],"is_preprint":false},{"year":2023,"finding":"MRGPRF protein is specifically expressed by entero-endocrine cells (EECs) in the mucosal biopsies of human terminal ileum and sigmoid colon, making it the only detectable MRGPR family member at these mucosal sites.","method":"RT-PCR on human GI mucosal biopsies; immunohistochemical staining for cell-type identification","journal":"Cell and tissue research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization with two methods (mRNA + protein IHC), replicated across two gut regions, identifies specific cell type","pmids":["37314493"],"is_preprint":false},{"year":2020,"finding":"In functional calcium mobilization assays, HEK293 cells transfected with dog MRGPRF did not respond to histamine-releasing agents (including fluoroquinolones), in contrast to dog or human MRGPRX2, indicating that MRGPRF does not mediate drug-induced pseudo-allergic calcium signaling in this heterologous expression system.","method":"Intracellular Ca2+ mobilization assay in HEK293 cells transiently transfected with dog MRGPRF; comparison with MRGPRX2, MRGPRD, MRGPRG","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — direct in vitro calcium assay, negative result explicitly reported, single lab","pmids":["32999394"],"is_preprint":false}],"current_model":"MRGPRF (GPR168/MrgprF) is an orphan Mas-related GPCR that functions as a tumor suppressor in melanoma by reducing PI3K p101/p110γ complex formation and suppressing PI3K/Akt signaling; its stability is controlled by USP45-mediated deubiquitination of K63-linked ubiquitin chains on its N-terminus. In the nervous system, MRGPRF modulates pain sensitivity and itch via its expression in DRG and spinal dorsal horn neurons—where protein levels are epigenetically regulated by gut-microbiota-driven RNA m6A methylation through the FTO/ETS1 axis. In the gut, it is specifically localized to entero-endocrine cells of the human ileum and colon. In adipocytes, it is essential for cell viability and regulates cAMP levels. Its expression in enteric neurons is negatively regulated by intestinal inflammation."},"narrative":{"mechanistic_narrative":"MRGPRF (GPR168/MrgprF) is an orphan Mas-related GPCR that acts as a tumor suppressor in cutaneous melanoma, where it reduces assembly of the PI3K p101/p110γ complex, limiting PIP2-to-PIP3 conversion and dampening downstream PI3K/Akt signaling; forced expression curbs proliferation, migration, and xenograft growth while knockdown is pro-tumorigenic, and the phenotype is reversed by the Akt agonist SC79 [PMID:35504869]. This anti-proliferative output is mediated through receptor functionality, as a blocking anti-GPR168 antibody restores melanoma proliferation and the Akt/GSK-3β/β-catenin/Myc/CyclinD1/CDK4 axis [PMID:38805406]. MRGPRF protein abundance is set post-translationally by USP45, which binds the receptor N-terminus through its catalytic domain and removes K63-linked ubiquitin chains to stabilize the protein, establishing a USP45→MRGPRF deubiquitination-dependent tumor-suppressor axis [PMID:40788071]. In the nervous system MRGPRF modulates somatosensation: global knockout reduces thermal and chemical nociceptive responses and downregulates DRG nociceptive genes including Nav1.7/1.8/1.9, c-fos, and Runx1 [PMID:40033362], and its dorsal-horn protein level—and consequent itch sensitivity—is governed by gut-microbiota-driven RNA m6A methylation acting through an ETS1/FTO axis [PMID:40289281]. The receptor shows restricted peripheral expression in entero-endocrine cells of human ileum and colon [PMID:37314493] and in enteric neuronal subpopulations whose numbers fall during intestinal inflammation [PMID:21912971]. A defined endogenous ligand has not been identified in the available corpus, and heterologous dog MRGPRF does not mediate histamine-releasing or fluoroquinolone-induced calcium signaling [PMID:32999394].","teleology":[{"year":2008,"claim":"First hint of regulatory wiring around MRGPRF in sensory neurons came from observing that loss of a related Mrg receptor selectively elevates MrgF, raising the question of how Mrg-family members cross-regulate.","evidence":"MrgE knockout mice with DRG gene expression profiling","pmids":["18197975"],"confidence":"Low","gaps":["single correlative expression observation with no mechanistic follow-up on MRGPRF","no causal link between MrgE loss and MrgF regulation established","no functional consequence tested"]},{"year":2011,"claim":"Established where MRGPRF acts in the gut and that its expression is dynamically controlled, showing it marks enteric neuronal subtypes whose numbers decline under inflammation.","evidence":"RT-PCR, qPCR, and IHC on murine ileum in schistosomiasis and chemical ileitis models","pmids":["21912971"],"confidence":"Medium","gaps":["mechanism linking inflammation to MrgF downregulation unknown","functional role of MrgF in enteric neurons not tested","no ligand or signaling readout"]},{"year":2020,"claim":"Addressed whether MRGPRF mediates the pseudo-allergic responses typical of its family, finding it does not signal to calcium in response to histamine-releasing agents, narrowing its candidate functions.","evidence":"Intracellular Ca2+ mobilization assay in HEK293 cells transfected with dog MRGPRF versus MRGPRX2","pmids":["32999394"],"confidence":"Medium","gaps":["negative result does not identify the true ligand or coupling","tested only dog ortholog in a heterologous system","did not survey other second messengers"]},{"year":2022,"claim":"Defined MRGPRF's first concrete molecular function: a melanoma tumor suppressor that blocks PI3K complex assembly and Akt signaling, answering what cellular process the receptor controls.","evidence":"Reciprocal overexpression/knockdown in melanoma lines, PI3K subunit Co-IP, Akt-agonist (SC79) epistasis rescue, and xenografts","pmids":["35504869"],"confidence":"High","gaps":["how a GPCR mechanistically reduces p101/p110γ assembly is unresolved","no endogenous ligand linking receptor activation to PI3K inhibition","G-protein coupling not defined"]},{"year":2024,"claim":"Confirmed that MRGPRF's anti-tumor effect requires intact receptor function and traced its output through the Akt/β-catenin/cell-cycle axis.","evidence":"GPR168 overexpression in B16-F10 cells, xenografts, western blot of pathway components, and blocking anti-GPR168 antibody rescue","pmids":["38805406"],"confidence":"Medium","gaps":["antibody blockade does not identify the natural agonist","single lab","does not connect to the PI3K complex mechanism biochemically"]},{"year":2025,"claim":"Identified the upstream control of MRGPRF protein stability, showing USP45 deubiquitinates K63 chains on the receptor N-terminus to maintain its tumor-suppressor levels.","evidence":"USP screen, Co-IP/pulldown, domain mapping, K63-linkage ubiquitination assays, and MRGPRF-rescue epistasis with xenografts","pmids":["40788071"],"confidence":"High","gaps":["E3 ligase placing K63 chains on MRGPRF unknown","whether deubiquitination alters trafficking versus degradation not resolved","USP45-MRGPRF axis tested only in melanoma"]},{"year":2025,"claim":"Demonstrated MRGPRF's causal role in somatosensation, with knockout blunting nociception and a gut-microbiota/m6A pathway tuning its dorsal-horn levels to set itch sensitivity.","evidence":"MrgF global knockout with behavioral nociception tests and DRG qPCR; germ-free/antibiotic mice, m6A mapping, ETS1/FTO analysis, and metabolite (ALC) rescue","pmids":["40033362","40289281"],"confidence":"Medium","gaps":["signaling mechanism by which MRGPRF modulates neuronal excitability unknown","ligand activating MRGPRF in DRG/dorsal horn not identified","link between m6A pathway and behavior is correlative across tissues"]},{"year":2023,"claim":"Extended MRGPRF function beyond tumor and nerve to metabolic tissue, indicating it supports adipocyte viability and regulates cAMP.","evidence":"GPCRome screen of adipose tissue with knockdown/overexpression in a (pre)adipocyte model and cAMP measurement","pmids":["37766984"],"confidence":"Low","gaps":["minimal methodological detail and no mutagenesis or reconstitution","G-protein coupling to cAMP not directly demonstrated","single lab, not independently replicated"]},{"year":null,"claim":"The endogenous ligand and G-protein coupling of MRGPRF remain unknown, leaving open how receptor activation is mechanistically transduced into PI3K suppression, cAMP regulation, and neuronal modulation.","evidence":"No deorphanization or defined transducer reported in the timeline","pmids":[],"confidence":"Low","gaps":["no endogenous agonist identified","G-protein/second-messenger coupling undefined","structural basis of signaling unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8,5]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,2]}],"complexes":[],"partners":["USP45"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96AM1","full_name":"Mas-related G-protein coupled receptor member F","aliases":["G-protein coupled receptor 140","G-protein coupled receptor 168"],"length_aa":343,"mass_kda":38.2,"function":"Orphan receptor. May bind to a neuropeptide and may regulate nociceptor function and/or development, including the sensation or modulation of pain (By similarity)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q96AM1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MRGPRF","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MRGPRF","total_profiled":1310},"omim":[{"mim_id":"607233","title":"MAS-RELATED G PROTEIN-COUPLED RECEPTOR FAMILY, MEMBER F; MRGPRF","url":"https://www.omim.org/entry/607233"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Plasma 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1","ntpm":169.7},{"tissue":"intestine","ntpm":162.2}],"url":"https://www.proteinatlas.org/search/MRGPRF"},"hgnc":{"alias_symbol":["MGC21621","mrgF"],"prev_symbol":["GPR168","GPR140"]},"alphafold":{"accession":"Q96AM1","domains":[{"cath_id":"1.20.1070.10","chopping":"47-227","consensus_level":"high","plddt":91.2464,"start":47,"end":227}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96AM1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96AM1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96AM1-F1-predicted_aligned_error_v6.png","plddt_mean":79.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MRGPRF","jax_strain_url":"https://www.jax.org/strain/search?query=MRGPRF"},"sequence":{"accession":"Q96AM1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96AM1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96AM1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96AM1"}},"corpus_meta":[{"pmid":"16906560","id":"PMC_16906560","title":"Comprehensive 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one","url":"https://pubmed.ncbi.nlm.nih.gov/38805406","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":13107,"output_tokens":2850,"usd":0.041036,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10137,"output_tokens":3077,"usd":0.063805,"stage2_stop_reason":"end_turn"},"total_usd":0.104841,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2022,\n      \"finding\": \"MrgprF reduces PI3K complex formation between p101 and p110γ subunits, thereby inhibiting the critical step of PIP2-to-PIP3 conversion and suppressing downstream PI3K/Akt signaling in cutaneous melanoma cells. Forced expression inhibits tumor cell proliferation, migration, xenograft growth, and metastasis; knockdown promotes proliferation and transformation. The Akt-specific agonist SC79 reverses these effects.\",\n      \"method\": \"Forced expression and knockdown in melanoma cell lines; xenograft tumor models; co-immunoprecipitation/pulldown of PI3K subunits; rescue with Akt agonist SC79; in vitro and in vivo functional assays\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional assays (OE + KD), epistasis rescue with Akt agonist, in vivo xenograft, and PI3K subunit interaction data in one study; replicated directionally by a second independent study (PMID:38805406)\",\n      \"pmids\": [\"35504869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GPR168 (MRGPRF) overexpression in mouse melanoma B16-F10 cells inhibits proliferation and migration through the Akt signaling pathway, evidenced by decreased p-Akt, p-GSK-3β, β-catenin, Myc, CyclinD1, and CDK4. A blocking anti-GPR168 polyclonal antibody restores proliferation and migration, confirming that signaling depends on GPR168 receptor functionality.\",\n      \"method\": \"GPR168 overexpression in B16-F10 cells; xenograft tumor model; western blot for pathway components; rescue with anti-GPR168 blocking antibody\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean OE with xenograft plus antibody rescue experiment, single lab, consistent with PMID:35504869\",\n      \"pmids\": [\"38805406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP45 directly binds the N-terminal domain of MRGPRF via its catalytic domain and stabilizes MRGPRF protein by removing K63-linked ubiquitination. USP45 knockdown destabilizes MRGPRF and promotes melanoma cell malignancy; these effects are rescued by MRGPRF overexpression, placing USP45 upstream of MRGPRF in a deubiquitination-dependent tumor suppressor axis.\",\n      \"method\": \"Screening of 40 USPs; Co-IP and pulldown of USP45-MRGPRF interaction; domain mapping (N-terminal MRGPRF); ubiquitination assays (K63-linkage); USP45 OE/KD functional assays; MRGPRF depletion/rescue epistasis; xenograft mouse model\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — biochemical deubiquitination assay with linkage specificity, domain mapping, reciprocal epistasis rescue, and in vivo xenograft in a single rigorous study\",\n      \"pmids\": [\"40788071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In the mouse spinal cord, MrgprF (MRGPRF) protein levels are regulated by RNA m6A methylation of MrgprF mRNA. Gut microbiota depletion reduces expression of the m6A demethylase FTO (via inactivation of the transcription factor ETS1 at the Fto promoter), leading to increased m6A methylation on MrgprF mRNA and decreased MRGPRF protein in the dorsal horn, resulting in reduced itch sensation. Oral administration of acetyl-L-carnitine (ALC) from Bacteroides fragilis restores FTO and MRGPRF levels.\",\n      \"method\": \"Antibiotic-treated and germ-free mouse models; oral gavage with B. fragilis and ALC; m6A site mapping on MrgprF mRNA; FTO expression analysis; ETS1 chromatin binding at Fto promoter; behavioral itch assays\",\n      \"journal\": \"Gut microbes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic pathway defined with multiple methods (m6A mapping, transcription factor binding, metabolite rescue) in a single lab study\",\n      \"pmids\": [\"40289281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Global knockout of MrgF (MRGPRF) in mice reduces pain-related behavioral responses to thermal and chemical (formalin) nociceptive stimuli, without affecting motor coordination or cerebellar histology. In MrgF-/- DRG, expression of nociceptive modulation genes c-fos, Runx1, Nav1.7, Nav1.8, and Nav1.9 is downregulated, placing MrgF in a pathway that modulates pain sensitivity.\",\n      \"method\": \"MrgF global knockout mouse model; behavioral tests (hot plate, tail flick, formalin, rotarod, pole, balance beam, treadmill); qPCR for nociceptive genes in DRG; cerebellar histology\",\n      \"journal\": \"Hereditas\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO model with specific pain phenotype and molecular readout (nociceptive gene downregulation in DRG), single lab\",\n      \"pmids\": [\"40033362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MrgF (MRGPRF) is expressed in distinct enteric neuronal subpopulations in the murine ileum, including sensory, secretomotor, and vasodilator neurons, and in nerve fibers of the tunica muscularis and lamina propria. The number of MrgF-expressing enteric neurons is significantly reduced during intestinal schistosomiasis and chemically induced ileitis, indicating that inflammation negatively regulates MrgF expression in the enteric nervous system.\",\n      \"method\": \"RT-PCR, quantitative PCR, and immunohistochemistry on murine ileum from control, schistosomiasis, and chemically induced ileitis models\",\n      \"journal\": \"Histochemistry and cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization by IHC with quantified loss-of-function in two independent inflammation models, single lab\",\n      \"pmids\": [\"21912971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Deletion of MrgE in mice results in upregulation of MrgF (MRGPRF) expression in DRG as the sole significantly affected Mrg family member, suggesting a regulatory relationship between MrgE and MrgF expression.\",\n      \"method\": \"MrgE knockout mice; gene expression analysis of Mrg family members and 84 sensory neuron genes in DRG\",\n      \"journal\": \"Molecular pain\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single gene expression observation from a KO study focused on MrgE, single lab, no mechanistic follow-up on MRGPRF\",\n      \"pmids\": [\"18197975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MRGPRF is essential for adipocyte viability and regulates intracellular cAMP levels in a (pre)adipocyte cell model, as determined by functional knockdown/overexpression experiments in the context of a GPCRome screen of adipose tissue.\",\n      \"method\": \"RNA-seq database (FATTLAS); functional assays in (pre)adipocyte cell model with MRGPRF manipulation; cAMP level measurement\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, limited methodological detail in abstract, no mutagenesis or reconstitution; cAMP regulation is a novel functional finding but minimally characterized\",\n      \"pmids\": [\"37766984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MRGPRF protein is specifically expressed by entero-endocrine cells (EECs) in the mucosal biopsies of human terminal ileum and sigmoid colon, making it the only detectable MRGPR family member at these mucosal sites.\",\n      \"method\": \"RT-PCR on human GI mucosal biopsies; immunohistochemical staining for cell-type identification\",\n      \"journal\": \"Cell and tissue research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization with two methods (mRNA + protein IHC), replicated across two gut regions, identifies specific cell type\",\n      \"pmids\": [\"37314493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In functional calcium mobilization assays, HEK293 cells transfected with dog MRGPRF did not respond to histamine-releasing agents (including fluoroquinolones), in contrast to dog or human MRGPRX2, indicating that MRGPRF does not mediate drug-induced pseudo-allergic calcium signaling in this heterologous expression system.\",\n      \"method\": \"Intracellular Ca2+ mobilization assay in HEK293 cells transiently transfected with dog MRGPRF; comparison with MRGPRX2, MRGPRD, MRGPRG\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — direct in vitro calcium assay, negative result explicitly reported, single lab\",\n      \"pmids\": [\"32999394\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MRGPRF (GPR168/MrgprF) is an orphan Mas-related GPCR that functions as a tumor suppressor in melanoma by reducing PI3K p101/p110γ complex formation and suppressing PI3K/Akt signaling; its stability is controlled by USP45-mediated deubiquitination of K63-linked ubiquitin chains on its N-terminus. In the nervous system, MRGPRF modulates pain sensitivity and itch via its expression in DRG and spinal dorsal horn neurons—where protein levels are epigenetically regulated by gut-microbiota-driven RNA m6A methylation through the FTO/ETS1 axis. In the gut, it is specifically localized to entero-endocrine cells of the human ileum and colon. In adipocytes, it is essential for cell viability and regulates cAMP levels. Its expression in enteric neurons is negatively regulated by intestinal inflammation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MRGPRF (GPR168/MrgprF) is an orphan Mas-related GPCR that acts as a tumor suppressor in cutaneous melanoma, where it reduces assembly of the PI3K p101/p110\\u03b3 complex, limiting PIP2-to-PIP3 conversion and dampening downstream PI3K/Akt signaling; forced expression curbs proliferation, migration, and xenograft growth while knockdown is pro-tumorigenic, and the phenotype is reversed by the Akt agonist SC79 [#0]. This anti-proliferative output is mediated through receptor functionality, as a blocking anti-GPR168 antibody restores melanoma proliferation and the Akt/GSK-3\\u03b2/\\u03b2-catenin/Myc/CyclinD1/CDK4 axis [#1]. MRGPRF protein abundance is set post-translationally by USP45, which binds the receptor N-terminus through its catalytic domain and removes K63-linked ubiquitin chains to stabilize the protein, establishing a USP45\\u2192MRGPRF deubiquitination-dependent tumor-suppressor axis [#2]. In the nervous system MRGPRF modulates somatosensation: global knockout reduces thermal and chemical nociceptive responses and downregulates DRG nociceptive genes including Nav1.7/1.8/1.9, c-fos, and Runx1 [#4], and its dorsal-horn protein level\\u2014and consequent itch sensitivity\\u2014is governed by gut-microbiota-driven RNA m6A methylation acting through an ETS1/FTO axis [#3]. The receptor shows restricted peripheral expression in entero-endocrine cells of human ileum and colon [#8] and in enteric neuronal subpopulations whose numbers fall during intestinal inflammation [#5]. A defined endogenous ligand has not been identified in the available corpus, and heterologous dog MRGPRF does not mediate histamine-releasing or fluoroquinolone-induced calcium signaling [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"First hint of regulatory wiring around MRGPRF in sensory neurons came from observing that loss of a related Mrg receptor selectively elevates MrgF, raising the question of how Mrg-family members cross-regulate.\",\n      \"evidence\": \"MrgE knockout mice with DRG gene expression profiling\",\n      \"pmids\": [\"18197975\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"single correlative expression observation with no mechanistic follow-up on MRGPRF\", \"no causal link between MrgE loss and MrgF regulation established\", \"no functional consequence tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established where MRGPRF acts in the gut and that its expression is dynamically controlled, showing it marks enteric neuronal subtypes whose numbers decline under inflammation.\",\n      \"evidence\": \"RT-PCR, qPCR, and IHC on murine ileum in schistosomiasis and chemical ileitis models\",\n      \"pmids\": [\"21912971\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"mechanism linking inflammation to MrgF downregulation unknown\", \"functional role of MrgF in enteric neurons not tested\", \"no ligand or signaling readout\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Addressed whether MRGPRF mediates the pseudo-allergic responses typical of its family, finding it does not signal to calcium in response to histamine-releasing agents, narrowing its candidate functions.\",\n      \"evidence\": \"Intracellular Ca2+ mobilization assay in HEK293 cells transfected with dog MRGPRF versus MRGPRX2\",\n      \"pmids\": [\"32999394\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"negative result does not identify the true ligand or coupling\", \"tested only dog ortholog in a heterologous system\", \"did not survey other second messengers\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined MRGPRF's first concrete molecular function: a melanoma tumor suppressor that blocks PI3K complex assembly and Akt signaling, answering what cellular process the receptor controls.\",\n      \"evidence\": \"Reciprocal overexpression/knockdown in melanoma lines, PI3K subunit Co-IP, Akt-agonist (SC79) epistasis rescue, and xenografts\",\n      \"pmids\": [\"35504869\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"how a GPCR mechanistically reduces p101/p110\\u03b3 assembly is unresolved\", \"no endogenous ligand linking receptor activation to PI3K inhibition\", \"G-protein coupling not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Confirmed that MRGPRF's anti-tumor effect requires intact receptor function and traced its output through the Akt/\\u03b2-catenin/cell-cycle axis.\",\n      \"evidence\": \"GPR168 overexpression in B16-F10 cells, xenografts, western blot of pathway components, and blocking anti-GPR168 antibody rescue\",\n      \"pmids\": [\"38805406\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"antibody blockade does not identify the natural agonist\", \"single lab\", \"does not connect to the PI3K complex mechanism biochemically\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified the upstream control of MRGPRF protein stability, showing USP45 deubiquitinates K63 chains on the receptor N-terminus to maintain its tumor-suppressor levels.\",\n      \"evidence\": \"USP screen, Co-IP/pulldown, domain mapping, K63-linkage ubiquitination assays, and MRGPRF-rescue epistasis with xenografts\",\n      \"pmids\": [\"40788071\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase placing K63 chains on MRGPRF unknown\", \"whether deubiquitination alters trafficking versus degradation not resolved\", \"USP45-MRGPRF axis tested only in melanoma\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated MRGPRF's causal role in somatosensation, with knockout blunting nociception and a gut-microbiota/m6A pathway tuning its dorsal-horn levels to set itch sensitivity.\",\n      \"evidence\": \"MrgF global knockout with behavioral nociception tests and DRG qPCR; germ-free/antibiotic mice, m6A mapping, ETS1/FTO analysis, and metabolite (ALC) rescue\",\n      \"pmids\": [\"40033362\", \"40289281\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"signaling mechanism by which MRGPRF modulates neuronal excitability unknown\", \"ligand activating MRGPRF in DRG/dorsal horn not identified\", \"link between m6A pathway and behavior is correlative across tissues\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended MRGPRF function beyond tumor and nerve to metabolic tissue, indicating it supports adipocyte viability and regulates cAMP.\",\n      \"evidence\": \"GPCRome screen of adipose tissue with knockdown/overexpression in a (pre)adipocyte model and cAMP measurement\",\n      \"pmids\": [\"37766984\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"minimal methodological detail and no mutagenesis or reconstitution\", \"G-protein coupling to cAMP not directly demonstrated\", \"single lab, not independently replicated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The endogenous ligand and G-protein coupling of MRGPRF remain unknown, leaving open how receptor activation is mechanistically transduced into PI3K suppression, cAMP regulation, and neuronal modulation.\",\n      \"evidence\": \"No deorphanization or defined transducer reported in the timeline\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"no endogenous agonist identified\", \"G-protein/second-messenger coupling undefined\", \"structural basis of signaling unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"USP45\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}