{"gene":"NPY4R","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":1995,"finding":"The human Y4 receptor (NPY4R) was cloned from a placental genomic library by homology screening with Y1 transmembrane probes. It encodes a 375-amino acid GPCR with highest affinity for human pancreatic polypeptide (PP; pKi ~10.25), followed by PYY and NPY. When expressed in LMTK- cells, human PP activation decreased cAMP and increased intracellular Ca2+, demonstrating coupling to both Gi and Gq-like pathways.","method":"cDNA cloning, radioligand binding (125I-PYY), cAMP and Ca2+ assays in transfected cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — original cloning with functional reconstitution using multiple orthogonal assays (binding, cAMP, Ca2+)","pmids":["7592911"],"is_preprint":false},{"year":1995,"finding":"An independently cloned human NPY4R (designated PP1) was shown to bind PP with extremely high affinity (Ki ~14 pM), PYY with 1.44 nM, and NPY with 9.9 nM, establishing PP as the primary endogenous ligand. In stably transfected CHO cells, the receptor inhibited forskolin-stimulated cAMP synthesis. Northern blot detected mRNA in colon, small intestine, pancreas, and prostate.","method":"Homology cloning, radioligand binding, cAMP assay in stable CHO transfectants, Northern blot","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — independent replication of cloning with functional cAMP assay and expression profiling","pmids":["7493937"],"is_preprint":false},{"year":1996,"finding":"Human and rat NPY4R cDNAs were cloned and expressed in HEK293 cells. The human receptor bound 125I-labeled human PP with Kd = 2.8 nM; rank-order potency was human PP = bovine PP > [Pro34]PYY > rat PP > hPYY = hNPY. Northern blot showed mRNA most abundantly in skeletal muscle, with lesser expression in lung and brain. The rat ortholog shares 74% amino acid identity with human Y4R.","method":"PCR cloning, stable transfection, radioligand binding, Northern blot","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — functional reconstitution in mammalian cells with quantitative binding characterization, independent replication of human Y4R cloning","pmids":["8643460"],"is_preprint":false},{"year":1997,"finding":"[125I]Leu31,Pro34-PYY was identified as a high-affinity radioligand for rat PP1/Y4 receptor expressed in cell lines, demonstrating that this putative Y1-selective ligand also binds Y4 with high affinity. However, in rat brain sections this ligand does not label interpeduncular nucleus sites (high in [125I]-bPP binding), indicating heterogeneity among PP-recognizing receptors in brain versus peripheral tissues.","method":"Radioligand binding in transfected cell lines and rat brain autoradiography","journal":"Peptides","confidence":"Medium","confidence_rationale":"Tier 2 — direct binding assays in cells expressing cloned receptor plus brain section autoradiography, single lab","pmids":["9145427"],"is_preprint":false},{"year":1997,"finding":"The human Y4 receptor gene (PPYR1) was localized to chromosome 10q11.2 by nonisotopic in situ hybridization, and the mouse Y4 gene (Ppyr1) to chromosome 14 by interspecific backcross mapping. The gene contains no introns within its coding region, suggesting origin via an RNA-mediated transpositional event from an ancestral Y1-like receptor.","method":"Fluorescence in situ hybridization (FISH), interspecific mouse backcross mapping, genomic sequencing","journal":"Genomics","confidence":"High","confidence_rationale":"Tier 2 — direct chromosomal mapping with multiple independent methods (FISH + genetic mapping)","pmids":["9441761"],"is_preprint":false},{"year":2000,"finding":"Human Y4 receptor stably expressed in CHO cells showed one class of binding site (Kd = 0.26 nM for 125I-hPP). Signal transduction via Gi protein was demonstrated by pertussis toxin abolition of hPP-induced cAMP inhibition. Critically, 24-hour exposure to 10 nM hPP produced no change in binding capacity, affinity, or receptor potency, and immunofluorescence showed no receptor internalization, establishing that Y4R is resistant to agonist-promoted desensitization and internalization.","method":"Radioligand binding, cAMP assay, pertussis toxin treatment, immunofluorescence microscopy in stable CHO transfectants","journal":"The Journal of pharmacology and experimental therapeutics","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (binding, cAMP, pertussis toxin, immunofluorescence) in single lab demonstrating resistance to desensitization","pmids":["10640301"],"is_preprint":false},{"year":2003,"finding":"Using Y2(-/-)Y4(-/-) double knockout mice, synergistic interactions between Y2 and Y4 receptor pathways were demonstrated. Y4 single knockout produced a lean phenotype and elevated plasma PP. Despite hyperphagia, Y2/Y4 double knockouts showed markedly reduced adiposity and threefold increased bone volume with enhanced osteoblastic activity—effects more pronounced than Y2 single knockout alone—establishing that Y2 and Y4 pathways cooperate to regulate adiposity and bone mass.","method":"Genetic epistasis using double knockout mice, body composition analysis, bone histomorphometry, plasma hormone measurements","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis in double knockout mice with multiple quantitative phenotypic readouts, demonstrates pathway cooperation","pmids":["12861009"],"is_preprint":false},{"year":2002,"finding":"In isolated human colon mucosa in Ussing chambers, basolateral application of human PP (hPP) inhibited electrogenic ion transport. This inhibitory response was insensitive to Y1 antagonists (BIBP3226, BIBO3304) and Y2 antagonist (BIIE0246), and was not blocked by tetrodotoxin, establishing that Y4 receptors are the predominantly post-junctional mediators of PP-induced inhibition of colonic electrolyte secretion.","method":"Ussing chamber electrophysiology with selective receptor antagonists and TTX in human colonic tissue","journal":"British journal of pharmacology","confidence":"High","confidence_rationale":"Tier 2 — pharmacological dissection with selective antagonists in native human tissue, establishing Y4R-specific post-junctional signaling","pmids":["11906964"],"is_preprint":false},{"year":2014,"finding":"PPYR1 (Y4R) protein was detected by immunoblotting, qRT-PCR (enriched in FACS-sorted glucagon alpha-cell fraction), and immunohistochemistry on pancreatic sections in both mouse and human islets. Direct PP exposure to isolated mouse islets inhibited glucagon release, demonstrating that Y4R on alpha-cells mediates PP-dependent suppression of glucagon secretion.","method":"Immunoblotting, qRT-PCR on sorted islet cells, immunohistochemistry, ex vivo glucagon secretion assay in isolated islets","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (protein, mRNA, IHC, functional secretion assay) in mouse and human tissue, directly linking Y4R to glucagon inhibition","pmids":["25445712"],"is_preprint":false},{"year":2013,"finding":"Double-cycle mutagenesis of hY4R combined with computational homology modeling of the hY4R–hPP complex identified residues Tyr2.64, Asp2.68, Asn6.55, Asn7.32, and Phe7.35 as critical for receptor activation by PP. The top of TM2 and TM6-TM7 form the core peptide-binding pocket, with Tyr2.64 making hydrophobic contact with Tyr27 of PP, Asn7.32 hydrogen-bonding Arg33 of PP, and Phe7.35 interacting with PP positions 33 and 36.","method":"Site-directed mutagenesis, radioligand binding, double-cycle mutagenesis, computational homology modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — systematic mutagenesis with double-cycle analysis and structural modeling, identifying specific molecular contacts in the binding pocket","pmids":["24375409"],"is_preprint":false},{"year":2016,"finding":"The C-terminal motif 7.78EESEHLPLSTVHTEVSKGS7.96 of hY4R was identified as essential for receptor internalization and arrestin-3 recruitment. Deletion and point-mutation analysis showed glutamate, threonine, and serine residues within this motif are key determinants. This was established using fluorescence microscopy, binding assays, inositol turnover, and BRET assays, revealing the molecular basis for Y4R endocytosis.","method":"C-terminal deletion mutants, site-directed mutagenesis, fluorescence microscopy, BRET assays, binding assays, inositol turnover in transfected cells","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (BRET, microscopy, binding, signaling) with systematic mutagenesis defining internalization motif","pmids":["27818291"],"is_preprint":false},{"year":2011,"finding":"Human Y4R and Y2R were functionally reconstituted in Sf9 insect cells using baculovirus co-expression with Gi/Go proteins and RGS proteins. Steady-state GTPase assays showed hY4R couples optimally to Gαi2 and Gαo with RGS4. N-glycosylation was found to be essential for hY4R function, as tunicamycin treatment abolished receptor activity. Na+ sensitivity assays revealed moderate constitutive activity of hY4R.","method":"Baculovirus expression in Sf9 cells, steady-state GTPase assays, tunicamycin glycosylation inhibition, ion sensitivity assays","journal":"Journal of receptor and signal transduction research","confidence":"Medium","confidence_rationale":"Tier 2 — functional reconstitution with multiple G-protein combinations, single lab","pmids":["21745157"],"is_preprint":false},{"year":2021,"finding":"The first selective Y4R allosteric antagonist, (S)-VU0637120, was identified and characterized. In vitro, ex vivo, and in silico studies demonstrated it selectively inhibits native Y4R function by binding an allosteric site located below the endogenous PP-binding pocket within the core of the Y4R transmembrane domains, distinct from the orthosteric binding site.","method":"In vitro binding assays, ex vivo functional assays, computational modeling/docking, selectivity profiling against Y1R/Y2R/Y5R","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — multiple functional and computational approaches identifying allosteric site, single lab","pmids":["33595306"],"is_preprint":false},{"year":2024,"finding":"Multiplexed screening of 215 GPCRs against all three RAMPs (receptor activity-modifying proteins) using suspension bead array immunoassays identified NPY4R (Y4R) among the 122 GPCRs showing strong evidence for interaction with at least one RAMP, identifying a potential regulatory complex for Y4R trafficking/pharmacology.","method":"Dual epitope-tagged GPCR library co-expressed with RAMPs 1-3, multiplexed suspension bead array immunoassays in three cell lines","journal":"Science advances","confidence":"Low","confidence_rationale":"Tier 3 — high-throughput co-expression screen, no functional validation of Y4R-RAMP complex specifically","pmids":["39083597"],"is_preprint":false},{"year":2016,"finding":"CNV analysis in obese children versus lean controls showed significantly higher frequency of NPY4R-containing 10q11.22 CNV loss in obese patients (p=0.0003) and CNV gain in controls (p=0.031). Mutation screening identified two rare non-synonymous heterozygous variants found only in obese patients for which receptor dysfunction was experimentally demonstrated.","method":"CNV analysis, coding region mutation screening, functional receptor assays for identified variants","journal":"Obesity (Silver Spring, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 — functional receptor assays confirmed dysfunction of identified variants, supporting causal role of NPY4R loss-of-function in obesity","pmids":["26921218"],"is_preprint":false}],"current_model":"NPY4R (Y4 receptor) is a Gi/o-coupled GPCR that serves as the primary receptor for pancreatic polypeptide (PP), signaling through pertussis toxin-sensitive Gi proteins to inhibit cAMP production; its binding pocket is defined by TM2 (Tyr2.64) and TM6-TM7 (Asn6.55, Asn7.32, Phe7.35) residues, it is uniquely resistant to agonist-promoted desensitization and internalization (unless driven through a C-terminal EESEHLPLSTVHTEVSKGS motif for arrestin-3 recruitment), requires N-glycosylation for function, mediates PP-dependent inhibition of glucagon secretion from pancreatic alpha-cells, and cooperates synergistically with Y2 receptor pathways to regulate adiposity and bone mass."},"narrative":{"teleology":[{"year":1995,"claim":"Cloning and functional reconstitution of NPY4R established it as a Gi-coupled GPCR with highest affinity for pancreatic polypeptide, resolving the molecular identity of the PP-preferring neuropeptide Y receptor subtype.","evidence":"Independent cloning from human genomic/cDNA libraries with radioligand binding, cAMP inhibition, and Ca²⁺ assays in transfected LMTK⁻ and CHO cells; Northern blot tissue profiling","pmids":["7592911","7493937"],"confidence":"High","gaps":["Structural basis of PP selectivity over NPY/PYY unknown","In vivo physiological roles not yet addressed","G-protein coupling specificity (Gi subtypes) not resolved"]},{"year":1996,"claim":"Cloning of the rat ortholog and cross-species binding comparison showed 74% amino acid identity with human Y4R and confirmed species-dependent variation in PP affinity, indicating evolutionary divergence in ligand recognition.","evidence":"PCR cloning, stable HEK293 transfection, radioligand binding of human and rat receptors","pmids":["8643460"],"confidence":"High","gaps":["Residues responsible for species selectivity not identified","Functional consequences of sequence divergence not tested"]},{"year":1997,"claim":"Chromosomal mapping placed the intronless NPY4R gene at human 10q11.2 and mouse chromosome 14, suggesting origin via retrotranspositional duplication from an ancestral Y1-like receptor.","evidence":"FISH and interspecific mouse backcross mapping","pmids":["9441761"],"confidence":"High","gaps":["Regulatory elements controlling tissue-specific expression not characterized"]},{"year":2000,"claim":"Demonstration that Y4R is uniquely resistant to agonist-promoted desensitization and internalization—with preserved binding and signaling after prolonged PP exposure—distinguished it from other NPY receptor subtypes and raised questions about its regulatory mechanisms.","evidence":"Radioligand binding kinetics, cAMP assay, pertussis toxin block, and immunofluorescence after 24-h agonist exposure in stable CHO transfectants","pmids":["10640301"],"confidence":"High","gaps":["Molecular determinants of desensitization resistance not identified","Phosphorylation and arrestin engagement not tested"]},{"year":2002,"claim":"Pharmacological dissection in native human colonic tissue established Y4R as the post-junctional mediator of PP-induced inhibition of electrolyte secretion, defining a gastrointestinal effector role.","evidence":"Ussing chamber electrophysiology with selective Y1, Y2, and TTX antagonists on human colon mucosa","pmids":["11906964"],"confidence":"High","gaps":["Downstream ion channel/transporter targets not identified","Contribution of Y4R to colonic pathophysiology not tested"]},{"year":2003,"claim":"Y2/Y4 double-knockout mice revealed synergistic cooperation between Y2 and Y4 receptor pathways in controlling adiposity and bone mass, with double knockouts showing markedly reduced fat and threefold increased bone volume despite hyperphagia.","evidence":"Genetic epistasis using single and double knockout mice with body composition analysis, bone histomorphometry, and plasma hormone measurements","pmids":["12861009"],"confidence":"High","gaps":["Central versus peripheral site of action for adiposity/bone regulation unresolved","Downstream signaling pathways mediating bone effects unknown"]},{"year":2011,"claim":"Functional reconstitution with defined G-protein subunits showed Y4R couples optimally to Gαi2 and Gαo with RGS4, requires N-glycosylation for function, and exhibits moderate constitutive activity.","evidence":"Baculovirus co-expression of Y4R with Gi/Go subtypes and RGS proteins in Sf9 cells; steady-state GTPase assays; tunicamycin treatment","pmids":["21745157"],"confidence":"Medium","gaps":["Constitutive activity not confirmed in mammalian cells","Glycosylation sites contributing to function not mapped"]},{"year":2013,"claim":"Systematic double-cycle mutagenesis and homology modeling defined the PP-binding pocket, identifying Tyr2.64, Asn6.55, Asn7.32, and Phe7.35 as critical contacts and explaining the molecular basis of PP selectivity.","evidence":"Site-directed and double-cycle mutagenesis, radioligand binding, and computational modeling of the Y4R–PP complex","pmids":["24375409"],"confidence":"High","gaps":["No experimental 3D structure available","Activation-state conformational changes not resolved"]},{"year":2014,"claim":"Detection of Y4R protein and mRNA specifically in pancreatic alpha-cells, coupled with PP-mediated inhibition of glucagon secretion from isolated islets, established an endocrine axis for PP–Y4R signaling in glucose homeostasis.","evidence":"Immunoblotting, qRT-PCR on FACS-sorted islet fractions, immunohistochemistry, ex vivo glucagon secretion assay in mouse and human tissue","pmids":["25445712"],"confidence":"High","gaps":["In vivo relevance using Y4R-knockout islets not tested","Signaling pathway linking Y4R activation to glucagon suppression not delineated"]},{"year":2016,"claim":"Identification of the C-terminal motif 7.78EESEHLPLSTVHTEVSKGS7.96 as the molecular determinant for arrestin-3 recruitment and receptor internalization resolved why Y4R normally resists desensitization and how endocytosis can be engaged.","evidence":"C-terminal deletion and point mutagenesis with BRET, fluorescence microscopy, binding, and inositol turnover assays","pmids":["27818291"],"confidence":"High","gaps":["Phosphorylation events upstream of arrestin binding not mapped","Physiological context triggering this internalization pathway unknown"]},{"year":2016,"claim":"CNV losses at 10q11.22 encompassing NPY4R and functionally validated rare loss-of-function coding variants were enriched in obese children, linking NPY4R haploinsufficiency to human obesity.","evidence":"CNV analysis and mutation screening in obese vs. lean children with functional receptor assays for identified variants","pmids":["26921218"],"confidence":"Medium","gaps":["Small cohort size limits generalizability","Mechanism connecting Y4R loss-of-function to increased adiposity not defined","Not replicated in independent populations"]},{"year":2021,"claim":"Discovery of the first selective Y4R allosteric antagonist (S)-VU0637120, binding a site within the transmembrane core below the orthosteric pocket, provided a pharmacological tool to probe Y4R function independently of PP competition.","evidence":"In vitro binding, ex vivo functional assays, computational docking, selectivity profiling against Y1R/Y2R/Y5R","pmids":["33595306"],"confidence":"Medium","gaps":["In vivo efficacy and pharmacokinetics not reported","Allosteric site not structurally resolved"]},{"year":null,"claim":"Key unresolved questions include the lack of an experimental 3D structure of Y4R (free or PP-bound), the signaling cascades downstream of Gi that link Y4R to bone and adiposity regulation, whether RAMP interactions modulate Y4R pharmacology, and the precise mechanism connecting Y4R loss-of-function to obesity in humans.","evidence":"","pmids":[],"confidence":"Low","gaps":["No cryo-EM or crystal structure available","RAMP interaction with Y4R not functionally validated","Central versus peripheral contributions to metabolic phenotypes not dissected"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,5,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,11]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,5,10]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,5,9,11]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-8963743","term_label":"Digestion and absorption","supporting_discovery_ids":[7]}],"complexes":[],"partners":["GNAI2","GNAO1","RGS4","ARRB2","PP"],"other_free_text":[]},"mechanistic_narrative":"NPY4R (Y4 receptor) is a Gi/o-coupled GPCR that serves as the primary high-affinity receptor for pancreatic polypeptide (PP), mediating inhibition of cAMP production, regulation of intracellular calcium, and suppression of electrolyte secretion in the colon and glucagon release from pancreatic alpha-cells [PMID:7592911, PMID:10640301, PMID:11906964, PMID:25445712]. Its PP-binding pocket is formed by residues in TM2 (Tyr2.64), TM6 (Asn6.55), and TM7 (Asn7.32, Phe7.35), it requires N-glycosylation for function, and it exhibits constitutive activity and notable resistance to agonist-promoted desensitization and internalization—properties governed by a C-terminal EESEHLPLSTVHTEVSKGS motif that controls arrestin-3 recruitment and endocytosis [PMID:24375409, PMID:21745157, PMID:27818291, PMID:10640301]. Genetic studies in Y2/Y4 double-knockout mice reveal synergistic cooperation between Y2 and Y4 pathways in regulating adiposity and bone mass, and copy-number losses at the NPY4R locus and rare loss-of-function coding variants are associated with childhood obesity [PMID:12861009, PMID:26921218]."},"prefetch_data":{"uniprot":{"accession":"P50391","full_name":"Neuropeptide Y receptor type 4","aliases":["Pancreatic polypeptide receptor 1","PP1"],"length_aa":375,"mass_kda":42.1,"function":"G protein-coupled receptor for PPY/pancreatic polypeptide/PP, NPY/neuropeptide Y and PYY/peptide YY that is negatively coupled to cAMP (PubMed:7493937, PubMed:7592911). The rank order of affinity for these polypeptides and their derivatives is PP, PP (2-36) and [Ile-31, Gln-34] PP > [Pro-34] PYY > PYY and [Leu-31, Pro-34] NPY > NPY > PYY (3-36) and NPY (2-36) > PP (13-36) > PP (31-36) > NPY free acid","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/P50391/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NPY4R","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":5,"dependency_fraction":0.4},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NPY4R","total_profiled":1310},"omim":[{"mim_id":"601790","title":"PANCREATIC POLYPEPTIDE RECEPTOR 1; PPYR1","url":"https://www.omim.org/entry/601790"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"intestine","ntpm":5.6},{"tissue":"skin 1","ntpm":2.8}],"url":"https://www.proteinatlas.org/search/NPY4R"},"hgnc":{"alias_symbol":["Y4","PP1"],"prev_symbol":["PPYR1"]},"alphafold":{"accession":"P50391","domains":[{"cath_id":"1.20.1070.10","chopping":"37-246_256-336","consensus_level":"high","plddt":90.6956,"start":37,"end":336}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P50391","model_url":"https://alphafold.ebi.ac.uk/files/AF-P50391-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P50391-F1-predicted_aligned_error_v6.png","plddt_mean":80.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NPY4R","jax_strain_url":"https://www.jax.org/strain/search?query=NPY4R"},"sequence":{"accession":"P50391","fasta_url":"https://rest.uniprot.org/uniprotkb/P50391.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P50391/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P50391"}},"corpus_meta":[{"pmid":"16262633","id":"PMC_16262633","title":"Contributions of protein 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letters","url":"https://pubmed.ncbi.nlm.nih.gov/21094159","citation_count":17,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12477932","id":"PMC_12477932","title":"Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.","date":"2002","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12477932","citation_count":1479,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21873635","id":"PMC_21873635","title":"Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium.","date":"2011","source":"Briefings in bioinformatics","url":"https://pubmed.ncbi.nlm.nih.gov/21873635","citation_count":656,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"28319085","id":"PMC_28319085","title":"Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions.","date":"2017","source":"Nature biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/28319085","citation_count":378,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"7592911","id":"PMC_7592911","title":"Cloning and functional expression of a human Y4 subtype receptor for pancreatic polypeptide, neuropeptide Y, and peptide YY.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7592911","citation_count":333,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"7493937","id":"PMC_7493937","title":"Cloning of a human receptor of the NPY receptor family with high affinity for pancreatic polypeptide and peptide YY.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7493937","citation_count":274,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"2834384","id":"PMC_2834384","title":"Presence of three distinct molecular species of Gi protein alpha subunit. Structure of rat cDNAs and human genomic DNAs.","date":"1988","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/2834384","citation_count":256,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20379614","id":"PMC_20379614","title":"Personalized smoking cessation: interactions between nicotine dose, dependence and quit-success genotype score.","date":"2010","source":"Molecular medicine (Cambridge, Mass.)","url":"https://pubmed.ncbi.nlm.nih.gov/20379614","citation_count":108,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11825645","id":"PMC_11825645","title":"Structure and receptor binding of PYY analogs.","date":"2002","source":"Peptides","url":"https://pubmed.ncbi.nlm.nih.gov/11825645","citation_count":93,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25324306","id":"PMC_25324306","title":"hnRNPA1 couples nuclear export and translation of specific mRNAs downstream of FGF-2/S6K2 signalling.","date":"2014","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/25324306","citation_count":78,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12007534","id":"PMC_12007534","title":"Neuropeptide Y receptors as targets for anti-obesity drug development: perspective and current status.","date":"2002","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/12007534","citation_count":71,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"18577758","id":"PMC_18577758","title":"Dissociation of heterotrimeric g proteins in cells.","date":"2008","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/18577758","citation_count":69,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"8643460","id":"PMC_8643460","title":"Cloning and functional expression of cDNAs encoding human and rat pancreatic polypeptide receptors.","date":"1996","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8643460","citation_count":67,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11739470","id":"PMC_11739470","title":"NPY regulates catecholamine secretion from human adrenal chromaffin cells.","date":"2001","source":"The Journal of clinical endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/11739470","citation_count":64,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19229253","id":"PMC_19229253","title":"Genome-wide association study suggested copy number variation may be associated with body mass index in the Chinese population.","date":"2009","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/19229253","citation_count":62,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11906964","id":"PMC_11906964","title":"Neuropeptide Y, Y1, Y2 and Y4 receptors mediate Y agonist responses in isolated human colon mucosa.","date":"2002","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/11906964","citation_count":54,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11975932","id":"PMC_11975932","title":"Clinical potentials of neuropeptide Y family of hormones.","date":"2002","source":"American journal of surgery","url":"https://pubmed.ncbi.nlm.nih.gov/11975932","citation_count":54,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26921218","id":"PMC_26921218","title":"CNV analysis and mutation screening indicate an important role for the NPY4R gene in human obesity.","date":"2016","source":"Obesity (Silver Spring, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/26921218","citation_count":29,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"24375409","id":"PMC_24375409","title":"Pancreatic polypeptide is recognized by two hydrophobic domains of the human Y4 receptor binding pocket.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24375409","citation_count":28,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26575790","id":"PMC_26575790","title":"Protein 4.1N acts as a potential tumor suppressor linking PP1 to JNK-c-Jun pathway regulation in NSCLC.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/26575790","citation_count":23,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"29621259","id":"PMC_29621259","title":"Copy number of pancreatic polypeptide receptor gene NPY4R correlates with body mass index and waist circumference.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/29621259","citation_count":17,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21745157","id":"PMC_21745157","title":"Functional reconstitution of human neuropeptide Y (NPY) Y(2) and Y(4) receptors in Sf9 insect cells.","date":"2011","source":"Journal of receptor and signal transduction research","url":"https://pubmed.ncbi.nlm.nih.gov/21745157","citation_count":10,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27818291","id":"PMC_27818291","title":"C-terminal motif of human neuropeptide Y4 receptor determines internalization and arrestin recruitment.","date":"2016","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/27818291","citation_count":8,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"18826257","id":"PMC_18826257","title":"Protein phosphatase-1 is targeted to DNA polymerase delta via an interaction with the p68 subunit.","date":"2008","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18826257","citation_count":7,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9441761","id":"PMC_9441761","title":"Assignment of the Y4 receptor gene (PPYR1) to human chromosome 10q11.2 and mouse chromosome 14.","date":"1997","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9441761","citation_count":7,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22947943","id":"PMC_22947943","title":"Comparison of fragments comprising the first two helices of the human Y4 and the yeast Ste2p G-protein-coupled receptors.","date":"2012","source":"Biophysical journal","url":"https://pubmed.ncbi.nlm.nih.gov/22947943","citation_count":6,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"33782920","id":"PMC_33782920","title":"Theoretical study of the interactions between peptide tyrosine tyrosine [PYY (1-36)], a newly identified modulator in type 2 diabetes pathophysiology, with receptors NPY1R and NPY4R.","date":"2021","source":"Hormones (Athens, Greece)","url":"https://pubmed.ncbi.nlm.nih.gov/33782920","citation_count":5,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"9417917","id":"PMC_9417917","title":"Neuropeptide Y receptor genes mapped in human and mouse: receptors with high affinity for pancreatic polypeptide are not clustered with receptors specific for neuropeptide Y and peptide YY.","date":"1997","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9417917","citation_count":5,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"39083597","id":"PMC_39083597","title":"Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.","date":"2024","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/39083597","citation_count":3,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19481128","id":"PMC_19481128","title":"Cloning and characterization of rabbit neuropeptide Y receptor subtypes.","date":"2009","source":"Peptides","url":"https://pubmed.ncbi.nlm.nih.gov/19481128","citation_count":2,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":45330,"output_tokens":1657,"usd":0.080422},"stage2":{"model":"claude-opus-4-6","input_tokens":4644,"output_tokens":2988,"usd":0.14688},"total_usd":0.51046,"stage1_batch_id":"msgbatch_012ZiiCUBfB62NCTQd4Bk1Jo","stage2_batch_id":"msgbatch_01Xuqq9aUHoQouS7UVGZNStZ","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":58232,"output_tokens":4202,"usd":0.118863},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":7636,"output_tokens":2854,"usd":0.164295}},"stage1_raw":"Looking at the 100 abstracts, I need to identify papers that are actually about NPY4R (the human neuropeptide Y receptor type 4, also known as PPYR1 or Y4 receptor). The vast majority of these papers are about PP1 (protein phosphatase 1) or MLO-Y4 osteocyte cell lines, which are not NPY4R.\n\nThe relevant NPY4R papers are:\n- PMID 9145427: [125I]Leu31,Pro34-PYY as radioligand for rat PP1/Y4 and Y1 receptors\n- PMID 12861009: Y2 and Y4 receptor knockout mice, adiposity and bone mass\n- PMID 25445712: PP regulates glucagon release through PPYR1 receptors in alpha-cells\n- PMID 10640301: Functional properties of human recombinant Y4 receptor\n- PMID 33595306: Highly selective Y4R allosteric antagonist\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"The rat PP1/Y4 receptor binds [125I]Leu31,Pro34-PYY with high affinity, establishing heterogeneity in pancreatic polypeptide receptors; the Y4 receptor is pharmacologically distinct from Y1 receptors in brain tissue despite shared radioligand binding in cell lines.\",\n      \"method\": \"Radioligand binding assay using [125I]Leu31,Pro34-PYY in cell lines expressing the cloned rat PP1/Y4 receptor and rat brain sections\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct radioligand binding in recombinant cell system; single lab, single method\",\n      \"pmids\": [\"9145427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The human Y4 receptor expressed in CHO cells signals through a pertussis toxin-sensitive Gi protein to inhibit forskolin-stimulated cAMP production; the receptor is resistant to agonist-promoted desensitization and internalization, as 24-hour exposure to 10 nM hPP did not alter binding capacity, affinity, or signaling potency, and immunofluorescence confirmed lack of receptor internalization.\",\n      \"method\": \"Radioligand binding, cAMP assay, pertussis toxin treatment, cross-linking, immunofluorescence in stably transfected CHO cells\",\n      \"journal\": \"The Journal of pharmacology and experimental therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (binding, cAMP, pertussis toxin, imaging) in a single rigorous study with stable recombinant expression\",\n      \"pmids\": [\"10640301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Genetic ablation of Y4 receptors in mice produces a lean phenotype; double knockout of Y2 and Y4 receptors (Y2−/−Y4−/−) results in a synergistically lean phenotype with reduced adipose tissue, leptin, and insulin levels despite hyperphagia, and a threefold increase in cancellous bone volume associated with enhanced osteoblastic activity, demonstrating that Y2 and Y4 receptor pathways cooperate to regulate adiposity and bone mass.\",\n      \"method\": \"Genetic epistasis using Y4 single knockout and Y2/Y4 double knockout mice; body composition, bone histomorphometry, plasma hormone measurements\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic loss-of-function with multiple defined phenotypic readouts; double-knockout epistasis experiment replicated across genotypes\",\n      \"pmids\": [\"12861009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PPYR1 (Y4 receptor) is expressed specifically in glucagon-secreting alpha-cells of mouse and human pancreatic islets; activation of Y4 receptors by pancreatic polypeptide (PP) inhibits glucagon release from isolated pancreatic islets, and glucose stimulation increases PP secretion, establishing a glucose-regulated PP/Y4R signaling axis that controls glucagon secretion.\",\n      \"method\": \"Radioligand binding, quantitative RT-PCR on FACS-sorted islet cell populations, immunohistochemistry on mouse and human pancreatic sections, glucagon and insulin secretion assays from isolated islets\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (receptor localization by IHC, cell-type specificity by sorted-cell RT-PCR, functional secretion assay) in a single study; validated in both mouse and human tissue\",\n      \"pmids\": [\"25445712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The Y4 receptor possesses an allosteric binding pocket located below the endogenous ligand (pancreatic polypeptide) binding site within the transmembrane domain core; the selective antagonist (S)-VU0637120 binds this allosteric site and inhibits native Y4R function, revealing a non-competitive inhibitory mechanism distinct from orthosteric ligand competition.\",\n      \"method\": \"In vitro pharmacological assays, ex vivo tissue assays, computational docking/in silico modeling of the allosteric pocket, medicinal chemistry structure-activity relationship\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — functional allosteric characterization with in vitro, ex vivo, and in silico methods; structural assignment is computational rather than crystallographic\",\n      \"pmids\": [\"33595306\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NPY4R (Y4 receptor) is a Gi protein-coupled receptor that preferentially binds pancreatic polypeptide (PP) with high affinity, signals through pertussis toxin-sensitive Gi to inhibit cAMP production, is expressed specifically on pancreatic islet alpha-cells where it mediates PP-induced inhibition of glucagon secretion, is resistant to agonist-promoted desensitization and internalization, and cooperates with Y2 receptors to regulate adiposity and bone mass in vivo; an allosteric binding pocket in its transmembrane core has been identified as a target for selective antagonism.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nLooking at the corpus, I need to classify papers as KEEP (about NPY4R/Y4 receptor) or EXCLUDE.\n\n**KEEP papers** (about NPY4R/Y4 receptor):\n- PMID:7592911, 7493937, 8643460, 9145427, 10640301, 12861009, 25445712, 11906964, 12007534, 33595306, 24375409, 27818291, 21745157, 33782920, 39083597, 19481128, 29621259, 26921218\n\n**EXCLUDE papers**: All the PP1/PP2A phosphatase papers (alias collision — \"PP1\" in those papers refers to the kinase inhibitor compound or protein phosphatase 1, not NPY4R). MLO-Y4 papers are about an osteocyte cell line, not NPY4R. All others are clearly off-target.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"The human Y4 receptor (NPY4R) was cloned from a placental genomic library by homology screening with Y1 transmembrane probes. It encodes a 375-amino acid GPCR with highest affinity for human pancreatic polypeptide (PP; pKi ~10.25), followed by PYY and NPY. When expressed in LMTK- cells, human PP activation decreased cAMP and increased intracellular Ca2+, demonstrating coupling to both Gi and Gq-like pathways.\",\n      \"method\": \"cDNA cloning, radioligand binding (125I-PYY), cAMP and Ca2+ assays in transfected cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original cloning with functional reconstitution using multiple orthogonal assays (binding, cAMP, Ca2+)\",\n      \"pmids\": [\"7592911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"An independently cloned human NPY4R (designated PP1) was shown to bind PP with extremely high affinity (Ki ~14 pM), PYY with 1.44 nM, and NPY with 9.9 nM, establishing PP as the primary endogenous ligand. In stably transfected CHO cells, the receptor inhibited forskolin-stimulated cAMP synthesis. Northern blot detected mRNA in colon, small intestine, pancreas, and prostate.\",\n      \"method\": \"Homology cloning, radioligand binding, cAMP assay in stable CHO transfectants, Northern blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — independent replication of cloning with functional cAMP assay and expression profiling\",\n      \"pmids\": [\"7493937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Human and rat NPY4R cDNAs were cloned and expressed in HEK293 cells. The human receptor bound 125I-labeled human PP with Kd = 2.8 nM; rank-order potency was human PP = bovine PP > [Pro34]PYY > rat PP > hPYY = hNPY. Northern blot showed mRNA most abundantly in skeletal muscle, with lesser expression in lung and brain. The rat ortholog shares 74% amino acid identity with human Y4R.\",\n      \"method\": \"PCR cloning, stable transfection, radioligand binding, Northern blot\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — functional reconstitution in mammalian cells with quantitative binding characterization, independent replication of human Y4R cloning\",\n      \"pmids\": [\"8643460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"[125I]Leu31,Pro34-PYY was identified as a high-affinity radioligand for rat PP1/Y4 receptor expressed in cell lines, demonstrating that this putative Y1-selective ligand also binds Y4 with high affinity. However, in rat brain sections this ligand does not label interpeduncular nucleus sites (high in [125I]-bPP binding), indicating heterogeneity among PP-recognizing receptors in brain versus peripheral tissues.\",\n      \"method\": \"Radioligand binding in transfected cell lines and rat brain autoradiography\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct binding assays in cells expressing cloned receptor plus brain section autoradiography, single lab\",\n      \"pmids\": [\"9145427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The human Y4 receptor gene (PPYR1) was localized to chromosome 10q11.2 by nonisotopic in situ hybridization, and the mouse Y4 gene (Ppyr1) to chromosome 14 by interspecific backcross mapping. The gene contains no introns within its coding region, suggesting origin via an RNA-mediated transpositional event from an ancestral Y1-like receptor.\",\n      \"method\": \"Fluorescence in situ hybridization (FISH), interspecific mouse backcross mapping, genomic sequencing\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct chromosomal mapping with multiple independent methods (FISH + genetic mapping)\",\n      \"pmids\": [\"9441761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Human Y4 receptor stably expressed in CHO cells showed one class of binding site (Kd = 0.26 nM for 125I-hPP). Signal transduction via Gi protein was demonstrated by pertussis toxin abolition of hPP-induced cAMP inhibition. Critically, 24-hour exposure to 10 nM hPP produced no change in binding capacity, affinity, or receptor potency, and immunofluorescence showed no receptor internalization, establishing that Y4R is resistant to agonist-promoted desensitization and internalization.\",\n      \"method\": \"Radioligand binding, cAMP assay, pertussis toxin treatment, immunofluorescence microscopy in stable CHO transfectants\",\n      \"journal\": \"The Journal of pharmacology and experimental therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (binding, cAMP, pertussis toxin, immunofluorescence) in single lab demonstrating resistance to desensitization\",\n      \"pmids\": [\"10640301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Using Y2(-/-)Y4(-/-) double knockout mice, synergistic interactions between Y2 and Y4 receptor pathways were demonstrated. Y4 single knockout produced a lean phenotype and elevated plasma PP. Despite hyperphagia, Y2/Y4 double knockouts showed markedly reduced adiposity and threefold increased bone volume with enhanced osteoblastic activity—effects more pronounced than Y2 single knockout alone—establishing that Y2 and Y4 pathways cooperate to regulate adiposity and bone mass.\",\n      \"method\": \"Genetic epistasis using double knockout mice, body composition analysis, bone histomorphometry, plasma hormone measurements\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in double knockout mice with multiple quantitative phenotypic readouts, demonstrates pathway cooperation\",\n      \"pmids\": [\"12861009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"In isolated human colon mucosa in Ussing chambers, basolateral application of human PP (hPP) inhibited electrogenic ion transport. This inhibitory response was insensitive to Y1 antagonists (BIBP3226, BIBO3304) and Y2 antagonist (BIIE0246), and was not blocked by tetrodotoxin, establishing that Y4 receptors are the predominantly post-junctional mediators of PP-induced inhibition of colonic electrolyte secretion.\",\n      \"method\": \"Ussing chamber electrophysiology with selective receptor antagonists and TTX in human colonic tissue\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological dissection with selective antagonists in native human tissue, establishing Y4R-specific post-junctional signaling\",\n      \"pmids\": [\"11906964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PPYR1 (Y4R) protein was detected by immunoblotting, qRT-PCR (enriched in FACS-sorted glucagon alpha-cell fraction), and immunohistochemistry on pancreatic sections in both mouse and human islets. Direct PP exposure to isolated mouse islets inhibited glucagon release, demonstrating that Y4R on alpha-cells mediates PP-dependent suppression of glucagon secretion.\",\n      \"method\": \"Immunoblotting, qRT-PCR on sorted islet cells, immunohistochemistry, ex vivo glucagon secretion assay in isolated islets\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (protein, mRNA, IHC, functional secretion assay) in mouse and human tissue, directly linking Y4R to glucagon inhibition\",\n      \"pmids\": [\"25445712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Double-cycle mutagenesis of hY4R combined with computational homology modeling of the hY4R–hPP complex identified residues Tyr2.64, Asp2.68, Asn6.55, Asn7.32, and Phe7.35 as critical for receptor activation by PP. The top of TM2 and TM6-TM7 form the core peptide-binding pocket, with Tyr2.64 making hydrophobic contact with Tyr27 of PP, Asn7.32 hydrogen-bonding Arg33 of PP, and Phe7.35 interacting with PP positions 33 and 36.\",\n      \"method\": \"Site-directed mutagenesis, radioligand binding, double-cycle mutagenesis, computational homology modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — systematic mutagenesis with double-cycle analysis and structural modeling, identifying specific molecular contacts in the binding pocket\",\n      \"pmids\": [\"24375409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The C-terminal motif 7.78EESEHLPLSTVHTEVSKGS7.96 of hY4R was identified as essential for receptor internalization and arrestin-3 recruitment. Deletion and point-mutation analysis showed glutamate, threonine, and serine residues within this motif are key determinants. This was established using fluorescence microscopy, binding assays, inositol turnover, and BRET assays, revealing the molecular basis for Y4R endocytosis.\",\n      \"method\": \"C-terminal deletion mutants, site-directed mutagenesis, fluorescence microscopy, BRET assays, binding assays, inositol turnover in transfected cells\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (BRET, microscopy, binding, signaling) with systematic mutagenesis defining internalization motif\",\n      \"pmids\": [\"27818291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Human Y4R and Y2R were functionally reconstituted in Sf9 insect cells using baculovirus co-expression with Gi/Go proteins and RGS proteins. Steady-state GTPase assays showed hY4R couples optimally to Gαi2 and Gαo with RGS4. N-glycosylation was found to be essential for hY4R function, as tunicamycin treatment abolished receptor activity. Na+ sensitivity assays revealed moderate constitutive activity of hY4R.\",\n      \"method\": \"Baculovirus expression in Sf9 cells, steady-state GTPase assays, tunicamycin glycosylation inhibition, ion sensitivity assays\",\n      \"journal\": \"Journal of receptor and signal transduction research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional reconstitution with multiple G-protein combinations, single lab\",\n      \"pmids\": [\"21745157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The first selective Y4R allosteric antagonist, (S)-VU0637120, was identified and characterized. In vitro, ex vivo, and in silico studies demonstrated it selectively inhibits native Y4R function by binding an allosteric site located below the endogenous PP-binding pocket within the core of the Y4R transmembrane domains, distinct from the orthosteric binding site.\",\n      \"method\": \"In vitro binding assays, ex vivo functional assays, computational modeling/docking, selectivity profiling against Y1R/Y2R/Y5R\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional and computational approaches identifying allosteric site, single lab\",\n      \"pmids\": [\"33595306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Multiplexed screening of 215 GPCRs against all three RAMPs (receptor activity-modifying proteins) using suspension bead array immunoassays identified NPY4R (Y4R) among the 122 GPCRs showing strong evidence for interaction with at least one RAMP, identifying a potential regulatory complex for Y4R trafficking/pharmacology.\",\n      \"method\": \"Dual epitope-tagged GPCR library co-expressed with RAMPs 1-3, multiplexed suspension bead array immunoassays in three cell lines\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — high-throughput co-expression screen, no functional validation of Y4R-RAMP complex specifically\",\n      \"pmids\": [\"39083597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CNV analysis in obese children versus lean controls showed significantly higher frequency of NPY4R-containing 10q11.22 CNV loss in obese patients (p=0.0003) and CNV gain in controls (p=0.031). Mutation screening identified two rare non-synonymous heterozygous variants found only in obese patients for which receptor dysfunction was experimentally demonstrated.\",\n      \"method\": \"CNV analysis, coding region mutation screening, functional receptor assays for identified variants\",\n      \"journal\": \"Obesity (Silver Spring, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional receptor assays confirmed dysfunction of identified variants, supporting causal role of NPY4R loss-of-function in obesity\",\n      \"pmids\": [\"26921218\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NPY4R (Y4 receptor) is a Gi/o-coupled GPCR that serves as the primary receptor for pancreatic polypeptide (PP), signaling through pertussis toxin-sensitive Gi proteins to inhibit cAMP production; its binding pocket is defined by TM2 (Tyr2.64) and TM6-TM7 (Asn6.55, Asn7.32, Phe7.35) residues, it is uniquely resistant to agonist-promoted desensitization and internalization (unless driven through a C-terminal EESEHLPLSTVHTEVSKGS motif for arrestin-3 recruitment), requires N-glycosylation for function, mediates PP-dependent inhibition of glucagon secretion from pancreatic alpha-cells, and cooperates synergistically with Y2 receptor pathways to regulate adiposity and bone mass.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NPY4R (Y4 receptor) is a Gi-coupled receptor that preferentially binds pancreatic polypeptide (PP) with high affinity and signals through pertussis toxin-sensitive Gi to inhibit cAMP production, with a distinctive resistance to agonist-promoted desensitization and internalization [PMID:10640301]. In pancreatic islets, NPY4R is expressed specifically on glucagon-secreting alpha cells, where PP activation of Y4R inhibits glucagon release as part of a glucose-regulated PP/Y4R signaling axis [PMID:25445712]. In vivo, Y4 receptor deletion produces a lean phenotype, and combined ablation of Y2 and Y4 receptors synergistically reduces adiposity while increasing cancellous bone volume through enhanced osteoblastic activity, establishing cooperative roles for these receptors in energy and bone homeostasis [PMID:12861009].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Demonstrating that the cloned Y4 receptor binds PP-related radioligands with high affinity and is pharmacologically distinct from Y1 receptors resolved the heterogeneity of PP receptor subtypes.\",\n      \"evidence\": \"Radioligand binding with [125I]Leu31,Pro34-PYY in recombinant cell lines and rat brain sections\",\n      \"pmids\": [\"9145427\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single radioligand approach\", \"No functional signaling readout in this study\", \"Brain versus peripheral receptor identity not further resolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defining Y4R coupling to pertussis toxin-sensitive Gi and its remarkable resistance to agonist-induced desensitization and internalization established a unique signaling profile among neuropeptide Y receptor family members.\",\n      \"evidence\": \"cAMP inhibition assay, pertussis toxin blockade, radioligand binding after prolonged agonist exposure, and immunofluorescence in stably transfected CHO cells\",\n      \"pmids\": [\"10640301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism underlying desensitization resistance (e.g., GRK/arrestin recruitment deficiency) not identified\", \"Signaling characterized only in heterologous CHO cells, not native tissue\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Genetic ablation of Y4R, alone and in combination with Y2R, revealed cooperative roles in regulating adiposity and bone mass in vivo, extending Y4R function beyond acute signaling to systemic metabolic and skeletal homeostasis.\",\n      \"evidence\": \"Y4 single-knockout and Y2/Y4 double-knockout mice with body composition analysis, bone histomorphometry, and plasma hormone measurements\",\n      \"pmids\": [\"12861009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific versus central contributions of Y4R to the phenotype not dissected\", \"Mechanism by which Y4R loss increases osteoblastic activity is unknown\", \"Hyperphagia coexisting with leanness not mechanistically explained\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Localizing Y4R specifically to islet alpha cells and showing that PP inhibits glucagon secretion via this receptor identified a glucose-regulated paracrine axis controlling glucagon output.\",\n      \"evidence\": \"RT-PCR on FACS-sorted islet cells, immunohistochemistry on mouse and human pancreas, and glucagon secretion assays from isolated islets\",\n      \"pmids\": [\"25445712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream intracellular pathway linking Gi inhibition of cAMP to glucagon granule exocytosis not characterized\", \"In vivo relevance of alpha-cell Y4R for systemic glucose homeostasis not tested with conditional knockouts\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of a transmembrane allosteric pocket in Y4R, distinct from the orthosteric PP-binding site, provided a structural basis for selective non-competitive antagonism.\",\n      \"evidence\": \"Pharmacological characterization with (S)-VU0637120, ex vivo tissue assays, and computational docking modeling\",\n      \"pmids\": [\"33595306\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Allosteric site assignment is based on computational docking rather than crystallographic or cryo-EM structure\", \"In vivo efficacy and selectivity of allosteric antagonists not yet demonstrated\", \"Whether endogenous allosteric modulators exist is unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of Y4R bound to PP and/or allosteric modulators, the molecular basis of desensitization resistance, and tissue-specific conditional knockout studies are needed to fully define Y4R mechanism and physiology.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No experimentally determined receptor structure\", \"Molecular basis for desensitization/internalization resistance unresolved\", \"Cell-type-specific contributions of Y4R to metabolic and bone phenotypes not dissected\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0162582\", \"supporting_discovery_ids\": [1, 3, 4]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"GNA1\", \"PP\"],\n    \"other_free_text\": []\n  }\n}\n```\n\nWait, let me fix the pathway term. I used an incorrect term_id. Let me correct:\n\n```json\n{\n  \"mechanistic_narrative\": \"NPY4R (Y4 receptor) is a Gi-coupled receptor that preferentially binds pancreatic polypeptide (PP) with high affinity and signals through pertussis toxin-sensitive Gi to inhibit cAMP production, with a distinctive resistance to agonist-promoted desensitization and internalization [PMID:10640301]. In pancreatic islets, NPY4R is expressed specifically on glucagon-secreting alpha cells, where PP activation of Y4R inhibits glucagon release as part of a glucose-regulated PP/Y4R signaling axis [PMID:25445712]. In vivo, Y4 receptor deletion produces a lean phenotype, and combined ablation of Y2 and Y4 receptors synergistically reduces adiposity while increasing cancellous bone volume through enhanced osteoblastic activity, establishing cooperative roles for these receptors in energy and bone homeostasis [PMID:12861009].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Demonstrating that the cloned Y4 receptor binds PP-related radioligands with high affinity and is pharmacologically distinct from Y1 receptors resolved the heterogeneity of PP receptor subtypes.\",\n      \"evidence\": \"Radioligand binding with [125I]Leu31,Pro34-PYY in recombinant cell lines and rat brain sections\",\n      \"pmids\": [\"9145427\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single radioligand approach\", \"No functional signaling readout in this study\", \"Brain versus peripheral receptor identity not further resolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defining Y4R coupling to pertussis toxin-sensitive Gi and its remarkable resistance to agonist-induced desensitization and internalization established a unique signaling profile among neuropeptide Y receptor family members.\",\n      \"evidence\": \"cAMP inhibition assay, pertussis toxin blockade, radioligand binding after prolonged agonist exposure, and immunofluorescence in stably transfected CHO cells\",\n      \"pmids\": [\"10640301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism underlying desensitization resistance (e.g., GRK/arrestin recruitment deficiency) not identified\", \"Signaling characterized only in heterologous CHO cells, not native tissue\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Genetic ablation of Y4R, alone and in combination with Y2R, revealed cooperative roles in regulating adiposity and bone mass in vivo, extending Y4R function beyond acute signaling to systemic metabolic and skeletal homeostasis.\",\n      \"evidence\": \"Y4 single-knockout and Y2/Y4 double-knockout mice with body composition analysis, bone histomorphometry, and plasma hormone measurements\",\n      \"pmids\": [\"12861009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific versus central contributions of Y4R to the phenotype not dissected\", \"Mechanism by which Y4R loss increases osteoblastic activity is unknown\", \"Hyperphagia coexisting with leanness not mechanistically explained\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Localizing Y4R specifically to islet alpha cells and showing that PP inhibits glucagon secretion via this receptor identified a glucose-regulated paracrine axis controlling glucagon output.\",\n      \"evidence\": \"RT-PCR on FACS-sorted islet cells, immunohistochemistry on mouse and human pancreas, and glucagon secretion assays from isolated islets\",\n      \"pmids\": [\"25445712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream intracellular pathway linking Gi inhibition of cAMP to glucagon granule exocytosis not characterized\", \"In vivo relevance of alpha-cell Y4R for systemic glucose homeostasis not tested with conditional knockouts\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of a transmembrane allosteric pocket in Y4R, distinct from the orthosteric PP-binding site, provided a structural basis for selective non-competitive antagonism.\",\n      \"evidence\": \"Pharmacological characterization with (S)-VU0637120, ex vivo tissue assays, and computational docking modeling\",\n      \"pmids\": [\"33595306\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Allosteric site assignment is based on computational docking rather than crystallographic or cryo-EM structure\", \"In vivo efficacy and selectivity of allosteric antagonists not yet demonstrated\", \"Whether endogenous allosteric modulators exist is unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of Y4R bound to PP and/or allosteric modulators, the molecular basis of desensitization resistance, and tissue-specific conditional knockout studies are needed to fully define Y4R mechanism and physiology.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No experimentally determined receptor structure\", \"Molecular basis for desensitization/internalization resistance unresolved\", \"Cell-type-specific contributions of Y4R to metabolic and bone phenotypes not dissected\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 3, 4]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NPY2R\", \"PPY\"],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"NPY4R (Y4 receptor) is a Gi/o-coupled GPCR that serves as the primary high-affinity receptor for pancreatic polypeptide (PP), mediating inhibition of cAMP production, regulation of intracellular calcium, and suppression of electrolyte secretion in the colon and glucagon release from pancreatic alpha-cells [PMID:7592911, PMID:10640301, PMID:11906964, PMID:25445712]. Its PP-binding pocket is formed by residues in TM2 (Tyr2.64), TM6 (Asn6.55), and TM7 (Asn7.32, Phe7.35), it requires N-glycosylation for function, and it exhibits constitutive activity and notable resistance to agonist-promoted desensitization and internalization—properties governed by a C-terminal EESEHLPLSTVHTEVSKGS motif that controls arrestin-3 recruitment and endocytosis [PMID:24375409, PMID:21745157, PMID:27818291, PMID:10640301]. Genetic studies in Y2/Y4 double-knockout mice reveal synergistic cooperation between Y2 and Y4 pathways in regulating adiposity and bone mass, and copy-number losses at the NPY4R locus and rare loss-of-function coding variants are associated with childhood obesity [PMID:12861009, PMID:26921218].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Cloning and functional reconstitution of NPY4R established it as a Gi-coupled GPCR with highest affinity for pancreatic polypeptide, resolving the molecular identity of the PP-preferring neuropeptide Y receptor subtype.\",\n      \"evidence\": \"Independent cloning from human genomic/cDNA libraries with radioligand binding, cAMP inhibition, and Ca²⁺ assays in transfected LMTK⁻ and CHO cells; Northern blot tissue profiling\",\n      \"pmids\": [\"7592911\", \"7493937\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PP selectivity over NPY/PYY unknown\", \"In vivo physiological roles not yet addressed\", \"G-protein coupling specificity (Gi subtypes) not resolved\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Cloning of the rat ortholog and cross-species binding comparison showed 74% amino acid identity with human Y4R and confirmed species-dependent variation in PP affinity, indicating evolutionary divergence in ligand recognition.\",\n      \"evidence\": \"PCR cloning, stable HEK293 transfection, radioligand binding of human and rat receptors\",\n      \"pmids\": [\"8643460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Residues responsible for species selectivity not identified\", \"Functional consequences of sequence divergence not tested\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Chromosomal mapping placed the intronless NPY4R gene at human 10q11.2 and mouse chromosome 14, suggesting origin via retrotranspositional duplication from an ancestral Y1-like receptor.\",\n      \"evidence\": \"FISH and interspecific mouse backcross mapping\",\n      \"pmids\": [\"9441761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulatory elements controlling tissue-specific expression not characterized\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstration that Y4R is uniquely resistant to agonist-promoted desensitization and internalization—with preserved binding and signaling after prolonged PP exposure—distinguished it from other NPY receptor subtypes and raised questions about its regulatory mechanisms.\",\n      \"evidence\": \"Radioligand binding kinetics, cAMP assay, pertussis toxin block, and immunofluorescence after 24-h agonist exposure in stable CHO transfectants\",\n      \"pmids\": [\"10640301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular determinants of desensitization resistance not identified\", \"Phosphorylation and arrestin engagement not tested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Pharmacological dissection in native human colonic tissue established Y4R as the post-junctional mediator of PP-induced inhibition of electrolyte secretion, defining a gastrointestinal effector role.\",\n      \"evidence\": \"Ussing chamber electrophysiology with selective Y1, Y2, and TTX antagonists on human colon mucosa\",\n      \"pmids\": [\"11906964\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream ion channel/transporter targets not identified\", \"Contribution of Y4R to colonic pathophysiology not tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Y2/Y4 double-knockout mice revealed synergistic cooperation between Y2 and Y4 receptor pathways in controlling adiposity and bone mass, with double knockouts showing markedly reduced fat and threefold increased bone volume despite hyperphagia.\",\n      \"evidence\": \"Genetic epistasis using single and double knockout mice with body composition analysis, bone histomorphometry, and plasma hormone measurements\",\n      \"pmids\": [\"12861009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Central versus peripheral site of action for adiposity/bone regulation unresolved\", \"Downstream signaling pathways mediating bone effects unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Functional reconstitution with defined G-protein subunits showed Y4R couples optimally to Gαi2 and Gαo with RGS4, requires N-glycosylation for function, and exhibits moderate constitutive activity.\",\n      \"evidence\": \"Baculovirus co-expression of Y4R with Gi/Go subtypes and RGS proteins in Sf9 cells; steady-state GTPase assays; tunicamycin treatment\",\n      \"pmids\": [\"21745157\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Constitutive activity not confirmed in mammalian cells\", \"Glycosylation sites contributing to function not mapped\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Systematic double-cycle mutagenesis and homology modeling defined the PP-binding pocket, identifying Tyr2.64, Asn6.55, Asn7.32, and Phe7.35 as critical contacts and explaining the molecular basis of PP selectivity.\",\n      \"evidence\": \"Site-directed and double-cycle mutagenesis, radioligand binding, and computational modeling of the Y4R–PP complex\",\n      \"pmids\": [\"24375409\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimental 3D structure available\", \"Activation-state conformational changes not resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Detection of Y4R protein and mRNA specifically in pancreatic alpha-cells, coupled with PP-mediated inhibition of glucagon secretion from isolated islets, established an endocrine axis for PP–Y4R signaling in glucose homeostasis.\",\n      \"evidence\": \"Immunoblotting, qRT-PCR on FACS-sorted islet fractions, immunohistochemistry, ex vivo glucagon secretion assay in mouse and human tissue\",\n      \"pmids\": [\"25445712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance using Y4R-knockout islets not tested\", \"Signaling pathway linking Y4R activation to glucagon suppression not delineated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of the C-terminal motif 7.78EESEHLPLSTVHTEVSKGS7.96 as the molecular determinant for arrestin-3 recruitment and receptor internalization resolved why Y4R normally resists desensitization and how endocytosis can be engaged.\",\n      \"evidence\": \"C-terminal deletion and point mutagenesis with BRET, fluorescence microscopy, binding, and inositol turnover assays\",\n      \"pmids\": [\"27818291\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphorylation events upstream of arrestin binding not mapped\", \"Physiological context triggering this internalization pathway unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"CNV losses at 10q11.22 encompassing NPY4R and functionally validated rare loss-of-function coding variants were enriched in obese children, linking NPY4R haploinsufficiency to human obesity.\",\n      \"evidence\": \"CNV analysis and mutation screening in obese vs. lean children with functional receptor assays for identified variants\",\n      \"pmids\": [\"26921218\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Small cohort size limits generalizability\", \"Mechanism connecting Y4R loss-of-function to increased adiposity not defined\", \"Not replicated in independent populations\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovery of the first selective Y4R allosteric antagonist (S)-VU0637120, binding a site within the transmembrane core below the orthosteric pocket, provided a pharmacological tool to probe Y4R function independently of PP competition.\",\n      \"evidence\": \"In vitro binding, ex vivo functional assays, computational docking, selectivity profiling against Y1R/Y2R/Y5R\",\n      \"pmids\": [\"33595306\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo efficacy and pharmacokinetics not reported\", \"Allosteric site not structurally resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the lack of an experimental 3D structure of Y4R (free or PP-bound), the signaling cascades downstream of Gi that link Y4R to bone and adiposity regulation, whether RAMP interactions modulate Y4R pharmacology, and the precise mechanism connecting Y4R loss-of-function to obesity in humans.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No cryo-EM or crystal structure available\", \"RAMP interaction with Y4R not functionally validated\", \"Central versus peripheral contributions to metabolic phenotypes not dissected\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 5, 9]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 11]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 5, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 5, 9, 11]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-8963743\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"GNAI2\",\n      \"GNAO1\",\n      \"RGS4\",\n      \"ARRB2\",\n      \"PP\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}