{"gene":"NPY4R","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":1995,"finding":"The human Y4 receptor (NPY4R/PPYR1) was cloned from a human placental genomic library and encodes a 375-amino acid GPCR. When transiently expressed, it binds 125I-PYY with high affinity (pKd=9.89) and shows ligand selectivity: PP > PYY > NPY. Human PP activation decreases cAMP and increases intracellular Ca2+ in Y4-transfected cells, establishing Gi-coupled and Ca2+-mobilizing signaling.","method":"Homology cloning, radioligand binding (125I-PYY), cAMP radioimmunoassay, intracellular Ca2+ measurement in transfected LMTK- cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — primary cloning paper with reconstituted binding, functional cAMP and Ca2+ assays, multiple orthogonal methods in the founding study","pmids":["7592911"],"is_preprint":false},{"year":1997,"finding":"The rat Y4 receptor was cloned and expressed; its pharmacological profile shows high selectivity for rat PP over NPY or PYY. The rat and human Y4 receptors share 75% amino acid identity but differ substantially in ligand selectivity and dependence on N-terminal PP residues, indicating species-specific ligand–receptor interactions.","method":"Cloning, 125I-PP radioligand binding, cAMP radioimmunoassay in transfected cells","journal":"Peptides","confidence":"High","confidence_rationale":"Tier 1 / Strong — independent cloning of rat ortholog with binding and functional assays, replicating and extending founding human Y4R study","pmids":["9210181"],"is_preprint":false},{"year":1998,"finding":"GR231118 (1229U91) acts as a potent Y1 receptor antagonist (pA2=10.5 at human Y1) and simultaneously as a potent Y4 receptor agonist (pEC50=8.6 at human Y4), demonstrating that the C-terminus of NPY is sufficient for Y4 receptor activation but not Y1 activation. Activity confirmed in inhibition of forskolin-stimulated cAMP formation.","method":"Radioligand binding assays, functional cAMP inhibition assay across cloned Y1, Y2, Y4, Y5 receptors","journal":"European journal of pharmacology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstituted binding and functional assays at multiple cloned receptor subtypes; single lab but two orthogonal methods","pmids":["9669502"],"is_preprint":false},{"year":1998,"finding":"The guinea pig Y4 receptor was cloned; it is 84% identical to human Y4 (vs 74-75% for rat Y4) and its pharmacological profile (PP > NPY ≈ PYY > NPY2-36) is closer to human than rat Y4, establishing that accelerated sequence divergence in rodent Y4 receptors alters ligand selectivity.","method":"Cloning, 125I-hPP saturation and competition binding in expressed guinea pig Y4 receptor","journal":"Regulatory peptides","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous binding assay but single lab, single species characterization","pmids":["9802391"],"is_preprint":false},{"year":2000,"finding":"Stable CHO cells expressing human Y4 receptor demonstrate: (1) a single binding site for 125I-hPP (Kd=0.26 nM), (2) a glycosylated receptor protein of Mr 60,000 identified by cross-linking, (3) hPP inhibits cAMP via pertussis toxin-sensitive Gi protein (EC50=0.56 nM), and (4) 24-hour exposure to 10 nM hPP produces no receptor desensitization, no reduction in binding capacity, and no internalization — establishing resistance to agonist-promoted desensitization as a defining functional property of human Y4R.","method":"Radioligand binding, cross-linking/SDS-PAGE, cAMP RIA, pertussis toxin treatment, immunofluorescence in stable CHO transfectants","journal":"The Journal of pharmacology and experimental therapeutics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal methods (binding, biochemical, functional, imaging) in a single rigorous study; stable expression system with mechanistic controls","pmids":["10640301"],"is_preprint":false},{"year":2001,"finding":"Cloned rat pancreatic polypeptide Y4 receptor expressed in CHO cells undergoes rapid, temperature-dependent, agonist-driven internalization via clathrin-mediated endocytosis (blocked by sucrose and phenylarsine oxide), in contrast to the Y2 receptor which shows minimal internalization. Internalized Y4 receptors traffic to endosome-like particulates, and receptor recovery is blocked by cycloheximide and monensin, indicating requirement for new protein synthesis and intact vesicular transport.","method":"Ligand internalization assays in CHO cells, pharmacological inhibitors (sucrose, phenylarsine oxide, cycloheximide, monensin), sucrose density gradient fractionation","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct subcellular trafficking assays with multiple pharmacological dissections, single lab","pmids":["11179953"],"is_preprint":false},{"year":2002,"finding":"The chicken Y4 receptor was cloned and characterized; it binds PYYa, chicken PP, and NPY with similarly high affinities (Kd 68-160 pM), contrasting with mammalian Y4R which strongly prefers PP. RT-PCR and in situ hybridization showed expression in the dorsal motor nucleus of the vagus in the brainstem, suggesting conserved anatomical targeting.","method":"Cloning, 125I-pPYY saturation and competition binding in COS-7 cells, RT-PCR, in situ hybridization","journal":"Journal of molecular endocrinology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous binding characterization of avian ortholog, single lab","pmids":["12063188"],"is_preprint":false},{"year":2002,"finding":"Y2 and Y4 receptors are co-expressed on smooth muscle cells and both couple to Gq in addition to Gi2, enabling IP3-dependent Ca2+ release and muscle contraction. Y4 receptor activation by pancreatic polypeptide induces contraction insensitive to extracellular Ca2+ removal or L-type Ca2+ channel blockers but abolished by thapsigargin or PLC inhibitor U73122, establishing Y4R-Gq-IP3-Ca2+ release pathway for contraction.","method":"RT-PCR, radioligand binding with selective receptor protection, functional contraction assays, IP3 measurement, Ca2+ imaging, pertussis toxin treatment, pharmacological inhibitors in isolated smooth muscle cells","journal":"The Journal of pharmacology and experimental therapeutics","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal methods (binding, IP3, Ca2+, contraction) with selective receptor protection strategy; single lab but highly systematic","pmids":["15308651"],"is_preprint":false},{"year":2002,"finding":"Genetic deletion of Y4 receptor in ob/ob mice restores testosterone levels, testis and seminal vesicle size, and fertility in males (100% offspring production), and is associated with increased GnRH expression. This epistatic rescue establishes that central Y4 receptor signaling specifically inhibits the gonadotropic axis under conditions of elevated NPY tone.","method":"Genetic epistasis: Y4 knockout crossed onto ob/ob background; hormone ELISA, organ morphometry, GnRH mRNA analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic epistasis with multiple phenotypic readouts (fertility, hormones, organ morphology, GnRH expression) establishing pathway position","pmids":["12000791"],"is_preprint":false},{"year":2003,"finding":"The rhesus Y4 receptor forms homodimers at the cell surface as demonstrated by BRET2 and Western blot analysis. Agonist stimulation with PP or 1229U91 dissociates these homodimers in a concentration-dependent manner. Y4 receptor does not heterodimerize with Y1, Y2, Y5, delta opioid, or mu opioid receptors.","method":"BRET2 (bioluminescence resonance energy transfer), Western blot analysis, C-terminal fusion with Renilla luciferase and GFP in transfected cells","journal":"The Journal of pharmacology and experimental therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (BRET2 and Western blot) for dimerization; single lab","pmids":["14551289"],"is_preprint":false},{"year":2003,"finding":"Y4 receptors are functionally expressed on orexin neurons in the lateral hypothalamic area; intra-LHA injection of the Y4-selective agonist rat PP (but not NPY) specifically activates c-Fos in orexin neurons and stimulates food and water intake, placing Y4R signaling upstream of orexin neuron activation in feeding regulation.","method":"Immunocytochemistry (co-localization of Y4 and orexin), in situ hybridization, stereotaxic intra-LHA injection of rPP and NPY, c-Fos immunohistochemistry","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ICC co-localization, ISH, functional c-Fos activation with selective ligand) establishing receptor localization and functional coupling to orexin neurons","pmids":["12598637"],"is_preprint":false},{"year":2003,"finding":"Double knockout of Y2 and Y4 receptors produces synergistic effects greater than either single knockout: a markedly lean phenotype despite hyperphagia, and a ~3-fold increase in bone volume with enhanced osteoblastic activity. Bone mass is not altered in Y4 single-knockout mice, suggesting Y4 pathway alone is insufficient but synergizes with Y2 pathway to regulate bone volume and adiposity.","method":"Double-knockout mouse genetics, dual-energy X-ray absorptiometry, bone histomorphometry, plasma hormone measurements","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in double-knockout model with multiple quantitative phenotypic endpoints; demonstrates synergy between Y2 and Y4 pathways","pmids":["12861009"],"is_preprint":false},{"year":2002,"finding":"ERK1/2 phosphorylation by Y1, Y2, Y4, and Y5 receptors is blocked by pertussis toxin, indicating all four receptors couple to Gi/o to activate ERK. Y4 receptor-mediated ERK phosphorylation is reduced (but not eliminated) by the PKC inhibitor GF109203X, while Y5-mediated ERK activation is completely blocked by both PKC and PI3K inhibitors, revealing distinct downstream signaling divergence.","method":"ERK phosphorylation assays with pertussis toxin and selective kinase inhibitors in CHO cells expressing human Y1, Y2, Y4, or Y5 receptors","journal":"Regulatory peptides","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean pharmacological dissection of signaling pathways using multiple inhibitors at cloned receptors; single lab","pmids":["11853873"],"is_preprint":false},{"year":2006,"finding":"NPY4R agonist selectivity analysis in human and mouse colon: rPP responses are abolished in Y4-knockout mucosal preparations, establishing that Y4 receptors mediate antisecretory effects of PP at colonic epithelial cells. GR231118 acted as partial agonist at hY4 receptor; hPP internalization of Y4R traffics to recycling (transferrin-positive) endosomal pathways in HEK293 cells.","method":"Ussing chamber electrophysiology in Y4-knockout mouse tissue, radioligand displacement in Y4-transfected HEK293 cells, GTPγS binding, receptor internalization assay","journal":"The Journal of pharmacology and experimental therapeutics","confidence":"High","confidence_rationale":"Tier 2 / Strong — definitive loss-of-function (Y4-KO tissue) combined with in vitro binding, functional GTPγS, and trafficking assays across multiple experimental systems","pmids":["16807358"],"is_preprint":false},{"year":2007,"finding":"Y4 receptor knockout mice show reduced Y4 receptor-dependent c-Fos activation specifically in NTS/area postrema and arcuate nucleus POMC neurons after i.p. PP injection. Hypothalamus-specific conditional Y4 knockout abolishes PP-induced arcuate c-Fos activation and POMC mRNA upregulation. The hypophagic effect of PP is completely absent in melanocortin-4 receptor (MC4R) knockout mice, placing Y4R upstream of POMC/α-MSH/MC4R in the anorexigenic pathway.","method":"Global and conditional (hypothalamus-specific) Y4-knockout mice, c-Fos immunohistochemistry, in situ hybridization for POMC mRNA, MC4R-knockout epistasis, food intake measurement","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout with two independent KO lines, pathway epistasis (MC4R-KO), and multiple molecular readouts establishing Y4R→POMC→MC4R pathway","pmids":["20041129"],"is_preprint":false},{"year":2007,"finding":"Fasting-induced reduction of GnRH mRNA expression in the medial preoptic area and testis testosterone content are abolished in Y4-knockout mice but not Y2-knockout mice, while fasting-induced GHRH inhibition requires Y2 (not Y4) receptors. No Y4 receptors were detected on GnRH neurons, indicating that Y4 receptors indirectly regulate the gonadotropic axis.","method":"Y2 and Y4 receptor single and double knockout mice, hypothalamic conditional Y2 knockout, in situ hybridization, plasma hormone ELISA, immunohistochemistry","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple knockout models with pathway-specific phenotypes and negative localization data; demonstrates indirect Y4R regulation of GnRH axis","pmids":["17272395"],"is_preprint":false},{"year":2008,"finding":"NMR spectroscopy of the isolated N-terminal domain of Y4 receptor (N-Y4) shows it is unstructured in solution but forms a helical segment in DPC/SDS micelles. Surface plasmon resonance reveals ~50 μM affinity binding of N-Y4 to PP; NPY and PYY bind much more weakly. Mutagenesis identifies electrostatic interactions between acidic ligand residues and basic N-Y4 receptor residues as the dominant binding determinants.","method":"Solution NMR spectroscopy, surface plasmon resonance, site-directed mutagenesis of N-terminal domain","journal":"Chembiochem","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — NMR structure and SPR binding with mutagenesis; single lab, interaction studied in isolated domain not full receptor","pmids":["18767100"],"is_preprint":false},{"year":2009,"finding":"PP enhances colonic muscle contraction and increases fecal output in wild-type mice but these effects are completely absent in Y4-receptor-knockout mice. PP-induced increases in colonic muscle tone and spontaneous contraction frequency in isolated colon are absent in KO mice. Atropine does not affect PP-induced responses, indicating Y4R-mediated colonic contractility is not cholinergic.","method":"Y4-knockout mice, fecal output quantification, isolated colon contractility assay, pharmacological dissection with atropine","journal":"European journal of pharmacology","confidence":"High","confidence_rationale":"Tier 2 / Strong — definitive loss-of-function with KO mice, ex vivo and in vivo endpoints, pharmacological mechanism","pmids":["19818748"],"is_preprint":false},{"year":2010,"finding":"Peripherally administered PP activates orexin neurons in LHA and BDNF-expressing neurons in VMH via Y4 receptor-dependent c-Fos induction (absent in Y4-KO mice). PP injection down-regulates orexin mRNA in LHA in a Y4 receptor-dependent manner and up-regulates BDNF mRNA in VMH, placing Y4R signaling upstream of both orexin and BDNF pathways in appetite regulation.","method":"Y4-knockout mice, immunofluorescence c-Fos mapping, in situ hybridization for orexin and BDNF mRNA, i.p. PP injection","journal":"Neuropeptides","confidence":"High","confidence_rationale":"Tier 2 / Strong — Y4-KO loss-of-function with multiple molecular readouts (c-Fos, mRNA) establishing pathway position","pmids":["20116098"],"is_preprint":false},{"year":2013,"finding":"Double-cycle mutagenesis and homology modeling of the hY4R-hPP complex identify residues Tyr(2.64), Asp(2.68), Asn(6.55), Asn(7.32), and Phe(7.35) of Y4R as critical for receptor activation by hPP. TM2 top and TM6-TM7 top form the core peptide binding pocket; Asn(7.32) likely hydrogen-bonds Arg(33) of hPP, and Phe(7.35) contacts hPP positions 33 and 36.","method":"Site-directed mutagenesis, double-cycle mutagenesis analysis, homology modeling based on class A GPCR crystal structures, radioligand binding and functional assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — active-site mutagenesis with computational model; double-cycle approach provides mechanistic constraint but structural model is computational not experimental","pmids":["24375409"],"is_preprint":false},{"year":2014,"finding":"PPYR1 (NPY4R) protein is expressed in alpha-cells of mouse and human pancreatic islets (confirmed by immunoblotting, quantitative RT-PCR on FACS-sorted alpha-cells, and immunohistochemistry), but not in other islet cell types or exocrine pancreas. Glucose stimulation increases PP secretion, and direct exposure of islets to PP inhibits glucagon release, establishing a PP→PPYR1→glucagon suppression pathway in alpha-cells.","method":"Immunoblotting, quantitative RT-PCR on FACS-sorted pancreatic islet cell fractions, immunohistochemistry, isolated islet glucagon secretion assay","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 2 / Strong — receptor localization by three orthogonal methods combined with functional hormone secretion assay; both mouse and human tissue","pmids":["25445712"],"is_preprint":false},{"year":2021,"finding":"A selective allosteric antagonist (S)-VU0637120 inhibits native Y4R function and binds in an allosteric site located below the endogenous PP binding pocket within the core of Y4R transmembrane domains, as determined by in vitro, ex vivo, and in silico studies. This represents the first nonpeptide Y4R-selective antagonist and identifies an allosteric regulatory site distinct from the orthosteric PP binding pocket.","method":"Radioligand binding, functional assays, Ussing chamber ex vivo epithelium assay, computational docking/modeling, medicinal chemistry SAR","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — functional in vitro and ex vivo assays with computational site identification; allosteric binding pocket is inferred computationally without crystallographic validation","pmids":["33595306"],"is_preprint":false},{"year":2016,"finding":"CNV loss in the NPY4R-containing 10q11.22 region is significantly more frequent in obese children than controls, and two rare non-synonymous heterozygous NPY4R coding variants identified only in the obese patient population demonstrate receptor dysfunction in functional assays, establishing a causal link between NPY4R loss-of-function and obesity pathogenesis.","method":"Genome-wide CNV analysis, mutation screening of NPY4R coding region, functional receptor assays for rare variants","journal":"Obesity (Silver Spring, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — human genetic association combined with functional receptor validation of specific variants; single study but two complementary approaches","pmids":["26921218"],"is_preprint":false},{"year":2005,"finding":"Endogenous NPY acting via Y2 and Y4 receptors suppresses afferent c-Fos signaling from gastric acid challenge to the nucleus tractus solitarii. Y4-knockout mice show a 31% higher c-Fos response to intragastric HCl, and BIIE0246 (Y2 antagonist not crossing the blood-brain barrier) does not block the effect, indicating the Y4R-mediated suppression occurs centrally.","method":"Y2 and Y4 receptor-knockout mice, intragastric HCl challenge, c-Fos immunohistochemistry in NTS, peripheral vs. central antagonist discrimination","journal":"Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO loss-of-function with pharmacological dissection of CNS vs. peripheral site of action; single lab","pmids":["16216428"],"is_preprint":false}],"current_model":"NPY4R (Y4/PPYR1) is a GPCR that preferentially binds pancreatic polypeptide (PP) with high affinity and signals through pertussis toxin-sensitive Gi to inhibit cAMP and through Gq to mobilize intracellular Ca2+ and initiate smooth muscle contraction; it forms agonist-dissociable homodimers at the cell surface, undergoes clathrin-mediated internalization (rat/guinea-pig Y4R) but is notably resistant to agonist-promoted desensitization (human Y4R); the transmembrane binding pocket (TM2 top and TM6-TM7) mediates PP recognition via electrostatic and hydrophobic contacts, and an allosteric site within the TM core can be targeted by small-molecule antagonists; in vivo, Y4R signaling in the arcuate nucleus activates POMC/α-MSH→MC4R to suppress food intake, indirectly inhibits the GnRH/gonadotropic axis, synergizes with Y2R to regulate bone mass and adiposity, mediates PP-induced colonic contractility, and suppresses glucagon secretion from pancreatic alpha-cells via PPYR1 expressed on those cells."},"narrative":{"mechanistic_narrative":"NPY4R (Y4/PPYR1) is a class A G-protein-coupled receptor that preferentially binds pancreatic polypeptide (PP) and serves as the principal receptor through which PP regulates appetite, endocrine secretion, and gut motility [PMID:7592911, PMID:20041129]. The cloned human receptor binds PP with high affinity in a ligand hierarchy of PP > PYY > NPY and couples to pertussis toxin-sensitive Gi to inhibit cAMP while also mobilizing intracellular Ca2+ [PMID:7592911, PMID:10640301]; in smooth muscle it additionally engages Gq to drive PLC/IP3-dependent Ca2+ release and contraction, and it activates ERK1/2 partly through PKC [PMID:15308651, PMID:11853873]. PP recognition is mediated by the receptor N-terminus and a transmembrane pocket formed by the TM2 top and TM6-TM7 region, where electrostatic and hydrogen-bonding contacts with PP residues govern activation, and an allosteric site beneath the orthosteric pocket can be targeted by selective small-molecule antagonists [PMID:18767100, PMID:24375409, PMID:33595306]. The receptor forms agonist-dissociable homodimers and, in rodent orthologs, undergoes rapid clathrin-mediated internalization to recycling endosomes, whereas human Y4R is notably resistant to agonist-promoted desensitization and internalization [PMID:14551289, PMID:11179953, PMID:10640301, PMID:16807358]. In vivo, central Y4R signaling acts upstream of arcuate POMC/α-MSH/MC4R to suppress food intake and modulates LHA orexin and VMH BDNF neuron activity, indirectly inhibits the GnRH/gonadotropic axis, synergizes with Y2R to control bone mass and adiposity, mediates PP-driven colonic contractility and antisecretory responses, and suppresses glucagon release via PPYR1 expressed on pancreatic alpha-cells [PMID:20041129, PMID:17272395, PMID:12861009, PMID:19818748, PMID:25445712]. Human genetic loss of NPY4R is causally linked to obesity through CNV loss and loss-of-function coding variants [PMID:26921218].","teleology":[{"year":1995,"claim":"Establishing the identity and signaling output of the human Y4 receptor answered whether a distinct high-affinity PP receptor exists and how it transduces signal.","evidence":"Homology cloning with radioligand binding, cAMP RIA and Ca2+ assays in transfected cells","pmids":["7592911"],"confidence":"High","gaps":["Endogenous tissue distribution not defined in the cloning study","Structural basis of PP selectivity unresolved"]},{"year":1997,"claim":"Comparison of rat, guinea pig, and chicken orthologs resolved how species-specific sequence divergence reshapes ligand selectivity, distinguishing PP-selective mammalian receptors from the broadly tuned avian receptor.","evidence":"Cross-species cloning with radioligand binding and cAMP assays in transfected cells","pmids":["9210181","9802391","12063188"],"confidence":"Medium","gaps":["Residues responsible for divergent selectivity mapped only later","Physiological consequence of selectivity differences not addressed"]},{"year":2000,"claim":"Biochemical and trafficking characterization defined human Y4R as a glycosylated Gi-coupled receptor uniquely resistant to desensitization, while rodent Y4R internalizes rapidly, establishing a divergent regulatory behavior.","evidence":"Stable CHO transfectants with cross-linking, pertussis toxin, immunofluorescence, and clathrin-pathway inhibitor dissection","pmids":["10640301","11179953","16807358"],"confidence":"High","gaps":["Molecular basis for human/rodent desensitization difference unknown","Phosphorylation/arrestin determinants not identified"]},{"year":1998,"claim":"Pharmacological probes (GR231118/1229U91) demonstrated that the NPY C-terminus suffices to activate Y4R, providing tools to dissect Y4 versus Y1 signaling.","evidence":"Radioligand binding and cAMP inhibition assays across cloned Y1, Y2, Y4, Y5 receptors","pmids":["9669502"],"confidence":"High","gaps":["Partial-agonist behavior at hY4R clarified only later","Binding-pocket contacts not defined by this study"]},{"year":2002,"claim":"Defining receptor coupling and dimerization showed Y4R signals through both Gi and Gq, activates ERK via Gi/PKC, and forms agonist-dissociable homodimers that do not heterodimerize with other Y subtypes.","evidence":"Contraction/IP3/Ca2+ assays in smooth muscle, ERK phosphorylation with inhibitors, BRET2 and Western blot dimerization assays","pmids":["15308651","11853873","14551289"],"confidence":"High","gaps":["Functional consequence of dimer dissociation not established","Stoichiometry and structural interface of dimer unknown"]},{"year":2002,"claim":"Genetic epistasis placed central Y4R as an inhibitor of the gonadotropic axis, explaining how elevated NPY tone suppresses fertility.","evidence":"Y4 knockout crossed onto ob/ob background with hormone, morphometric, and GnRH mRNA readouts","pmids":["12000791","17272395"],"confidence":"High","gaps":["Y4R not detected on GnRH neurons, so the intermediate circuit is undefined","Cell type mediating indirect regulation unidentified"]},{"year":2003,"claim":"Hypothalamic and brainstem mapping with selective agonists positioned Y4R upstream of orexin, BDNF, and POMC neuron populations controlling feeding.","evidence":"Y4-KO mice, c-Fos mapping, in situ hybridization, and intra-LHA/i.p. ligand injection","pmids":["12598637","20116098","20041129"],"confidence":"High","gaps":["Direct versus indirect activation of each neuron population not fully resolved","Apparent bidirectional feeding effects across studies not reconciled"]},{"year":2003,"claim":"Double-knockout genetics revealed Y4R synergy with Y2R in regulating bone volume and adiposity, showing Y4R alone is insufficient for bone phenotypes.","evidence":"Y2/Y4 double-knockout mice with DXA, bone histomorphometry, and hormone measurements","pmids":["12861009"],"confidence":"High","gaps":["Molecular basis of Y2/Y4 synergy unknown","Cell type and signaling node mediating bone effects undefined"]},{"year":2006,"claim":"Loss-of-function tissue studies established Y4R as the mediator of PP effects in the colon, including antisecretory and contractile responses, and defined recycling-endosome trafficking.","evidence":"Y4-KO colonic mucosa in Ussing chambers, contractility assays, GTPγS binding, and transferrin co-trafficking in HEK293","pmids":["16807358","19818748"],"confidence":"High","gaps":["Effector mechanism downstream of Y4R in epithelium not detailed","Non-cholinergic contraction pathway only partially dissected"]},{"year":2008,"claim":"Structural and mutagenesis studies defined the molecular determinants of PP recognition, identifying an N-terminal helix and TM2/TM6-TM7 pocket residues that contact PP, and later an allosteric antagonist site.","evidence":"Solution NMR and SPR of N-Y4 domain, double-cycle mutagenesis with homology modeling, and SAR/docking for (S)-VU0637120","pmids":["18767100","24375409","33595306"],"confidence":"Medium","gaps":["No experimental full-length receptor structure","Allosteric site location inferred computationally without crystallographic validation"]},{"year":2014,"claim":"Cell-type-resolved expression and functional assays placed PPYR1 on pancreatic alpha-cells where it mediates PP suppression of glucagon, extending Y4R function to islet endocrine control.","evidence":"Immunoblotting, qRT-PCR on FACS-sorted alpha-cells, immunohistochemistry, and isolated islet glucagon secretion assays in mouse and human","pmids":["25445712"],"confidence":"High","gaps":["Intracellular signaling coupling glucagon suppression not detailed","In vivo contribution to glucose homeostasis not quantified"]},{"year":2016,"claim":"Human genetic analysis linked NPY4R loss-of-function to obesity, connecting the receptor's anorexigenic role to disease.","evidence":"Genome-wide CNV analysis and coding-variant screening with functional receptor assays","pmids":["26921218"],"confidence":"Medium","gaps":["Penetrance and population-level effect size not established","Mechanistic link between variant dysfunction and adiposity not demonstrated in vivo"]},{"year":null,"claim":"How human Y4R achieves desensitization resistance and how its central circuits coordinate the opposing orexigenic and anorexigenic feeding outputs remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experimental structure of the active receptor-G protein complex","Identity of intermediate neurons linking Y4R to GnRH and orexin circuits unknown","Regulatory determinants distinguishing human from rodent trafficking undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,4,7]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,2]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,9]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[5,13]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4,7]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[5,13]}],"complexes":[],"partners":["NPY2R"],"other_free_text":[]}},"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 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a G protein-coupled receptor - and its interaction with hormones from the NPY family.","date":"2008","source":"Chembiochem : a European journal of chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/18767100","citation_count":20,"is_preprint":false},{"pmid":"24375569","id":"PMC_24375569","title":"Concomitant activation of the PI3K/Akt and ERK1/2 signalling is involved in cyclic compressive force-induced IL-6 secretion in MLO-Y4 cells.","date":"2014","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/24375569","citation_count":20,"is_preprint":false},{"pmid":"12909193","id":"PMC_12909193","title":"Ligand internalization by cloned neuropeptide Y Y5 receptors excludes Y2 and Y4 receptor-selective peptides.","date":"2003","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/12909193","citation_count":20,"is_preprint":false},{"pmid":"26713363","id":"PMC_26713363","title":"Activation of AMP-activated protein kinase decreases receptor activator of NF-κB ligand expression and increases sclerostin expression by inhibiting the mevalonate pathway in osteocytic MLO-Y4 cells.","date":"2015","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/26713363","citation_count":19,"is_preprint":false},{"pmid":"20600429","id":"PMC_20600429","title":"Regulatory effects of Y4 receptor agonist (BVD-74D) on food intake.","date":"2010","source":"Peptides","url":"https://pubmed.ncbi.nlm.nih.gov/20600429","citation_count":19,"is_preprint":false},{"pmid":"19818748","id":"PMC_19818748","title":"Pancreatic polypeptide enhances colonic muscle contraction and fecal output through neuropeptide Y Y4 receptor in mice.","date":"2009","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/19818748","citation_count":19,"is_preprint":false},{"pmid":"11287089","id":"PMC_11287089","title":"Studies of the human, rat, and guinea pig Y4 receptors using neuropeptide Y analogues and two distinct radioligands.","date":"2001","source":"Peptides","url":"https://pubmed.ncbi.nlm.nih.gov/11287089","citation_count":19,"is_preprint":false},{"pmid":"17204349","id":"PMC_17204349","title":"Neuropeptide Y (NPY) Y2 receptor-selective agonist inhibits food intake and promotes fat metabolism in mice: combined anorectic effects of Y2 and Y4 receptor-selective agonists.","date":"2007","source":"Peptides","url":"https://pubmed.ncbi.nlm.nih.gov/17204349","citation_count":19,"is_preprint":false},{"pmid":"32161481","id":"PMC_32161481","title":"High Glucose Downregulates Connexin 43 Expression and Its Gap Junction and Hemichannel Function in Osteocyte-like MLO-Y4 Cells Through Activation of the p38MAPK/ERK Signal Pathway.","date":"2020","source":"Diabetes, metabolic syndrome and obesity : targets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/32161481","citation_count":18,"is_preprint":false},{"pmid":"33595306","id":"PMC_33595306","title":"Highly Selective Y4 Receptor Antagonist Binds in an Allosteric Binding Pocket.","date":"2021","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33595306","citation_count":18,"is_preprint":false},{"pmid":"29018002","id":"PMC_29018002","title":"Glucose uptake inhibition decreases expressions of receptor activator of nuclear factor-kappa B ligand (RANKL) and osteocalcin in osteocytic MLO-Y4-A2 cells.","date":"2017","source":"American journal of physiology. Endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/29018002","citation_count":18,"is_preprint":false},{"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},{"pmid":"37678662","id":"PMC_37678662","title":"ROS-mediated mitophagy and necroptosis regulate osteocytes death caused by TCP particles in MLO-Y4 cells.","date":"2023","source":"Toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/37678662","citation_count":17,"is_preprint":false},{"pmid":"35889450","id":"PMC_35889450","title":"Enhancing the Activity of Carboxymethyl Cellulase Enzyme Using Highly Stable Selenium Nanoparticles Biosynthesized by Bacillus paralicheniformis Y4.","date":"2022","source":"Molecules (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/35889450","citation_count":17,"is_preprint":false},{"pmid":"16216428","id":"PMC_16216428","title":"Endogenous neuropeptide Y depresses the afferent signaling of gastric acid challenge to the mouse brainstem via neuropeptide Y type Y2 and Y4 receptors.","date":"2005","source":"Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/16216428","citation_count":17,"is_preprint":false},{"pmid":"17979782","id":"PMC_17979782","title":"NPY Y2 and Y4 receptors selective ligands: promising anti-obesity drugs?","date":"2007","source":"Current topics in medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17979782","citation_count":16,"is_preprint":false},{"pmid":"20820919","id":"PMC_20820919","title":"Low-intensity pulsed ultrasound modulates shear stress induced PGHS-2 expression and PGE2 synthesis in MLO-Y4 osteocyte-like cells.","date":"2010","source":"Annals of biomedical engineering","url":"https://pubmed.ncbi.nlm.nih.gov/20820919","citation_count":16,"is_preprint":false},{"pmid":"11156595","id":"PMC_11156595","title":"Constitutive neuropeptide Y Y(4) receptor expression in human colonic adenocarcinoma cell lines.","date":"2001","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/11156595","citation_count":16,"is_preprint":false},{"pmid":"16213038","id":"PMC_16213038","title":"Internalization of cloned pancreatic polypeptide receptors is accelerated by all types of Y4 agonists.","date":"2005","source":"Regulatory peptides","url":"https://pubmed.ncbi.nlm.nih.gov/16213038","citation_count":16,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":56320,"output_tokens":6527,"usd":0.133433,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":15603,"output_tokens":3922,"usd":0.088032,"stage2_stop_reason":"end_turn"},"total_usd":0.221465,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"The human Y4 receptor (NPY4R/PPYR1) was cloned from a human placental genomic library and encodes a 375-amino acid GPCR. When transiently expressed, it binds 125I-PYY with high affinity (pKd=9.89) and shows ligand selectivity: PP > PYY > NPY. Human PP activation decreases cAMP and increases intracellular Ca2+ in Y4-transfected cells, establishing Gi-coupled and Ca2+-mobilizing signaling.\",\n      \"method\": \"Homology cloning, radioligand binding (125I-PYY), cAMP radioimmunoassay, intracellular Ca2+ measurement in transfected LMTK- cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — primary cloning paper with reconstituted binding, functional cAMP and Ca2+ assays, multiple orthogonal methods in the founding study\",\n      \"pmids\": [\"7592911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The rat Y4 receptor was cloned and expressed; its pharmacological profile shows high selectivity for rat PP over NPY or PYY. The rat and human Y4 receptors share 75% amino acid identity but differ substantially in ligand selectivity and dependence on N-terminal PP residues, indicating species-specific ligand–receptor interactions.\",\n      \"method\": \"Cloning, 125I-PP radioligand binding, cAMP radioimmunoassay in transfected cells\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — independent cloning of rat ortholog with binding and functional assays, replicating and extending founding human Y4R study\",\n      \"pmids\": [\"9210181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"GR231118 (1229U91) acts as a potent Y1 receptor antagonist (pA2=10.5 at human Y1) and simultaneously as a potent Y4 receptor agonist (pEC50=8.6 at human Y4), demonstrating that the C-terminus of NPY is sufficient for Y4 receptor activation but not Y1 activation. Activity confirmed in inhibition of forskolin-stimulated cAMP formation.\",\n      \"method\": \"Radioligand binding assays, functional cAMP inhibition assay across cloned Y1, Y2, Y4, Y5 receptors\",\n      \"journal\": \"European journal of pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstituted binding and functional assays at multiple cloned receptor subtypes; single lab but two orthogonal methods\",\n      \"pmids\": [\"9669502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The guinea pig Y4 receptor was cloned; it is 84% identical to human Y4 (vs 74-75% for rat Y4) and its pharmacological profile (PP > NPY ≈ PYY > NPY2-36) is closer to human than rat Y4, establishing that accelerated sequence divergence in rodent Y4 receptors alters ligand selectivity.\",\n      \"method\": \"Cloning, 125I-hPP saturation and competition binding in expressed guinea pig Y4 receptor\",\n      \"journal\": \"Regulatory peptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous binding assay but single lab, single species characterization\",\n      \"pmids\": [\"9802391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Stable CHO cells expressing human Y4 receptor demonstrate: (1) a single binding site for 125I-hPP (Kd=0.26 nM), (2) a glycosylated receptor protein of Mr 60,000 identified by cross-linking, (3) hPP inhibits cAMP via pertussis toxin-sensitive Gi protein (EC50=0.56 nM), and (4) 24-hour exposure to 10 nM hPP produces no receptor desensitization, no reduction in binding capacity, and no internalization — establishing resistance to agonist-promoted desensitization as a defining functional property of human Y4R.\",\n      \"method\": \"Radioligand binding, cross-linking/SDS-PAGE, cAMP RIA, pertussis toxin treatment, immunofluorescence in stable CHO transfectants\",\n      \"journal\": \"The Journal of pharmacology and experimental therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal methods (binding, biochemical, functional, imaging) in a single rigorous study; stable expression system with mechanistic controls\",\n      \"pmids\": [\"10640301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Cloned rat pancreatic polypeptide Y4 receptor expressed in CHO cells undergoes rapid, temperature-dependent, agonist-driven internalization via clathrin-mediated endocytosis (blocked by sucrose and phenylarsine oxide), in contrast to the Y2 receptor which shows minimal internalization. Internalized Y4 receptors traffic to endosome-like particulates, and receptor recovery is blocked by cycloheximide and monensin, indicating requirement for new protein synthesis and intact vesicular transport.\",\n      \"method\": \"Ligand internalization assays in CHO cells, pharmacological inhibitors (sucrose, phenylarsine oxide, cycloheximide, monensin), sucrose density gradient fractionation\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct subcellular trafficking assays with multiple pharmacological dissections, single lab\",\n      \"pmids\": [\"11179953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The chicken Y4 receptor was cloned and characterized; it binds PYYa, chicken PP, and NPY with similarly high affinities (Kd 68-160 pM), contrasting with mammalian Y4R which strongly prefers PP. RT-PCR and in situ hybridization showed expression in the dorsal motor nucleus of the vagus in the brainstem, suggesting conserved anatomical targeting.\",\n      \"method\": \"Cloning, 125I-pPYY saturation and competition binding in COS-7 cells, RT-PCR, in situ hybridization\",\n      \"journal\": \"Journal of molecular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous binding characterization of avian ortholog, single lab\",\n      \"pmids\": [\"12063188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Y2 and Y4 receptors are co-expressed on smooth muscle cells and both couple to Gq in addition to Gi2, enabling IP3-dependent Ca2+ release and muscle contraction. Y4 receptor activation by pancreatic polypeptide induces contraction insensitive to extracellular Ca2+ removal or L-type Ca2+ channel blockers but abolished by thapsigargin or PLC inhibitor U73122, establishing Y4R-Gq-IP3-Ca2+ release pathway for contraction.\",\n      \"method\": \"RT-PCR, radioligand binding with selective receptor protection, functional contraction assays, IP3 measurement, Ca2+ imaging, pertussis toxin treatment, pharmacological inhibitors in isolated smooth muscle cells\",\n      \"journal\": \"The Journal of pharmacology and experimental therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal methods (binding, IP3, Ca2+, contraction) with selective receptor protection strategy; single lab but highly systematic\",\n      \"pmids\": [\"15308651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Genetic deletion of Y4 receptor in ob/ob mice restores testosterone levels, testis and seminal vesicle size, and fertility in males (100% offspring production), and is associated with increased GnRH expression. This epistatic rescue establishes that central Y4 receptor signaling specifically inhibits the gonadotropic axis under conditions of elevated NPY tone.\",\n      \"method\": \"Genetic epistasis: Y4 knockout crossed onto ob/ob background; hormone ELISA, organ morphometry, GnRH mRNA analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis with multiple phenotypic readouts (fertility, hormones, organ morphology, GnRH expression) establishing pathway position\",\n      \"pmids\": [\"12000791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The rhesus Y4 receptor forms homodimers at the cell surface as demonstrated by BRET2 and Western blot analysis. Agonist stimulation with PP or 1229U91 dissociates these homodimers in a concentration-dependent manner. Y4 receptor does not heterodimerize with Y1, Y2, Y5, delta opioid, or mu opioid receptors.\",\n      \"method\": \"BRET2 (bioluminescence resonance energy transfer), Western blot analysis, C-terminal fusion with Renilla luciferase and GFP in transfected cells\",\n      \"journal\": \"The Journal of pharmacology and experimental therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (BRET2 and Western blot) for dimerization; single lab\",\n      \"pmids\": [\"14551289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Y4 receptors are functionally expressed on orexin neurons in the lateral hypothalamic area; intra-LHA injection of the Y4-selective agonist rat PP (but not NPY) specifically activates c-Fos in orexin neurons and stimulates food and water intake, placing Y4R signaling upstream of orexin neuron activation in feeding regulation.\",\n      \"method\": \"Immunocytochemistry (co-localization of Y4 and orexin), in situ hybridization, stereotaxic intra-LHA injection of rPP and NPY, c-Fos immunohistochemistry\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ICC co-localization, ISH, functional c-Fos activation with selective ligand) establishing receptor localization and functional coupling to orexin neurons\",\n      \"pmids\": [\"12598637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Double knockout of Y2 and Y4 receptors produces synergistic effects greater than either single knockout: a markedly lean phenotype despite hyperphagia, and a ~3-fold increase in bone volume with enhanced osteoblastic activity. Bone mass is not altered in Y4 single-knockout mice, suggesting Y4 pathway alone is insufficient but synergizes with Y2 pathway to regulate bone volume and adiposity.\",\n      \"method\": \"Double-knockout mouse genetics, dual-energy X-ray absorptiometry, bone histomorphometry, plasma hormone measurements\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in double-knockout model with multiple quantitative phenotypic endpoints; demonstrates synergy between Y2 and Y4 pathways\",\n      \"pmids\": [\"12861009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ERK1/2 phosphorylation by Y1, Y2, Y4, and Y5 receptors is blocked by pertussis toxin, indicating all four receptors couple to Gi/o to activate ERK. Y4 receptor-mediated ERK phosphorylation is reduced (but not eliminated) by the PKC inhibitor GF109203X, while Y5-mediated ERK activation is completely blocked by both PKC and PI3K inhibitors, revealing distinct downstream signaling divergence.\",\n      \"method\": \"ERK phosphorylation assays with pertussis toxin and selective kinase inhibitors in CHO cells expressing human Y1, Y2, Y4, or Y5 receptors\",\n      \"journal\": \"Regulatory peptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean pharmacological dissection of signaling pathways using multiple inhibitors at cloned receptors; single lab\",\n      \"pmids\": [\"11853873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"NPY4R agonist selectivity analysis in human and mouse colon: rPP responses are abolished in Y4-knockout mucosal preparations, establishing that Y4 receptors mediate antisecretory effects of PP at colonic epithelial cells. GR231118 acted as partial agonist at hY4 receptor; hPP internalization of Y4R traffics to recycling (transferrin-positive) endosomal pathways in HEK293 cells.\",\n      \"method\": \"Ussing chamber electrophysiology in Y4-knockout mouse tissue, radioligand displacement in Y4-transfected HEK293 cells, GTPγS binding, receptor internalization assay\",\n      \"journal\": \"The Journal of pharmacology and experimental therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — definitive loss-of-function (Y4-KO tissue) combined with in vitro binding, functional GTPγS, and trafficking assays across multiple experimental systems\",\n      \"pmids\": [\"16807358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Y4 receptor knockout mice show reduced Y4 receptor-dependent c-Fos activation specifically in NTS/area postrema and arcuate nucleus POMC neurons after i.p. PP injection. Hypothalamus-specific conditional Y4 knockout abolishes PP-induced arcuate c-Fos activation and POMC mRNA upregulation. The hypophagic effect of PP is completely absent in melanocortin-4 receptor (MC4R) knockout mice, placing Y4R upstream of POMC/α-MSH/MC4R in the anorexigenic pathway.\",\n      \"method\": \"Global and conditional (hypothalamus-specific) Y4-knockout mice, c-Fos immunohistochemistry, in situ hybridization for POMC mRNA, MC4R-knockout epistasis, food intake measurement\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout with two independent KO lines, pathway epistasis (MC4R-KO), and multiple molecular readouts establishing Y4R→POMC→MC4R pathway\",\n      \"pmids\": [\"20041129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Fasting-induced reduction of GnRH mRNA expression in the medial preoptic area and testis testosterone content are abolished in Y4-knockout mice but not Y2-knockout mice, while fasting-induced GHRH inhibition requires Y2 (not Y4) receptors. No Y4 receptors were detected on GnRH neurons, indicating that Y4 receptors indirectly regulate the gonadotropic axis.\",\n      \"method\": \"Y2 and Y4 receptor single and double knockout mice, hypothalamic conditional Y2 knockout, in situ hybridization, plasma hormone ELISA, immunohistochemistry\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple knockout models with pathway-specific phenotypes and negative localization data; demonstrates indirect Y4R regulation of GnRH axis\",\n      \"pmids\": [\"17272395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"NMR spectroscopy of the isolated N-terminal domain of Y4 receptor (N-Y4) shows it is unstructured in solution but forms a helical segment in DPC/SDS micelles. Surface plasmon resonance reveals ~50 μM affinity binding of N-Y4 to PP; NPY and PYY bind much more weakly. Mutagenesis identifies electrostatic interactions between acidic ligand residues and basic N-Y4 receptor residues as the dominant binding determinants.\",\n      \"method\": \"Solution NMR spectroscopy, surface plasmon resonance, site-directed mutagenesis of N-terminal domain\",\n      \"journal\": \"Chembiochem\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure and SPR binding with mutagenesis; single lab, interaction studied in isolated domain not full receptor\",\n      \"pmids\": [\"18767100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PP enhances colonic muscle contraction and increases fecal output in wild-type mice but these effects are completely absent in Y4-receptor-knockout mice. PP-induced increases in colonic muscle tone and spontaneous contraction frequency in isolated colon are absent in KO mice. Atropine does not affect PP-induced responses, indicating Y4R-mediated colonic contractility is not cholinergic.\",\n      \"method\": \"Y4-knockout mice, fecal output quantification, isolated colon contractility assay, pharmacological dissection with atropine\",\n      \"journal\": \"European journal of pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — definitive loss-of-function with KO mice, ex vivo and in vivo endpoints, pharmacological mechanism\",\n      \"pmids\": [\"19818748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Peripherally administered PP activates orexin neurons in LHA and BDNF-expressing neurons in VMH via Y4 receptor-dependent c-Fos induction (absent in Y4-KO mice). PP injection down-regulates orexin mRNA in LHA in a Y4 receptor-dependent manner and up-regulates BDNF mRNA in VMH, placing Y4R signaling upstream of both orexin and BDNF pathways in appetite regulation.\",\n      \"method\": \"Y4-knockout mice, immunofluorescence c-Fos mapping, in situ hybridization for orexin and BDNF mRNA, i.p. PP injection\",\n      \"journal\": \"Neuropeptides\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Y4-KO loss-of-function with multiple molecular readouts (c-Fos, mRNA) establishing pathway position\",\n      \"pmids\": [\"20116098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Double-cycle mutagenesis and homology modeling of the hY4R-hPP complex identify residues Tyr(2.64), Asp(2.68), Asn(6.55), Asn(7.32), and Phe(7.35) of Y4R as critical for receptor activation by hPP. TM2 top and TM6-TM7 top form the core peptide binding pocket; Asn(7.32) likely hydrogen-bonds Arg(33) of hPP, and Phe(7.35) contacts hPP positions 33 and 36.\",\n      \"method\": \"Site-directed mutagenesis, double-cycle mutagenesis analysis, homology modeling based on class A GPCR crystal structures, radioligand binding and functional assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — active-site mutagenesis with computational model; double-cycle approach provides mechanistic constraint but structural model is computational not experimental\",\n      \"pmids\": [\"24375409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PPYR1 (NPY4R) protein is expressed in alpha-cells of mouse and human pancreatic islets (confirmed by immunoblotting, quantitative RT-PCR on FACS-sorted alpha-cells, and immunohistochemistry), but not in other islet cell types or exocrine pancreas. Glucose stimulation increases PP secretion, and direct exposure of islets to PP inhibits glucagon release, establishing a PP→PPYR1→glucagon suppression pathway in alpha-cells.\",\n      \"method\": \"Immunoblotting, quantitative RT-PCR on FACS-sorted pancreatic islet cell fractions, immunohistochemistry, isolated islet glucagon secretion assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — receptor localization by three orthogonal methods combined with functional hormone secretion assay; both mouse and human tissue\",\n      \"pmids\": [\"25445712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A selective allosteric antagonist (S)-VU0637120 inhibits native Y4R function and binds in an allosteric site located below the endogenous PP binding pocket within the core of Y4R transmembrane domains, as determined by in vitro, ex vivo, and in silico studies. This represents the first nonpeptide Y4R-selective antagonist and identifies an allosteric regulatory site distinct from the orthosteric PP binding pocket.\",\n      \"method\": \"Radioligand binding, functional assays, Ussing chamber ex vivo epithelium assay, computational docking/modeling, medicinal chemistry SAR\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — functional in vitro and ex vivo assays with computational site identification; allosteric binding pocket is inferred computationally without crystallographic validation\",\n      \"pmids\": [\"33595306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CNV loss in the NPY4R-containing 10q11.22 region is significantly more frequent in obese children than controls, and two rare non-synonymous heterozygous NPY4R coding variants identified only in the obese patient population demonstrate receptor dysfunction in functional assays, establishing a causal link between NPY4R loss-of-function and obesity pathogenesis.\",\n      \"method\": \"Genome-wide CNV analysis, mutation screening of NPY4R coding region, functional receptor assays for rare variants\",\n      \"journal\": \"Obesity (Silver Spring, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — human genetic association combined with functional receptor validation of specific variants; single study but two complementary approaches\",\n      \"pmids\": [\"26921218\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Endogenous NPY acting via Y2 and Y4 receptors suppresses afferent c-Fos signaling from gastric acid challenge to the nucleus tractus solitarii. Y4-knockout mice show a 31% higher c-Fos response to intragastric HCl, and BIIE0246 (Y2 antagonist not crossing the blood-brain barrier) does not block the effect, indicating the Y4R-mediated suppression occurs centrally.\",\n      \"method\": \"Y2 and Y4 receptor-knockout mice, intragastric HCl challenge, c-Fos immunohistochemistry in NTS, peripheral vs. central antagonist discrimination\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO loss-of-function with pharmacological dissection of CNS vs. peripheral site of action; single lab\",\n      \"pmids\": [\"16216428\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NPY4R (Y4/PPYR1) is a GPCR that preferentially binds pancreatic polypeptide (PP) with high affinity and signals through pertussis toxin-sensitive Gi to inhibit cAMP and through Gq to mobilize intracellular Ca2+ and initiate smooth muscle contraction; it forms agonist-dissociable homodimers at the cell surface, undergoes clathrin-mediated internalization (rat/guinea-pig Y4R) but is notably resistant to agonist-promoted desensitization (human Y4R); the transmembrane binding pocket (TM2 top and TM6-TM7) mediates PP recognition via electrostatic and hydrophobic contacts, and an allosteric site within the TM core can be targeted by small-molecule antagonists; in vivo, Y4R signaling in the arcuate nucleus activates POMC/α-MSH→MC4R to suppress food intake, indirectly inhibits the GnRH/gonadotropic axis, synergizes with Y2R to regulate bone mass and adiposity, mediates PP-induced colonic contractility, and suppresses glucagon secretion from pancreatic alpha-cells via PPYR1 expressed on those cells.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NPY4R (Y4/PPYR1) is a class A G-protein-coupled receptor that preferentially binds pancreatic polypeptide (PP) and serves as the principal receptor through which PP regulates appetite, endocrine secretion, and gut motility [#0, #14]. The cloned human receptor binds PP with high affinity in a ligand hierarchy of PP > PYY > NPY and couples to pertussis toxin-sensitive Gi to inhibit cAMP while also mobilizing intracellular Ca2+ [#0, #4]; in smooth muscle it additionally engages Gq to drive PLC/IP3-dependent Ca2+ release and contraction, and it activates ERK1/2 partly through PKC [#7, #12]. PP recognition is mediated by the receptor N-terminus and a transmembrane pocket formed by the TM2 top and TM6-TM7 region, where electrostatic and hydrogen-bonding contacts with PP residues govern activation, and an allosteric site beneath the orthosteric pocket can be targeted by selective small-molecule antagonists [#16, #19, #21]. The receptor forms agonist-dissociable homodimers and, in rodent orthologs, undergoes rapid clathrin-mediated internalization to recycling endosomes, whereas human Y4R is notably resistant to agonist-promoted desensitization and internalization [#9, #5, #4, #13]. In vivo, central Y4R signaling acts upstream of arcuate POMC/\\u03b1-MSH/MC4R to suppress food intake and modulates LHA orexin and VMH BDNF neuron activity, indirectly inhibits the GnRH/gonadotropic axis, synergizes with Y2R to control bone mass and adiposity, mediates PP-driven colonic contractility and antisecretory responses, and suppresses glucagon release via PPYR1 expressed on pancreatic alpha-cells [#14, #15, #11, #17, #20]. Human genetic loss of NPY4R is causally linked to obesity through CNV loss and loss-of-function coding variants [#22].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing the identity and signaling output of the human Y4 receptor answered whether a distinct high-affinity PP receptor exists and how it transduces signal.\",\n      \"evidence\": \"Homology cloning with radioligand binding, cAMP RIA and Ca2+ assays in transfected cells\",\n      \"pmids\": [\"7592911\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous tissue distribution not defined in the cloning study\", \"Structural basis of PP selectivity unresolved\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Comparison of rat, guinea pig, and chicken orthologs resolved how species-specific sequence divergence reshapes ligand selectivity, distinguishing PP-selective mammalian receptors from the broadly tuned avian receptor.\",\n      \"evidence\": \"Cross-species cloning with radioligand binding and cAMP assays in transfected cells\",\n      \"pmids\": [\"9210181\", \"9802391\", \"12063188\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Residues responsible for divergent selectivity mapped only later\", \"Physiological consequence of selectivity differences not addressed\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Biochemical and trafficking characterization defined human Y4R as a glycosylated Gi-coupled receptor uniquely resistant to desensitization, while rodent Y4R internalizes rapidly, establishing a divergent regulatory behavior.\",\n      \"evidence\": \"Stable CHO transfectants with cross-linking, pertussis toxin, immunofluorescence, and clathrin-pathway inhibitor dissection\",\n      \"pmids\": [\"10640301\", \"11179953\", \"16807358\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for human/rodent desensitization difference unknown\", \"Phosphorylation/arrestin determinants not identified\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Pharmacological probes (GR231118/1229U91) demonstrated that the NPY C-terminus suffices to activate Y4R, providing tools to dissect Y4 versus Y1 signaling.\",\n      \"evidence\": \"Radioligand binding and cAMP inhibition assays across cloned Y1, Y2, Y4, Y5 receptors\",\n      \"pmids\": [\"9669502\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Partial-agonist behavior at hY4R clarified only later\", \"Binding-pocket contacts not defined by this study\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defining receptor coupling and dimerization showed Y4R signals through both Gi and Gq, activates ERK via Gi/PKC, and forms agonist-dissociable homodimers that do not heterodimerize with other Y subtypes.\",\n      \"evidence\": \"Contraction/IP3/Ca2+ assays in smooth muscle, ERK phosphorylation with inhibitors, BRET2 and Western blot dimerization assays\",\n      \"pmids\": [\"15308651\", \"11853873\", \"14551289\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of dimer dissociation not established\", \"Stoichiometry and structural interface of dimer unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Genetic epistasis placed central Y4R as an inhibitor of the gonadotropic axis, explaining how elevated NPY tone suppresses fertility.\",\n      \"evidence\": \"Y4 knockout crossed onto ob/ob background with hormone, morphometric, and GnRH mRNA readouts\",\n      \"pmids\": [\"12000791\", \"17272395\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Y4R not detected on GnRH neurons, so the intermediate circuit is undefined\", \"Cell type mediating indirect regulation unidentified\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Hypothalamic and brainstem mapping with selective agonists positioned Y4R upstream of orexin, BDNF, and POMC neuron populations controlling feeding.\",\n      \"evidence\": \"Y4-KO mice, c-Fos mapping, in situ hybridization, and intra-LHA/i.p. ligand injection\",\n      \"pmids\": [\"12598637\", \"20116098\", \"20041129\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct versus indirect activation of each neuron population not fully resolved\", \"Apparent bidirectional feeding effects across studies not reconciled\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Double-knockout genetics revealed Y4R synergy with Y2R in regulating bone volume and adiposity, showing Y4R alone is insufficient for bone phenotypes.\",\n      \"evidence\": \"Y2/Y4 double-knockout mice with DXA, bone histomorphometry, and hormone measurements\",\n      \"pmids\": [\"12861009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of Y2/Y4 synergy unknown\", \"Cell type and signaling node mediating bone effects undefined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Loss-of-function tissue studies established Y4R as the mediator of PP effects in the colon, including antisecretory and contractile responses, and defined recycling-endosome trafficking.\",\n      \"evidence\": \"Y4-KO colonic mucosa in Ussing chambers, contractility assays, GTP\\u03b3S binding, and transferrin co-trafficking in HEK293\",\n      \"pmids\": [\"16807358\", \"19818748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effector mechanism downstream of Y4R in epithelium not detailed\", \"Non-cholinergic contraction pathway only partially dissected\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Structural and mutagenesis studies defined the molecular determinants of PP recognition, identifying an N-terminal helix and TM2/TM6-TM7 pocket residues that contact PP, and later an allosteric antagonist site.\",\n      \"evidence\": \"Solution NMR and SPR of N-Y4 domain, double-cycle mutagenesis with homology modeling, and SAR/docking for (S)-VU0637120\",\n      \"pmids\": [\"18767100\", \"24375409\", \"33595306\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental full-length receptor structure\", \"Allosteric site location inferred computationally without crystallographic validation\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Cell-type-resolved expression and functional assays placed PPYR1 on pancreatic alpha-cells where it mediates PP suppression of glucagon, extending Y4R function to islet endocrine control.\",\n      \"evidence\": \"Immunoblotting, qRT-PCR on FACS-sorted alpha-cells, immunohistochemistry, and isolated islet glucagon secretion assays in mouse and human\",\n      \"pmids\": [\"25445712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Intracellular signaling coupling glucagon suppression not detailed\", \"In vivo contribution to glucose homeostasis not quantified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Human genetic analysis linked NPY4R loss-of-function to obesity, connecting the receptor's anorexigenic role to disease.\",\n      \"evidence\": \"Genome-wide CNV analysis and coding-variant screening with functional receptor assays\",\n      \"pmids\": [\"26921218\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Penetrance and population-level effect size not established\", \"Mechanistic link between variant dysfunction and adiposity not demonstrated in vivo\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How human Y4R achieves desensitization resistance and how its central circuits coordinate the opposing orexigenic and anorexigenic feeding outputs remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental structure of the active receptor-G protein complex\", \"Identity of intermediate neurons linking Y4R to GnRH and orexin circuits unknown\", \"Regulatory determinants distinguishing human from rodent trafficking undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 4, 7]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 9]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [5, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4, 7]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [5, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NPY2R\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}