{"gene":"TAC4","run_date":"2026-04-28T21:42:58","timeline":{"discoveries":[{"year":2000,"finding":"A new preprotachykinin gene (Pptc/TAC4) encodes hemokinin-1 (HK-1), a tachykinin peptide primarily expressed in hematopoietic cells (unlike neuronal Ppta/Pptb). HK-1 stimulated proliferation of IL-7-expanded B cell precursors and promoted survival of bone marrow B lineage cells, whereas substance P had no effect on these cells. In vivo administration of a tachykinin receptor antagonist specifically reduced the pre-B cell compartment (B220lowCD43) in bone marrow and newly generated B cells in spleen, establishing HK-1 as an autocrine survival factor for B cell precursors.","method":"Molecular cloning, in vitro B cell proliferation/survival assays, in vivo antagonist administration with flow cytometric readouts","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — original discovery paper with multiple orthogonal in vitro and in vivo methods, foundational study with 241 citations","pmids":["11062498"],"is_preprint":false},{"year":2002,"finding":"Human and rat orthologs of PPT-C (TAC4) were isolated and characterized. Human PPT-C can generate full-length HK-1 and a truncated form HK-1(4-11) due to a monobasic rather than dibasic N-terminal cleavage site. Both human and mouse HK-1 bind the NK1 receptor with high affinity (comparable to substance P) and act as agonists at NK1, NK2, and NK3 receptors, with strongest selectivity for NK1.","method":"PCR expression analysis, radioligand binding assays, functional receptor assays in transfected cells","journal":"Gene","confidence":"High","confidence_rationale":"Tier 1-2 — multiple binding and functional assays across species, replicated receptor pharmacology with 134 citations","pmids":["12383518"],"is_preprint":false},{"year":2002,"finding":"Hemokinin-1 (HEK-1) is a full agonist at tachykinin NK1, NK2, and NK3 receptors. It inhibits [3H]-substance P binding to the human NK1 receptor with Ki=0.175 nM (comparable to SP Ki=0.13 nM), while its affinity for NK2 is markedly lower (Ki=560 nM). In vivo, intravenous HK-1 produces dose-related decreases in blood pressure and salivary secretion in anaesthetized animals, effects fully blocked by the selective NK1 antagonist SR 140333.","method":"Radioligand binding, isolated organ bioassays (rat urinary bladder, rabbit pulmonary artery, guinea pig ileum), in vivo pharmacology with selective antagonists","journal":"British journal of pharmacology","confidence":"High","confidence_rationale":"Tier 1 — in vitro binding and functional assays with mutagenesis-level pharmacological dissection, replicated across multiple tissue preparations","pmids":["11786503"],"is_preprint":false},{"year":2003,"finding":"Four human tachykinins encoded by the TAC4 gene — endokinins A, B, C, and D (EKA-D) — were identified from four alternative mRNAs (alpha, beta, gamma, delta). TAC4 expression was detected primarily in adrenal gland and placenta. EKA/B 10-mers displayed equivalent affinity for NK1, NK2, and NK3 receptors as substance P, whereas EKC/D (possessing a novel FQGLL-NH2 motif instead of FXGLM) had low potency. EKA/B produced the same hemodynamic effects as SP in rats (fall in mean arterial blood pressure, tachycardia, mesenteric vasoconstriction, hindquarter vasodilatation), establishing EKA/B as endocrine/paracrine agonists at peripheral SP receptors.","method":"cDNA cloning, RT-PCR tissue expression, radioligand binding, in vivo hemodynamic measurements in rats","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — original discovery of endokinins with molecular cloning, receptor pharmacology, and in vivo functional validation; 121 citations","pmids":["12716968"],"is_preprint":false},{"year":2003,"finding":"Centrally administered HK-1 acts as a functional agonist at NK1 receptors in the CNS. HK-1 competed with substance P for binding to mouse and human NK1 receptors and induced calcium release in CHO cells transfected with human NK1 receptor. In vivo, intracerebroventricular HK-1 induced foot-tapping (gerbils) and scratching (mice) behaviors identical to those induced by substance P or NK1 agonist GR-73632; these were blocked by the selective NK1 antagonist MK-869.","method":"Radioligand competition binding, intracellular calcium release assay in transfected CHO cells, in vivo behavioral pharmacology with selective antagonist","journal":"Neuropharmacology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (binding, functional cell assay, in vivo behavior with antagonist blockade)","pmids":["12842130"],"is_preprint":false},{"year":2004,"finding":"The TAC4 gene encodes multiple species-divergent tachykinin peptides: hemokinin-1 (HK-1) in mouse and rat, endokinin-1 (EK-1) in rabbit, and EKA, EKB, human HK-1 (hHK-1), and hHK(4-11) in humans, plus three orphan tachykinin gene-related peptides (EK-2 in rabbit; EKC and EKD in humans). All TAC4-encoded tachykinins exhibit remarkable selectivity and potency for the NK1 receptor similar to substance P, establishing them as the endogenous peripheral SP-like agonists in tissues where SP is not expressed.","method":"Comparative genomic and peptide sequence analysis, receptor pharmacology review integrating published data","journal":"Cellular and molecular life sciences : CMLS","confidence":"Medium","confidence_rationale":"Tier 3 — comprehensive synthesis/review paper consolidating experimental findings from multiple labs; 133 citations","pmids":["15224188"],"is_preprint":false},{"year":2006,"finding":"TAC4-encoded endokinin peptides EKA/B and EKC/D have distinct roles in spinal pain processing. Intrathecal EKA/B (common C-terminal decapeptide) evoked scratching behavior and thermal hyperalgesia in rats via the NK1 receptor, whereas EKC/D (common C-terminal duodecapeptide) did not. These effects of EKA/B were blocked by NK1 receptor antagonists, placing EKA/B as NK1 agonists in spinal nociception.","method":"Intrathecal administration, behavioral assays (scratching, paw withdrawal latency), NK1 receptor antagonist pharmacology in rats","journal":"Neuroscience letters","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo functional assay with receptor-selective antagonist blockade, single lab","pmids":["17101218"],"is_preprint":false},{"year":2007,"finding":"EKC/D (C-terminal duodecapeptide common to endokinins C and D, TAC4 products) acts as an antagonist of substance P in rat spinal pain processing. EKC/D pretreatment prevented EKA/B- and SP-induced scratching behavior and thermal hyperalgesia. The antagonistic effect depends on leucine at the carboxyl-terminus of EKC/D: replacing leucine with methionine ([Met12]-EKC/D) abolished inhibition and instead caused thermal hyperalgesia.","method":"Intrathecal administration of peptides and analogs, behavioral assays (scratching, thermal hyperalgesia), c-Fos immunohistochemistry in spinal cord, structure-activity mutagenesis (leucine-to-methionine substitution)","journal":"Brain research","confidence":"High","confidence_rationale":"Tier 1-2 — structure-activity mutagenesis combined with in vivo functional readouts and histological validation","pmids":["17655832"],"is_preprint":false},{"year":2007,"finding":"TAC4 mRNA (encoding hHK-1) is expressed in human spermatozoa, and hHK-1 along with other tachykinins produces concentration-dependent increases in sperm progressive motility. These effects are antagonized by selective NK1, NK2, and NK3 receptor antagonists, and immunocytochemistry confirmed expression of all three tachykinin receptor proteins in spermatozoa at distinct subcellular localizations.","method":"RT-PCR, Western blotting, immunocytochemistry, WHO-guideline motility analysis with selective receptor antagonists","journal":"Human reproduction (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (RT-PCR, Western blot, ICC, functional assay with pharmacological dissection), single lab","pmids":["17437961"],"is_preprint":false},{"year":2008,"finding":"TAC4 gene transcription is initiated from multiple start sites through a TATA-less promoter, a mechanism distinct from the substance P promoter. The 5' non-coding region is conserved across species. NFκB was identified as a transcription factor that drives increased TAC4 transcription upon PMA stimulation in a T cell line, providing a molecular basis for HK-1's immune cell-specific expression.","method":"Promoter analysis, 5' RACE, reporter assays, NFκB pathway manipulation in T cell line","journal":"Neuropeptides","confidence":"Medium","confidence_rationale":"Tier 2 — direct promoter functional analysis with transcription factor identification, single lab","pmids":["19081134"],"is_preprint":false},{"year":2010,"finding":"TAC4-/- mice show an accumulation of CD19+CD117+HSA+BP.1- 'fraction B' pro-B cells in bone marrow, with normal pre-B, immature, and mature B cell numbers. In vitro cultures from TAC4-/- bone marrow or sorted pro-B cells generated significantly more pro-B cells than controls. Exogenous HK-1 added to long-term and intermediate-term reconstituting stem cell cultures significantly decreased de novo generated pro-B cells, establishing an inhibitory role for HK-1 in pro-B cell development.","method":"Gene knockout (TAC4-/-), flow cytometry, in vitro bone marrow cultures with exogenous peptide addition","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — clean genetic knockout with defined cellular phenotype, confirmed by exogenous peptide rescue experiments","pmids":["20660792"],"is_preprint":false},{"year":2010,"finding":"hHK-1 expression and TAC4 mRNA were detected in human bronchi (including airway macrophages). Exogenous hHK-1 caused contractile responses in human bronchi primarily through NK2 receptors, with unmasked NK1 receptor involvement (subject to rapid desensitization) when NK2 is blocked. In guinea pig trachea, hHK-1 contraction was mainly NK1-mediated. Endokinins A/B had similar effects; endokinins C/D were inactive.","method":"RT-PCR, enzyme immunoassay, isolated tissue pharmacology (human bronchi, guinea pig trachea) with selective NK1/NK2 receptor antagonists","journal":"Respiratory research","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (RT-PCR, EIA, functional organ assays with pharmacological dissection), single lab","pmids":["20929541"],"is_preprint":false},{"year":2010,"finding":"The N-terminal domain of hHK-1 confers functional selectivity at the NK1 receptor: hHK-1 and its C-terminal fragments independently activate Gs (adenylate cyclase/cAMP) and Gq (intracellular Ca2+ release) pathways, with a relative bias toward Gq over Gs. Residues T1, K3, and Q6 in the N-terminus of hHK-1 contribute specifically to Gs/adenylate cyclase activation without affecting Gq-mediated calcium release. Stepwise N-terminal truncation progressively decreased ERK1/2 phosphorylation and NF-κB activity without affecting NK1 receptor desensitization or internalization.","method":"Peptide truncation structure-activity analysis, cAMP assay, intracellular Ca2+ measurement, ERK1/2 phosphorylation, NF-κB reporter assay, receptor internalization assay","journal":"Biochemical pharmacology","confidence":"High","confidence_rationale":"Tier 1-2 — systematic structure-activity mutagenesis with multiple downstream signaling readouts and functional selectivity characterization","pmids":["21168392"],"is_preprint":false},{"year":2011,"finding":"HK-1 (encoded by TAC4) and substance P promote differentiation of human memory CD4+ T cells into Th17 cells by acting on monocytes via NK1 receptors: both peptides triggered IL-1β, IL-6, TNF-α production, upregulated IL-23, and enhanced TNF-like 1A expression on monocyte surfaces. Neutralization experiments demonstrated that IL-1β, IL-23, and TNF-like 1A are required intermediaries for HK-1-induced Th17 generation. Neurokinins A and B had no effect, identifying this as a specific property of SP/HK-1.","method":"In vitro human PBMC culture system, cytokine neutralization experiments, flow cytometry, ELISA, monocyte depletion assays","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 2 — epistatic pathway dissection via neutralization experiments with multiple orthogonal readouts, replicated across multiple donors","pmids":["21368235"],"is_preprint":false},{"year":2012,"finding":"Hemokinins (rat/mouse HK-1, human HK-1, hHK(4-11)) dose-dependently stimulated proliferation, migration, adhesion, and tube formation of human umbilical vein endothelial cells (HUVECs), and exhibited in vivo angiogenic activity in the chick embryo chorioallantoic membrane model. These angiogenic effects were inhibited by selective NK1 (but not NK2) receptor antagonist. Mechanistically, HKs activated ERK1/2 phosphorylation, stimulated nitric oxide production, and upregulated eNOS and VEGF expression in HUVECs.","method":"HUVEC proliferation, migration, adhesion, and tube formation assays; chick embryo CAM model; NK1/NK2 selective antagonists; ERK1/2 phosphorylation; NO production assay; Western blotting for eNOS and VEGF","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal in vitro and in vivo functional assays with mechanistic pathway dissection, single lab","pmids":["22554585"],"is_preprint":false},{"year":2016,"finding":"TAC4-encoded HK-1 and its receptor NK1R-Tr (truncated isoform) are expressed in human mural granulosa cells (MGCs) and cumulus cells. Kisspeptin treatment modulated HK-1, NK3R, and KISS1R mRNA expression in these cells, and substance P regulated kisspeptin mRNA levels and attenuated kisspeptin-induced intracellular Ca2+ responses, revealing a reciprocal interaction between the tachykinin and kisspeptin systems in regulating human ovarian granulosa cell function.","method":"RT-PCR, quantitative RT-PCR, immunocytochemistry, Western blotting, intracellular Ca2+ measurement in primary human granulosa cells","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple detection methods in primary cells with functional Ca2+ readout, but mechanistic pathway not fully resolved, single lab","pmids":["27146034"],"is_preprint":false},{"year":2016,"finding":"hHK-1 promotes migration of melanoma cells via NK1 receptor activation. NK1 receptor expression correlated with melanoma metastatic potential. hHK-1 treatment upregulated MMP-2 and MT1-MMP expression in A375 and B16F10 melanoma cells and induced phosphorylation of ERK1/2, JNK, and p38 via PKC and PKA pathways; an NK1 receptor antagonist (L732138) blocked migration.","method":"Cell migration assay, NK1 receptor antagonist pharmacology, Western blotting for MMP-2/MT1-MMP and kinase phosphorylation, kinase inhibitor dissection","journal":"Peptides","confidence":"Medium","confidence_rationale":"Tier 2-3 — functional migration assay with receptor and kinase pathway dissection, single lab","pmids":["27458061"],"is_preprint":false},{"year":2020,"finding":"Human HK-1 (encoded by TAC4) stimulates production and release of multiple inflammatory cytokines and chemokines (MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, IL-1β, IL-6) from human colonic mucosal explants. These effects were mediated through both NK1 and NK2 tachykinin receptors, as separate NK1 (SR140333) and NK2 (SR48968) antagonists each partially inhibited the responses, distinguishing hHK-1's receptor usage profile from that of substance P (which did not affect MCP-1 or RANTES).","method":"Human colonic mucosal explant system, Procarta multiplex cytokine assay, QuantiGene mRNA assay, selective NK1/NK2 receptor antagonists","journal":"Neuropeptides","confidence":"Medium","confidence_rationale":"Tier 2 — human tissue explant with receptor pharmacological dissection and multiplex cytokine quantification, single lab","pmids":["32600668"],"is_preprint":false},{"year":2021,"finding":"In teleost (grass carp), TAC4 encodes two mature peptides: HK1 (containing a mutant VFGLM motif) and HK2 (containing canonical FXGLM motif). HK2 activated all 6 grass carp neurokinin receptors with highest activity at NK2R, whereas HK1 showed very weak activation of each NKR. In grass carp pituitary cells, HK2 induced prolactin, somatolactin α, and other neuropeptide mRNA expression via NK2R and NK3R through cAMP/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMKII cascades. A phenylalanine-to-valine substitution in the signature motif of HK1 accounts for its weak agonistic activity.","method":"cDNA cloning, receptor activation assays (all 6 NKR isoforms), pituitary cell culture with peptide treatment and receptor-selective antagonists, signaling pathway inhibitors, RT-qPCR for pituitary gene expression","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — ortholog study with systematic receptor profiling and signaling pathway dissection, structure-activity insight from natural variant","pmids":["34884698"],"is_preprint":false},{"year":2021,"finding":"In healthy subjects, serum HK-1 (encoded by TAC4) is present at high concentrations that can desensitize MRGPRX2 on mast cells, thereby preventing mast cell degranulation by substance P. HK-1 induced histamine release from skin-derived mast cells with an EC50 12-fold higher than SP (5056 nM vs 426 nM). Brief pre-incubation of mast cells with HK-1 at 3-10 µM significantly reduced subsequent histamine release by SP without causing MRGPRX2 internalization, suggesting a competitive or allosteric desensitization mechanism distinct from receptor internalization.","method":"ELISA for serum HK-1, histamine release assay from primary skin-derived mast cells, lentiviral shRNA knockdown of MRGPRX2, receptor internalization assay","journal":"Allergology international : official journal of the Japanese Society of Allergology","confidence":"Medium","confidence_rationale":"Tier 2-3 — functional mast cell assay with shRNA knockdown and receptor internalization dissection, single lab","pmids":["34090787"],"is_preprint":false}],"current_model":"TAC4 encodes the preprotachykinin-C precursor that is processed into multiple bioactive peptides — hemokinin-1 (HK-1) in rodents and human HK-1/endokinins A-D in humans — which act primarily as high-affinity NK1 receptor agonists (with additional NK2/NK3 activity) in non-neuronal peripheral tissues, mediating diverse functions including promotion of B cell precursor survival (with inhibitory feedback on pro-B cell expansion), Th17 cell polarization via monocyte-derived IL-1β/IL-23/TNF-like 1A, airway and vascular smooth muscle contraction, angiogenesis through NK1/ERK1/2/eNOS/VEGF signaling, sperm motility regulation, melanoma cell migration via MMP-2/MT1-MMP upregulation, and mast cell MRGPRX2 desensitization; its non-neuronal immune expression is driven by a TATA-less promoter with NFκB-dependent induction, and the N-terminal domain of hHK-1 confers biased agonism at NK1 receptors, selectively engaging Gs over Gq and modulating ERK1/2 and NF-κB downstream signaling."},"narrative":{"teleology":[{"year":2000,"claim":"The discovery of TAC4 (Pptc) and its product HK-1 established that a non-neuronal tachykinin gene exists with a dedicated role in hematopoiesis, resolving how B cell precursors receive tachykinin survival signals in the absence of neuronal substance P expression.","evidence":"Molecular cloning of TAC4, in vitro B cell proliferation/survival assays, in vivo NK1 antagonist administration with flow cytometric analysis of bone marrow and spleen B cell compartments","pmids":["11062498"],"confidence":"High","gaps":["Mechanism by which HK-1 promotes B cell precursor survival (downstream intracellular signaling) was not defined","Whether HK-1 acts on B cells directly or via stromal intermediaries was not resolved"]},{"year":2002,"claim":"Receptor pharmacology studies demonstrated that HK-1 and the human TAC4 products bind NK1 receptors with affinity comparable to substance P and also activate NK2 and NK3 receptors, establishing the receptor selectivity profile of this new tachykinin family.","evidence":"Radioligand competition binding at NK1/NK2/NK3 receptors, functional organ bioassays (rat bladder, rabbit pulmonary artery, guinea pig ileum), in vivo cardiovascular pharmacology with selective NK1 antagonist blockade","pmids":["12383518","11786503"],"confidence":"High","gaps":["Whether different TAC4 splice products have distinct receptor selectivity profiles was not yet addressed","Signaling bias among G-protein pathways at NK1 was unknown"]},{"year":2003,"claim":"Identification of endokinins A–D from four alternative TAC4 mRNAs revealed that the human gene produces a diverse set of tachykinin peptides with divergent receptor activities — EKA/B acting as potent NK1 agonists and EKC/D having a novel C-terminal motif with low receptor potency — expanding the functional repertoire beyond a single HK-1 peptide.","evidence":"cDNA cloning of four human TAC4 splice variants, RT-PCR tissue expression profiling, radioligand binding, in vivo hemodynamic measurements in rats","pmids":["12716968"],"confidence":"High","gaps":["Tissue-specific splice variant regulation was not characterized","Whether EKC/D have a dedicated receptor or function remained unknown"]},{"year":2007,"claim":"Structure-activity analysis of EKC/D revealed that it acts as a functional antagonist of substance P in spinal pain processing, with the C-terminal leucine residue being critical — resolving the paradox of why TAC4 encodes peptides with weak agonist activity and demonstrating that a single gene can produce both agonists and antagonists at tachykinin receptors.","evidence":"Intrathecal peptide and analog administration, behavioral assays, c-Fos immunohistochemistry, leucine-to-methionine substitution mutagenesis","pmids":["17655832"],"confidence":"High","gaps":["Molecular mechanism of EKC/D antagonism (competitive vs. allosteric) at the receptor level was not determined","Relevance of EKC/D antagonism outside the spinal cord was untested"]},{"year":2008,"claim":"Characterization of the TAC4 promoter revealed a TATA-less architecture with NFκB-dependent transcriptional induction, explaining the immune-cell-enriched expression pattern that distinguishes TAC4 from neuronal tachykinin genes.","evidence":"5' RACE, promoter-reporter assays, NFκB pathway manipulation in a T cell line","pmids":["19081134"],"confidence":"Medium","gaps":["Additional transcription factors regulating tissue-specific TAC4 expression were not identified","Chromatin-level regulation and epigenetic control were not examined"]},{"year":2010,"claim":"TAC4 knockout mice revealed that HK-1 has an unexpected inhibitory role in pro-B cell expansion — TAC4-/- mice accumulated fraction B pro-B cells — refining the initial model from pure survival factor to a dual role as a survival signal for pre-B cells and a negative regulator of pro-B cell proliferation.","evidence":"TAC4-/- mice, flow cytometric bone marrow analysis, in vitro bone marrow and sorted pro-B cell cultures with exogenous HK-1 addition","pmids":["20660792"],"confidence":"High","gaps":["Receptor and signaling pathway mediating pro-B cell inhibition were not identified","Whether the knockout phenotype has functional immunological consequences (e.g., antibody responses) was not tested"]},{"year":2010,"claim":"Systematic truncation and structure-activity studies demonstrated that the N-terminal domain of human HK-1 confers biased agonism at NK1 receptors, preferentially engaging Gs/cAMP over Gq/Ca2+ signaling and modulating ERK1/2 phosphorylation and NF-κB activation, providing a molecular basis for functional selectivity among TAC4-derived peptides.","evidence":"Peptide truncation series, cAMP and intracellular Ca2+ assays, ERK1/2 phosphorylation, NF-κB reporter assay, receptor internalization assay in NK1-expressing cells","pmids":["21168392"],"confidence":"High","gaps":["Structural basis of biased agonism (receptor conformation) was not resolved","In vivo consequences of biased signaling in immune or vascular contexts were not tested"]},{"year":2011,"claim":"HK-1 was shown to drive Th17 cell differentiation through an indirect monocyte-dependent mechanism involving NK1 receptor–triggered IL-1β, IL-23, and TNF-like 1A production, establishing TAC4-derived peptides as regulators of adaptive immune polarization beyond their known innate/B cell roles.","evidence":"Human PBMC co-culture, cytokine neutralization experiments, monocyte depletion, flow cytometry and ELISA across multiple donors","pmids":["21368235"],"confidence":"High","gaps":["Whether HK-1-driven Th17 polarization operates in vivo in autoimmune or inflammatory contexts was not demonstrated","Relative contribution of HK-1 versus substance P to Th17 generation in tissues was not defined"]},{"year":2012,"claim":"TAC4-encoded hemokinins were established as pro-angiogenic factors acting through NK1 receptors to activate ERK1/2, stimulate eNOS/NO production, and upregulate VEGF expression — extending the functional scope of TAC4 products from immune regulation to vascular biology.","evidence":"HUVEC proliferation, migration, adhesion, tube formation assays; chick embryo CAM model; NK1/NK2 antagonists; ERK1/2, eNOS, and VEGF Western blotting","pmids":["22554585"],"confidence":"Medium","gaps":["In vivo angiogenic role in mammalian pathological contexts (tumor angiogenesis, wound healing) was not tested","Whether this pathway operates independently of VEGF receptor signaling was unclear"]},{"year":2016,"claim":"The functional repertoire of TAC4 products was further extended to include promotion of melanoma cell migration via NK1 receptor–dependent upregulation of MMP-2 and MT1-MMP through PKC/PKA and MAPK cascades, and regulation of human ovarian granulosa cell function through reciprocal signaling with the kisspeptin system.","evidence":"Melanoma cell migration assays with NK1 antagonist and kinase inhibitors; primary human granulosa cell RT-PCR, Western blot, and Ca2+ assays with kisspeptin/SP co-treatment","pmids":["27458061","27146034"],"confidence":"Medium","gaps":["In vivo relevance of HK-1-driven melanoma metastasis was not established","Molecular mechanism linking TAC4 and kisspeptin signaling was not fully resolved"]},{"year":2021,"claim":"Circulating HK-1 was found to desensitize MRGPRX2 on mast cells without inducing receptor internalization, providing a mechanism by which TAC4 products modulate innate immune responses by cross-regulating a non-tachykinin receptor.","evidence":"Serum HK-1 ELISA, primary skin mast cell histamine release assays, MRGPRX2 shRNA knockdown, receptor internalization assay","pmids":["34090787"],"confidence":"Medium","gaps":["Mechanism of MRGPRX2 desensitization (competitive binding vs. allosteric modulation) was not resolved","In vivo relevance of this HK-1/MRGPRX2 axis in allergic or inflammatory disease was not tested"]},{"year":null,"claim":"Major unresolved questions include the structural basis for HK-1's biased agonism at NK1 receptors, the in vivo significance of TAC4 products in adaptive immunity and cancer metastasis, the precise mechanism of EKC/D antagonism, and whether TAC4-derived peptides have dedicated non-NK receptors.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal structure or cryo-EM structure of HK-1 bound to NK1R","In vivo role of TAC4 in tumor angiogenesis and metastasis lacks genetic evidence","Potential non-NK receptor targets for EKC/D remain unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,1,2,3,4,12,13,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,19]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[0,2,3,14,17,19]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,10,13,17]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[10,15]}],"complexes":[],"partners":["TACR1","TACR2","TACR3","MRGPRX2"],"other_free_text":[]},"mechanistic_narrative":"TAC4 encodes preprotachykinin-C, the precursor of hemokinin-1 (HK-1) and endokinins A–D, which function as high-affinity NK1 receptor agonists (with additional NK2/NK3 activity) predominantly in non-neuronal peripheral tissues including hematopoietic, immune, airway, vascular, and reproductive cells [PMID:11062498, PMID:12383518, PMID:12716968]. In the immune system, HK-1 acts as an autocrine survival factor for B cell precursors while exerting inhibitory feedback on pro-B cell expansion, and drives Th17 polarization via monocyte-derived IL-1β, IL-23, and TNF-like 1A through NK1 receptor signaling [PMID:20660792, PMID:21368235]. TAC4 transcription is controlled by a TATA-less promoter with NFκB-dependent induction in immune cells, and the N-terminal domain of human HK-1 confers biased agonism at NK1 receptors, selectively engaging Gs over Gq to differentially modulate ERK1/2 and NF-κB signaling [PMID:19081134, PMID:21168392]. Beyond hematopoiesis, TAC4-derived peptides mediate airway and vascular smooth muscle contraction, promote angiogenesis via NK1/ERK1/2/eNOS/VEGF signaling, regulate sperm motility, and facilitate melanoma cell migration through MMP-2/MT1-MMP upregulation [PMID:20929541, PMID:22554585, PMID:17437961, PMID:27458061]."},"prefetch_data":{"uniprot":{"accession":"Q86UU9","full_name":"Tachykinin-4","aliases":["Preprotachykinin-C","PPT-C"],"length_aa":113,"mass_kda":12.3,"function":"Tachykinins are active peptides which excite neurons, evoke behavioral responses, are potent vasodilators and secretagogues, and contract (directly or indirectly) many smooth muscles. Endokinin-A induces thermal hyperalgesia and pain-related behavior such as scratching following intrathecal administration in rats. These effects are suppressed by treatment with endokinin-C. Endokinin-A/B reduces arterial blood pressure and increases sperm motility","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/Q86UU9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TAC4","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TAC4","total_profiled":1310},"omim":[{"mim_id":"607833","title":"TACHYKININ 4; TAC4","url":"https://www.omim.org/entry/607833"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"choroid plexus","ntpm":2.5},{"tissue":"pituitary gland","ntpm":5.1}],"url":"https://www.proteinatlas.org/search/TAC4"},"hgnc":{"alias_symbol":["HK-1","Pptc","PPT-C"],"prev_symbol":[]},"alphafold":{"accession":"Q86UU9","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UU9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UU9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86UU9-F1-predicted_aligned_error_v6.png","plddt_mean":56.22},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TAC4","jax_strain_url":"https://www.jax.org/strain/search?query=TAC4"},"sequence":{"accession":"Q86UU9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86UU9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86UU9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86UU9"}},"corpus_meta":[{"pmid":"25296002","id":"PMC_25296002","title":"Cr(VI) reduction and Cr(III) immobilization by Acinetobacter sp. HK-1 with the assistance of a novel quinone/graphene oxide composite.","date":"2014","source":"Environmental science & technology","url":"https://pubmed.ncbi.nlm.nih.gov/25296002","citation_count":80,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12842130","id":"PMC_12842130","title":"Centrally administered hemokinin-1 (HK-1), a neurokinin NK1 receptor agonist, produces substance P-like behavioral effects in mice and gerbils.","date":"2003","source":"Neuropharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/12842130","citation_count":69,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"32628357","id":"PMC_32628357","title":"TAC4 controls tiller angle by regulating the endogenous auxin content and distribution in rice.","date":"2020","source":"Plant biotechnology journal","url":"https://pubmed.ncbi.nlm.nih.gov/32628357","citation_count":48,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"22427645","id":"PMC_22427645","title":"Mapping of clinical and expression quantitative trait loci in a sex-dependent effect of host susceptibility to mouse-adapted influenza H3N2/HK/1/68.","date":"2012","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/22427645","citation_count":45,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12417948","id":"PMC_12417948","title":"Recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus partially protects mice from challenge with heterologous virus: A/HK/1/68 (H3N2).","date":"2002","source":"Archives of virology","url":"https://pubmed.ncbi.nlm.nih.gov/12417948","citation_count":36,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"24480425","id":"PMC_24480425","title":"Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill.","date":"2014","source":"Chemosphere","url":"https://pubmed.ncbi.nlm.nih.gov/24480425","citation_count":23,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11783948","id":"PMC_11783948","title":"Downeast anemia (dea), a new mouse model of severe nonspherocytic hemolytic anemia caused by hexokinase (HK(1)) deficiency.","date":"2001","source":"Blood cells, molecules & diseases","url":"https://pubmed.ncbi.nlm.nih.gov/11783948","citation_count":21,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20660792","id":"PMC_20660792","title":"Targeted deletion of the tachykinin 4 gene (TAC4-/-) influences the early stages of B lymphocyte development.","date":"2010","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/20660792","citation_count":20,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"6971618","id":"PMC_6971618","title":"Evidence of gene dosage effect for HK 1 in the red cells of a patient with trisomy 10pter leads to p13.","date":"1981","source":"Annales de genetique","url":"https://pubmed.ncbi.nlm.nih.gov/6971618","citation_count":10,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17175032","id":"PMC_17175032","title":"The neuropeptide genes TAC1, TAC3, TAC4, VIP and PACAP(ADCYAP1), and susceptibility to multiple sclerosis.","date":"2006","source":"Journal of neuroimmunology","url":"https://pubmed.ncbi.nlm.nih.gov/17175032","citation_count":8,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35628116","id":"PMC_35628116","title":"BRITTLE CULM17, a Novel Allele of TAC4, Affects the Mechanical Properties of Rice Plants.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35628116","citation_count":7,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"37999059","id":"PMC_37999059","title":"Insecticidal Effect of the Entomopathogenic Fungus Lecanicillium araneicola HK-1 in Aphis craccivora (Hemiptera: Aphididae).","date":"2023","source":"Insects","url":"https://pubmed.ncbi.nlm.nih.gov/37999059","citation_count":6,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"38346469","id":"PMC_38346469","title":"PARP-1 inhibitor alleviates cerebral ischemia/reperfusion injury by reducing PARylation of HK-1 and LDH in mice.","date":"2024","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/38346469","citation_count":6,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"30507306","id":"PMC_30507306","title":"In vitro cytotoxicity evaluation of thiourea derivatives bearing Salix sp. constituent against HK-1 cell lines.","date":"2018","source":"Natural product research","url":"https://pubmed.ncbi.nlm.nih.gov/30507306","citation_count":5,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"34884698","id":"PMC_34884698","title":"Novel Pituitary Actions of TAC4 Gene Products in Teleost.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34884698","citation_count":3,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"41097514","id":"PMC_41097514","title":"Characterization of Lactiplantibacillus paraplantarum HK-1 and GABA Synthesis Under Simulated Gastrointestinal Conditions.","date":"2025","source":"Foods (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/41097514","citation_count":1,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"36445858","id":"PMC_36445858","title":"The deletion of HK-1 gene affects the bacterial virulence of Pseudomonas stutzeri LH-42.","date":"2022","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/36445858","citation_count":0,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17886721","id":"PMC_17886721","title":"[Construction and sequencing of full-length cDNA clone of swine vesicular disease virus strain HK'1/70].","date":"2007","source":"Bing du xue bao = Chinese journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/17886721","citation_count":0,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"38527673","id":"PMC_38527673","title":"Genetic association of novel SNPs in HK-1 (rs201626997) and HK-3 (rs143604141) with type 2 diabetes mellitus in Bangladeshi population.","date":"2024","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/38527673","citation_count":0,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"38618297","id":"PMC_38618297","title":"Mast Cells and Their Related Gene HK-1 are Closely Associated with Discogenic Low Back Pain: A Bioinformatics and Clinical Sample Study.","date":"2024","source":"Journal of pain research","url":"https://pubmed.ncbi.nlm.nih.gov/38618297","citation_count":0,"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":"34237774","id":"PMC_34237774","title":"Mapping the human genetic architecture of COVID-19.","date":"2021","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/34237774","citation_count":734,"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":"11062498","id":"PMC_11062498","title":"Hemokinin is a hematopoietic-specific tachykinin that regulates B lymphopoiesis.","date":"2000","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/11062498","citation_count":241,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"10737800","id":"PMC_10737800","title":"Shotgun sequencing of the human transcriptome with ORF expressed sequence tags.","date":"2000","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/10737800","citation_count":135,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12383518","id":"PMC_12383518","title":"Identification, localization and receptor characterization of novel mammalian substance P-like peptides.","date":"2002","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/12383518","citation_count":134,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15224188","id":"PMC_15224188","title":"Hemokinins and endokinins.","date":"2004","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/15224188","citation_count":133,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12716968","id":"PMC_12716968","title":"Characterization of the endokinins: human tachykinins with cardiovascular activity.","date":"2003","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12716968","citation_count":121,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11786503","id":"PMC_11786503","title":"Pharmacological profile of the novel mammalian tachykinin, hemokinin 1.","date":"2002","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/11786503","citation_count":87,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21368235","id":"PMC_21368235","title":"The tachykinins substance P and hemokinin-1 favor the generation of human memory Th17 cells by inducing IL-1β, IL-23, and TNF-like 1A expression by monocytes.","date":"2011","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/21368235","citation_count":85,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17437961","id":"PMC_17437961","title":"A role for tachykinins in the regulation of human sperm motility.","date":"2007","source":"Human reproduction (Oxford, England)","url":"https://pubmed.ncbi.nlm.nih.gov/17437961","citation_count":44,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16931167","id":"PMC_16931167","title":"Characterization of the gene structures, precursor processing and pharmacology of the endokinin peptides.","date":"2006","source":"Vascular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/16931167","citation_count":41,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21342363","id":"PMC_21342363","title":"Distinct differences in tachykinin gene expression in ulcerative colitis, Crohn's disease and diverticular disease: a role for hemokinin-1?","date":"2011","source":"Neurogastroenterology and motility","url":"https://pubmed.ncbi.nlm.nih.gov/21342363","citation_count":25,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17655832","id":"PMC_17655832","title":"Leucine at the carboxyl-terminal of endokinins C and D contributes to elicitation of the antagonistic effect on substance P in rat pain processing.","date":"2007","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/17655832","citation_count":21,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17101218","id":"PMC_17101218","title":"Intrathecal administration of the common carboxyl-terminal decapeptide in endokinin A and endokinin B evokes scratching behavior and thermal hyperalgesia in the rat.","date":"2006","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/17101218","citation_count":20,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20929541","id":"PMC_20929541","title":"Expression and function of human hemokinin-1 in human and guinea pig airways.","date":"2010","source":"Respiratory research","url":"https://pubmed.ncbi.nlm.nih.gov/20929541","citation_count":18,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22554585","id":"PMC_22554585","title":"Hemokinins modulate endothelium function and promote angiogenesis through neurokinin-1 receptor.","date":"2012","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/22554585","citation_count":17,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27146034","id":"PMC_27146034","title":"Expression of Tachykinins and Tachykinin Receptors and Interaction with Kisspeptin in Human Granulosa and Cumulus Cells.","date":"2016","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/27146034","citation_count":17,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21168392","id":"PMC_21168392","title":"The N-terminal domain of human hemokinin-1 influences functional selectivity property for tachykinin receptor neurokinin-1.","date":"2010","source":"Biochemical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/21168392","citation_count":16,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"19081134","id":"PMC_19081134","title":"Regulatory mechanisms in the differential expression of Hemokinin-1.","date":"2008","source":"Neuropeptides","url":"https://pubmed.ncbi.nlm.nih.gov/19081134","citation_count":13,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27456549","id":"PMC_27456549","title":"Altered expression of the tachykinins substance P/neurokinin A/hemokinin-1 and their preferred neurokinin 1/neurokinin 2 receptors in uterine leiomyomata.","date":"2016","source":"Fertility and sterility","url":"https://pubmed.ncbi.nlm.nih.gov/27456549","citation_count":10,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27458061","id":"PMC_27458061","title":"Human hemokinin-1 promotes migration of melanoma cells and increases MMP-2 and MT1-MMP expression by activating tumor cell NK1 receptors.","date":"2016","source":"Peptides","url":"https://pubmed.ncbi.nlm.nih.gov/27458061","citation_count":9,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"34090787","id":"PMC_34090787","title":"Serum level of hemokinin-1 is significantly lower in patients with chronic spontaneous urticaria than in healthy subjects.","date":"2021","source":"Allergology international : official journal of the Japanese Society of Allergology","url":"https://pubmed.ncbi.nlm.nih.gov/34090787","citation_count":8,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"32600668","id":"PMC_32600668","title":"Hemokinin-1 and substance P stimulate production of inflammatory cytokines and chemokines in human colonic mucosa via both NK1 and NK2 tachykinin receptors.","date":"2020","source":"Neuropeptides","url":"https://pubmed.ncbi.nlm.nih.gov/32600668","citation_count":7,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20580442","id":"PMC_20580442","title":"Common variants of the neuropeptide expressing tachykinin genes and susceptibility to asthma: a case-control study.","date":"2010","source":"Journal of neuroimmunology","url":"https://pubmed.ncbi.nlm.nih.gov/20580442","citation_count":4,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11141,"output_tokens":1174,"usd":0.025517},"stage2":{"model":"claude-opus-4-6","input_tokens":4376,"output_tokens":1545,"usd":0.090758},"total_usd":0.405929,"stage1_batch_id":"msgbatch_01RPbVc9PDH9zQmYK5Hs6srm","stage2_batch_id":"msgbatch_01JVbVLzeKN2yVrsGp3zC8aM","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":23308,"output_tokens":7459,"usd":0.090904},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":9905,"output_tokens":3319,"usd":0.19875}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"Hemokinin-1 (HK-1), encoded by TAC4, is an endogenous agonist at NK1 receptors: it competes with substance P for binding to mouse and human NK1 receptors, stimulates intracellular calcium release in CHO cells transfected with human NK1 receptor, and induces NK1-dependent behaviors (foot-tapping, scratching) in vivo that are blocked by the NK1 antagonist MK-869.\",\n      \"method\": \"Radioligand competition binding, calcium mobilization assay in transfected CHO cells, in vivo intracerebroventricular administration with pharmacological antagonism\",\n      \"journal\": \"Neuropharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (binding, functional calcium assay, in vivo pharmacology) in a single study with antagonist controls\",\n      \"pmids\": [\"12842130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TAC4-encoded HK-1 plays an inhibitory role in early B lymphocyte development: TAC4-/- mice show accumulation of 'fraction B' pro-B cells in bone marrow, and exogenous HK-1 addition to stem cell cultures significantly decreases de novo generated pro-B cells, indicating HK-1 negatively regulates pro-B cell generation.\",\n      \"method\": \"Targeted gene knockout (TAC4-/-), flow cytometry, in vitro bone marrow culture with sorted stem cells, exogenous peptide addition\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean knockout with defined cellular phenotype replicated in multiple culture systems and confirmed by peptide-addition rescue experiments\",\n      \"pmids\": [\"20660792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In teleost (grass carp), TAC4 encodes two mature peptides (HK1 and HK2); HK2 activates all six neurokinin receptors with highest potency at NK2R, and stimulates pituitary gene expression (PRL, SLα, UTS1, NMB1, CART2 mRNA) via NK2R and NK3R through cAMP/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMKII cascades; a phenylalanine-to-valine substitution in HK1's C-terminal FXGLM motif accounts for its markedly weaker agonist activity.\",\n      \"method\": \"Ligand-receptor activation assays, receptor-specific pharmacological blockade, mRNA expression analysis in primary pituitary cells, sequence/structural analysis with site-directed mutagenesis rationale\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple receptor and signaling pathway assays in primary cells; single lab, teleost ortholog\",\n      \"pmids\": [\"34884698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HK-1 (encoded by TAC4) is expressed in mast cells within nucleus pulposus tissue of intervertebral disc degeneration patients, and its expression correlates with mast cell activation and elevated blood IL-12p70 levels, suggesting HK-1 activates mast cells and promotes IL-12p70 release to drive discogenic low back pain.\",\n      \"method\": \"Immunohistochemistry, Western blot, RT-PCR, cytokine multiplex assay on clinical samples\",\n      \"journal\": \"Journal of pain research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — correlative clinical data with IHC/WB, no direct functional manipulation of TAC4/HK-1\",\n      \"pmids\": [\"38618297\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TAC4 encodes hemokinin-1 (HK-1), a tachykinin peptide that acts as an endogenous agonist preferentially at the NK1 receptor (stimulating Gq-coupled calcium signaling), is broadly expressed in immune and neural cells, and negatively regulates early B lymphocyte (pro-B cell) development in bone marrow, with additional evidence for pituitary neuroendocrine regulation via NK2R/NK3R-coupled cAMP/PKA, PLC/PKC, and Ca2+/CaMKII pathways in teleosts.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEPT papers.\n\n**Classification:**\n\n**KEEP (about TAC4/hemokinin-1/endokinins - the canonical human/mammalian gene):**\n- PMID:12842130 - HK-1 mouse tachykinin, NK1 receptor agonist behavior (KEEP)\n- PMID:20660792 - TAC4 gene knockout, B lymphocyte development (KEEP)\n- PMID:11062498 - Hemokinin cloning, B cell development (KEEP)\n- PMID:12383518 - Human/rat PPT-C orthologs, NK receptor characterization (KEEP)\n- PMID:15224188 - TAC4 gene products review (KEEP)\n- PMID:12716968 - Endokinins A-D from TAC4 gene (KEEP)\n- PMID:11786503 - Hemokinin-1 pharmacological profile (KEEP)\n- PMID:21368235 - SP/HK-1 Th17 cell generation via monocytes (KEEP)\n- PMID:17437961 - TAC4 expression in sperm, motility (KEEP)\n- PMID:16931167 - TAC4 gene structure, endokinin processing (KEEP)\n- PMID:17655832 - EKC/D antagonistic effect on pain (KEEP)\n- PMID:17101218 - EKA/B intrathecal pain behavior (KEEP)\n- PMID:20929541 - hHK-1 expression/function in airways (KEEP)\n- PMID:22554585 - Hemokinins angiogenesis via NK1 receptor (KEEP)\n- PMID:27146034 - TAC4/HK-1 in granulosa cells, kisspeptin interaction (KEEP)\n- PMID:21168392 - hHK-1 N-terminal domain, functional selectivity NK1 (KEEP)\n- PMID:19081134 - TAC4 promoter, NFκB regulation (KEEP)\n- PMID:27456549 - Tachykinin expression in leiomyomata (expression only - EXCLUDE)\n- PMID:27458061 - hHK-1 melanoma migration, MMP-2, ERK/JNK/p38 (KEEP)\n- PMID:34090787 - HK-1 mast cell MRGPRX2 desensitization (KEEP)\n- PMID:32600668 - HK-1 cytokines via NK1/NK2 in colon (KEEP)\n- PMID:21342363 - TAC4 expression in IBD (expression - EXCLUDE mechanistic findings minimal)\n- PMID:34884698 - Grass carp TAC4, HK1/HK2 receptor selectivity, pituitary signaling (KEEP - ortholog)\n- PMID:20580442 - SNP association only (EXCLUDE)\n- PMID:17175032 - SNP association only (EXCLUDE)\n\n**EXCLUDE (alias collisions - HK-1 = hexokinase-1, or unrelated organisms):**\n- PMID:25296002 - Acinetobacter HK-1 (bacteria, chromium reduction)\n- PMID:32628357 - Rice TAC4 (Tiller Angle Control 4 - plant gene, SYMBOL COLLISION)\n- PMID:22427645 - Mouse influenza A/HK/1/68\n- PMID:12417948 - Influenza A/HK/1/68\n- PMID:24480425 - Morganella sp. HK-1 (bacteria)\n- PMID:11783948 - Hexokinase-1 (HK1) mouse model\n- PMID:6971618 - HK1 hexokinase gene dosage\n- PMID:30507306 - HK-1 nasopharyngeal carcinoma cell line\n- PMID:38346469 - HK-1 hexokinase-1 PARylation\n- PMID:37999059 - Lecanicillium HK-1 fungus\n- PMID:36445858 - Pseudomonas HK-1 two-component system\n- PMID:17886721 - Swine vesicular disease virus HK'1/70\n- PMID:41097514 - Lactiplantibacillus HK-1 bacteria\n- PMID:38527673 - Hexokinase-1 SNPs\n- PMID:38618297 - HK-1 mast cells/discogenic pain (hemokinin-1 context - actually KEEP for mechanistic finding)\n- PMID:35628116 - Rice OsBC17/TAC4 (plant SYMBOL COLLISION)\n- PMID:12477932 - MGC cDNA (no mechanistic findings)\n- PMID:34237774 - COVID GWAS (no mechanistic findings)\n- PMID:21873635 - GO annotation (no mechanistic findings)\n- PMID:10737800 - EST sequencing (no mechanistic findings)\n\nRe-checking PMID:38618297: This describes hemokinin-1 (HK-1, encoded by TAC4) and mast cell activation via MRGPRX2 - KEEP.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"A new preprotachykinin gene (Pptc/TAC4) encodes hemokinin-1 (HK-1), a tachykinin peptide primarily expressed in hematopoietic cells (unlike neuronal Ppta/Pptb). HK-1 stimulated proliferation of IL-7-expanded B cell precursors and promoted survival of bone marrow B lineage cells, whereas substance P had no effect on these cells. In vivo administration of a tachykinin receptor antagonist specifically reduced the pre-B cell compartment (B220lowCD43) in bone marrow and newly generated B cells in spleen, establishing HK-1 as an autocrine survival factor for B cell precursors.\",\n      \"method\": \"Molecular cloning, in vitro B cell proliferation/survival assays, in vivo antagonist administration with flow cytometric readouts\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — original discovery paper with multiple orthogonal in vitro and in vivo methods, foundational study with 241 citations\",\n      \"pmids\": [\"11062498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human and rat orthologs of PPT-C (TAC4) were isolated and characterized. Human PPT-C can generate full-length HK-1 and a truncated form HK-1(4-11) due to a monobasic rather than dibasic N-terminal cleavage site. Both human and mouse HK-1 bind the NK1 receptor with high affinity (comparable to substance P) and act as agonists at NK1, NK2, and NK3 receptors, with strongest selectivity for NK1.\",\n      \"method\": \"PCR expression analysis, radioligand binding assays, functional receptor assays in transfected cells\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple binding and functional assays across species, replicated receptor pharmacology with 134 citations\",\n      \"pmids\": [\"12383518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Hemokinin-1 (HEK-1) is a full agonist at tachykinin NK1, NK2, and NK3 receptors. It inhibits [3H]-substance P binding to the human NK1 receptor with Ki=0.175 nM (comparable to SP Ki=0.13 nM), while its affinity for NK2 is markedly lower (Ki=560 nM). In vivo, intravenous HK-1 produces dose-related decreases in blood pressure and salivary secretion in anaesthetized animals, effects fully blocked by the selective NK1 antagonist SR 140333.\",\n      \"method\": \"Radioligand binding, isolated organ bioassays (rat urinary bladder, rabbit pulmonary artery, guinea pig ileum), in vivo pharmacology with selective antagonists\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro binding and functional assays with mutagenesis-level pharmacological dissection, replicated across multiple tissue preparations\",\n      \"pmids\": [\"11786503\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Four human tachykinins encoded by the TAC4 gene — endokinins A, B, C, and D (EKA-D) — were identified from four alternative mRNAs (alpha, beta, gamma, delta). TAC4 expression was detected primarily in adrenal gland and placenta. EKA/B 10-mers displayed equivalent affinity for NK1, NK2, and NK3 receptors as substance P, whereas EKC/D (possessing a novel FQGLL-NH2 motif instead of FXGLM) had low potency. EKA/B produced the same hemodynamic effects as SP in rats (fall in mean arterial blood pressure, tachycardia, mesenteric vasoconstriction, hindquarter vasodilatation), establishing EKA/B as endocrine/paracrine agonists at peripheral SP receptors.\",\n      \"method\": \"cDNA cloning, RT-PCR tissue expression, radioligand binding, in vivo hemodynamic measurements in rats\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — original discovery of endokinins with molecular cloning, receptor pharmacology, and in vivo functional validation; 121 citations\",\n      \"pmids\": [\"12716968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Centrally administered HK-1 acts as a functional agonist at NK1 receptors in the CNS. HK-1 competed with substance P for binding to mouse and human NK1 receptors and induced calcium release in CHO cells transfected with human NK1 receptor. In vivo, intracerebroventricular HK-1 induced foot-tapping (gerbils) and scratching (mice) behaviors identical to those induced by substance P or NK1 agonist GR-73632; these were blocked by the selective NK1 antagonist MK-869.\",\n      \"method\": \"Radioligand competition binding, intracellular calcium release assay in transfected CHO cells, in vivo behavioral pharmacology with selective antagonist\",\n      \"journal\": \"Neuropharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (binding, functional cell assay, in vivo behavior with antagonist blockade)\",\n      \"pmids\": [\"12842130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The TAC4 gene encodes multiple species-divergent tachykinin peptides: hemokinin-1 (HK-1) in mouse and rat, endokinin-1 (EK-1) in rabbit, and EKA, EKB, human HK-1 (hHK-1), and hHK(4-11) in humans, plus three orphan tachykinin gene-related peptides (EK-2 in rabbit; EKC and EKD in humans). All TAC4-encoded tachykinins exhibit remarkable selectivity and potency for the NK1 receptor similar to substance P, establishing them as the endogenous peripheral SP-like agonists in tissues where SP is not expressed.\",\n      \"method\": \"Comparative genomic and peptide sequence analysis, receptor pharmacology review integrating published data\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — comprehensive synthesis/review paper consolidating experimental findings from multiple labs; 133 citations\",\n      \"pmids\": [\"15224188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TAC4-encoded endokinin peptides EKA/B and EKC/D have distinct roles in spinal pain processing. Intrathecal EKA/B (common C-terminal decapeptide) evoked scratching behavior and thermal hyperalgesia in rats via the NK1 receptor, whereas EKC/D (common C-terminal duodecapeptide) did not. These effects of EKA/B were blocked by NK1 receptor antagonists, placing EKA/B as NK1 agonists in spinal nociception.\",\n      \"method\": \"Intrathecal administration, behavioral assays (scratching, paw withdrawal latency), NK1 receptor antagonist pharmacology in rats\",\n      \"journal\": \"Neuroscience letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo functional assay with receptor-selective antagonist blockade, single lab\",\n      \"pmids\": [\"17101218\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"EKC/D (C-terminal duodecapeptide common to endokinins C and D, TAC4 products) acts as an antagonist of substance P in rat spinal pain processing. EKC/D pretreatment prevented EKA/B- and SP-induced scratching behavior and thermal hyperalgesia. The antagonistic effect depends on leucine at the carboxyl-terminus of EKC/D: replacing leucine with methionine ([Met12]-EKC/D) abolished inhibition and instead caused thermal hyperalgesia.\",\n      \"method\": \"Intrathecal administration of peptides and analogs, behavioral assays (scratching, thermal hyperalgesia), c-Fos immunohistochemistry in spinal cord, structure-activity mutagenesis (leucine-to-methionine substitution)\",\n      \"journal\": \"Brain research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — structure-activity mutagenesis combined with in vivo functional readouts and histological validation\",\n      \"pmids\": [\"17655832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TAC4 mRNA (encoding hHK-1) is expressed in human spermatozoa, and hHK-1 along with other tachykinins produces concentration-dependent increases in sperm progressive motility. These effects are antagonized by selective NK1, NK2, and NK3 receptor antagonists, and immunocytochemistry confirmed expression of all three tachykinin receptor proteins in spermatozoa at distinct subcellular localizations.\",\n      \"method\": \"RT-PCR, Western blotting, immunocytochemistry, WHO-guideline motility analysis with selective receptor antagonists\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (RT-PCR, Western blot, ICC, functional assay with pharmacological dissection), single lab\",\n      \"pmids\": [\"17437961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TAC4 gene transcription is initiated from multiple start sites through a TATA-less promoter, a mechanism distinct from the substance P promoter. The 5' non-coding region is conserved across species. NFκB was identified as a transcription factor that drives increased TAC4 transcription upon PMA stimulation in a T cell line, providing a molecular basis for HK-1's immune cell-specific expression.\",\n      \"method\": \"Promoter analysis, 5' RACE, reporter assays, NFκB pathway manipulation in T cell line\",\n      \"journal\": \"Neuropeptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct promoter functional analysis with transcription factor identification, single lab\",\n      \"pmids\": [\"19081134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TAC4-/- mice show an accumulation of CD19+CD117+HSA+BP.1- 'fraction B' pro-B cells in bone marrow, with normal pre-B, immature, and mature B cell numbers. In vitro cultures from TAC4-/- bone marrow or sorted pro-B cells generated significantly more pro-B cells than controls. Exogenous HK-1 added to long-term and intermediate-term reconstituting stem cell cultures significantly decreased de novo generated pro-B cells, establishing an inhibitory role for HK-1 in pro-B cell development.\",\n      \"method\": \"Gene knockout (TAC4-/-), flow cytometry, in vitro bone marrow cultures with exogenous peptide addition\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic knockout with defined cellular phenotype, confirmed by exogenous peptide rescue experiments\",\n      \"pmids\": [\"20660792\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"hHK-1 expression and TAC4 mRNA were detected in human bronchi (including airway macrophages). Exogenous hHK-1 caused contractile responses in human bronchi primarily through NK2 receptors, with unmasked NK1 receptor involvement (subject to rapid desensitization) when NK2 is blocked. In guinea pig trachea, hHK-1 contraction was mainly NK1-mediated. Endokinins A/B had similar effects; endokinins C/D were inactive.\",\n      \"method\": \"RT-PCR, enzyme immunoassay, isolated tissue pharmacology (human bronchi, guinea pig trachea) with selective NK1/NK2 receptor antagonists\",\n      \"journal\": \"Respiratory research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (RT-PCR, EIA, functional organ assays with pharmacological dissection), single lab\",\n      \"pmids\": [\"20929541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The N-terminal domain of hHK-1 confers functional selectivity at the NK1 receptor: hHK-1 and its C-terminal fragments independently activate Gs (adenylate cyclase/cAMP) and Gq (intracellular Ca2+ release) pathways, with a relative bias toward Gq over Gs. Residues T1, K3, and Q6 in the N-terminus of hHK-1 contribute specifically to Gs/adenylate cyclase activation without affecting Gq-mediated calcium release. Stepwise N-terminal truncation progressively decreased ERK1/2 phosphorylation and NF-κB activity without affecting NK1 receptor desensitization or internalization.\",\n      \"method\": \"Peptide truncation structure-activity analysis, cAMP assay, intracellular Ca2+ measurement, ERK1/2 phosphorylation, NF-κB reporter assay, receptor internalization assay\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — systematic structure-activity mutagenesis with multiple downstream signaling readouts and functional selectivity characterization\",\n      \"pmids\": [\"21168392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"HK-1 (encoded by TAC4) and substance P promote differentiation of human memory CD4+ T cells into Th17 cells by acting on monocytes via NK1 receptors: both peptides triggered IL-1β, IL-6, TNF-α production, upregulated IL-23, and enhanced TNF-like 1A expression on monocyte surfaces. Neutralization experiments demonstrated that IL-1β, IL-23, and TNF-like 1A are required intermediaries for HK-1-induced Th17 generation. Neurokinins A and B had no effect, identifying this as a specific property of SP/HK-1.\",\n      \"method\": \"In vitro human PBMC culture system, cytokine neutralization experiments, flow cytometry, ELISA, monocyte depletion assays\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistatic pathway dissection via neutralization experiments with multiple orthogonal readouts, replicated across multiple donors\",\n      \"pmids\": [\"21368235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Hemokinins (rat/mouse HK-1, human HK-1, hHK(4-11)) dose-dependently stimulated proliferation, migration, adhesion, and tube formation of human umbilical vein endothelial cells (HUVECs), and exhibited in vivo angiogenic activity in the chick embryo chorioallantoic membrane model. These angiogenic effects were inhibited by selective NK1 (but not NK2) receptor antagonist. Mechanistically, HKs activated ERK1/2 phosphorylation, stimulated nitric oxide production, and upregulated eNOS and VEGF expression in HUVECs.\",\n      \"method\": \"HUVEC proliferation, migration, adhesion, and tube formation assays; chick embryo CAM model; NK1/NK2 selective antagonists; ERK1/2 phosphorylation; NO production assay; Western blotting for eNOS and VEGF\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal in vitro and in vivo functional assays with mechanistic pathway dissection, single lab\",\n      \"pmids\": [\"22554585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TAC4-encoded HK-1 and its receptor NK1R-Tr (truncated isoform) are expressed in human mural granulosa cells (MGCs) and cumulus cells. Kisspeptin treatment modulated HK-1, NK3R, and KISS1R mRNA expression in these cells, and substance P regulated kisspeptin mRNA levels and attenuated kisspeptin-induced intracellular Ca2+ responses, revealing a reciprocal interaction between the tachykinin and kisspeptin systems in regulating human ovarian granulosa cell function.\",\n      \"method\": \"RT-PCR, quantitative RT-PCR, immunocytochemistry, Western blotting, intracellular Ca2+ measurement in primary human granulosa cells\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple detection methods in primary cells with functional Ca2+ readout, but mechanistic pathway not fully resolved, single lab\",\n      \"pmids\": [\"27146034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"hHK-1 promotes migration of melanoma cells via NK1 receptor activation. NK1 receptor expression correlated with melanoma metastatic potential. hHK-1 treatment upregulated MMP-2 and MT1-MMP expression in A375 and B16F10 melanoma cells and induced phosphorylation of ERK1/2, JNK, and p38 via PKC and PKA pathways; an NK1 receptor antagonist (L732138) blocked migration.\",\n      \"method\": \"Cell migration assay, NK1 receptor antagonist pharmacology, Western blotting for MMP-2/MT1-MMP and kinase phosphorylation, kinase inhibitor dissection\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — functional migration assay with receptor and kinase pathway dissection, single lab\",\n      \"pmids\": [\"27458061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Human HK-1 (encoded by TAC4) stimulates production and release of multiple inflammatory cytokines and chemokines (MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, IL-1β, IL-6) from human colonic mucosal explants. These effects were mediated through both NK1 and NK2 tachykinin receptors, as separate NK1 (SR140333) and NK2 (SR48968) antagonists each partially inhibited the responses, distinguishing hHK-1's receptor usage profile from that of substance P (which did not affect MCP-1 or RANTES).\",\n      \"method\": \"Human colonic mucosal explant system, Procarta multiplex cytokine assay, QuantiGene mRNA assay, selective NK1/NK2 receptor antagonists\",\n      \"journal\": \"Neuropeptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — human tissue explant with receptor pharmacological dissection and multiplex cytokine quantification, single lab\",\n      \"pmids\": [\"32600668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In teleost (grass carp), TAC4 encodes two mature peptides: HK1 (containing a mutant VFGLM motif) and HK2 (containing canonical FXGLM motif). HK2 activated all 6 grass carp neurokinin receptors with highest activity at NK2R, whereas HK1 showed very weak activation of each NKR. In grass carp pituitary cells, HK2 induced prolactin, somatolactin α, and other neuropeptide mRNA expression via NK2R and NK3R through cAMP/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMKII cascades. A phenylalanine-to-valine substitution in the signature motif of HK1 accounts for its weak agonistic activity.\",\n      \"method\": \"cDNA cloning, receptor activation assays (all 6 NKR isoforms), pituitary cell culture with peptide treatment and receptor-selective antagonists, signaling pathway inhibitors, RT-qPCR for pituitary gene expression\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ortholog study with systematic receptor profiling and signaling pathway dissection, structure-activity insight from natural variant\",\n      \"pmids\": [\"34884698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In healthy subjects, serum HK-1 (encoded by TAC4) is present at high concentrations that can desensitize MRGPRX2 on mast cells, thereby preventing mast cell degranulation by substance P. HK-1 induced histamine release from skin-derived mast cells with an EC50 12-fold higher than SP (5056 nM vs 426 nM). Brief pre-incubation of mast cells with HK-1 at 3-10 µM significantly reduced subsequent histamine release by SP without causing MRGPRX2 internalization, suggesting a competitive or allosteric desensitization mechanism distinct from receptor internalization.\",\n      \"method\": \"ELISA for serum HK-1, histamine release assay from primary skin-derived mast cells, lentiviral shRNA knockdown of MRGPRX2, receptor internalization assay\",\n      \"journal\": \"Allergology international : official journal of the Japanese Society of Allergology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — functional mast cell assay with shRNA knockdown and receptor internalization dissection, single lab\",\n      \"pmids\": [\"34090787\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TAC4 encodes the preprotachykinin-C precursor that is processed into multiple bioactive peptides — hemokinin-1 (HK-1) in rodents and human HK-1/endokinins A-D in humans — which act primarily as high-affinity NK1 receptor agonists (with additional NK2/NK3 activity) in non-neuronal peripheral tissues, mediating diverse functions including promotion of B cell precursor survival (with inhibitory feedback on pro-B cell expansion), Th17 cell polarization via monocyte-derived IL-1β/IL-23/TNF-like 1A, airway and vascular smooth muscle contraction, angiogenesis through NK1/ERK1/2/eNOS/VEGF signaling, sperm motility regulation, melanoma cell migration via MMP-2/MT1-MMP upregulation, and mast cell MRGPRX2 desensitization; its non-neuronal immune expression is driven by a TATA-less promoter with NFκB-dependent induction, and the N-terminal domain of hHK-1 confers biased agonism at NK1 receptors, selectively engaging Gs over Gq and modulating ERK1/2 and NF-κB downstream signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"TAC4 encodes hemokinin-1 (HK-1), a tachykinin peptide that functions as an endogenous agonist at the neurokinin-1 (NK1) receptor, competing with substance P for binding and stimulating Gq-coupled intracellular calcium release [PMID:12842130]. HK-1 negatively regulates early B lymphopoiesis, as TAC4-knockout mice accumulate pro-B cells in bone marrow and exogenous HK-1 suppresses de novo pro-B cell generation from stem cell cultures [PMID:20660792]. In mast cells, HK-1 expression correlates with mast cell activation and IL-12p70 release in human intervertebral disc tissue [PMID:38618297].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing that the TAC4-encoded peptide HK-1 is a bona fide NK1 receptor agonist resolved the question of whether a non-substance P endogenous ligand exists for this receptor and defined HK-1's primary signaling mode as Gq-coupled calcium mobilization.\",\n      \"evidence\": \"Radioligand competition binding, calcium mobilization in NK1R-transfected CHO cells, and in vivo behavioral assays with NK1 antagonist blockade\",\n      \"pmids\": [\"12842130\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Selectivity profile across NK2R and NK3R was not characterized in mammalian systems\",\n        \"Endogenous tissue sources of HK-1 were not mapped\",\n        \"Downstream transcriptional or signaling consequences beyond calcium flux were not explored\"\n      ]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating that TAC4 knockout causes pro-B cell accumulation in bone marrow established HK-1 as a negative regulator of early B lymphopoiesis, providing the first defined physiological role for this gene in immune cell development.\",\n      \"evidence\": \"TAC4−/− knockout mice analyzed by flow cytometry plus in vitro bone marrow stem cell cultures with exogenous HK-1 peptide addition\",\n      \"pmids\": [\"20660792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Receptor(s) mediating the inhibitory effect on pro-B cells were not identified\",\n        \"Mechanism of HK-1's inhibitory action (apoptosis, differentiation block, proliferation arrest) was not resolved\",\n        \"Impact on later stages of B cell maturation and peripheral immune function was not assessed\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Characterization of teleost TAC4-derived peptides showed that a C-terminal phenylalanine-to-valine substitution in the FXGLM motif determines receptor potency, and that the HK2 peptide activates NK2R/NK3R to drive pituitary gene expression through cAMP/PKA, PLC/PKC, and Ca²⁺/CaMKII cascades, broadening TAC4's signaling repertoire beyond NK1R.\",\n      \"evidence\": \"Ligand-receptor activation assays with receptor-specific antagonists and signaling pathway inhibitors in primary grass carp pituitary cells\",\n      \"pmids\": [\"34884698\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Findings are from a teleost ortholog and have not been confirmed in mammalian systems\",\n        \"Whether mammalian TAC4 splice variants produce functional equivalents of HK2 is unknown\",\n        \"In vivo neuroendocrine relevance of TAC4-derived peptides at the pituitary level is untested in mammals\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Detection of HK-1 in mast cells within degenerating human intervertebral disc tissue, correlated with mast cell activation and IL-12p70 elevation, implicated TAC4 in neuroinflammatory pain signaling in a clinical context.\",\n      \"evidence\": \"Immunohistochemistry, Western blot, RT-PCR, and cytokine multiplex analysis on clinical disc samples\",\n      \"pmids\": [\"38618297\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No direct functional manipulation of TAC4/HK-1 was performed; evidence is correlative\",\n        \"Receptor and signaling pathway mediating mast cell activation by HK-1 were not identified\",\n        \"Causal relationship between HK-1 and IL-12p70 release requires interventional confirmation\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The receptor(s) and intracellular pathways through which HK-1 regulates mammalian B lymphopoiesis remain unidentified, and whether TAC4's neuroendocrine functions observed in teleosts are conserved in mammals is unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No receptor-specific antagonist studies in the bone marrow pro-B cell system\",\n        \"No mammalian pituitary functional data for TAC4-derived peptides\",\n        \"Structural basis for HK-1 selectivity at mammalian NK1R versus NK2R/NK3R is unresolved\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0162582\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"TACR1\",\n      \"TACR2\",\n      \"TACR3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"TAC4 encodes preprotachykinin-C, the precursor of hemokinin-1 (HK-1) and endokinins A–D, which function as high-affinity NK1 receptor agonists (with additional NK2/NK3 activity) predominantly in non-neuronal peripheral tissues including hematopoietic, immune, airway, vascular, and reproductive cells [PMID:11062498, PMID:12383518, PMID:12716968]. In the immune system, HK-1 acts as an autocrine survival factor for B cell precursors while exerting inhibitory feedback on pro-B cell expansion, and drives Th17 polarization via monocyte-derived IL-1β, IL-23, and TNF-like 1A through NK1 receptor signaling [PMID:20660792, PMID:21368235]. TAC4 transcription is controlled by a TATA-less promoter with NFκB-dependent induction in immune cells, and the N-terminal domain of human HK-1 confers biased agonism at NK1 receptors, selectively engaging Gs over Gq to differentially modulate ERK1/2 and NF-κB signaling [PMID:19081134, PMID:21168392]. Beyond hematopoiesis, TAC4-derived peptides mediate airway and vascular smooth muscle contraction, promote angiogenesis via NK1/ERK1/2/eNOS/VEGF signaling, regulate sperm motility, and facilitate melanoma cell migration through MMP-2/MT1-MMP upregulation [PMID:20929541, PMID:22554585, PMID:17437961, PMID:27458061].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"The discovery of TAC4 (Pptc) and its product HK-1 established that a non-neuronal tachykinin gene exists with a dedicated role in hematopoiesis, resolving how B cell precursors receive tachykinin survival signals in the absence of neuronal substance P expression.\",\n      \"evidence\": \"Molecular cloning of TAC4, in vitro B cell proliferation/survival assays, in vivo NK1 antagonist administration with flow cytometric analysis of bone marrow and spleen B cell compartments\",\n      \"pmids\": [\"11062498\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which HK-1 promotes B cell precursor survival (downstream intracellular signaling) was not defined\", \"Whether HK-1 acts on B cells directly or via stromal intermediaries was not resolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Receptor pharmacology studies demonstrated that HK-1 and the human TAC4 products bind NK1 receptors with affinity comparable to substance P and also activate NK2 and NK3 receptors, establishing the receptor selectivity profile of this new tachykinin family.\",\n      \"evidence\": \"Radioligand competition binding at NK1/NK2/NK3 receptors, functional organ bioassays (rat bladder, rabbit pulmonary artery, guinea pig ileum), in vivo cardiovascular pharmacology with selective NK1 antagonist blockade\",\n      \"pmids\": [\"12383518\", \"11786503\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether different TAC4 splice products have distinct receptor selectivity profiles was not yet addressed\", \"Signaling bias among G-protein pathways at NK1 was unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identification of endokinins A–D from four alternative TAC4 mRNAs revealed that the human gene produces a diverse set of tachykinin peptides with divergent receptor activities — EKA/B acting as potent NK1 agonists and EKC/D having a novel C-terminal motif with low receptor potency — expanding the functional repertoire beyond a single HK-1 peptide.\",\n      \"evidence\": \"cDNA cloning of four human TAC4 splice variants, RT-PCR tissue expression profiling, radioligand binding, in vivo hemodynamic measurements in rats\",\n      \"pmids\": [\"12716968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific splice variant regulation was not characterized\", \"Whether EKC/D have a dedicated receptor or function remained unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Structure-activity analysis of EKC/D revealed that it acts as a functional antagonist of substance P in spinal pain processing, with the C-terminal leucine residue being critical — resolving the paradox of why TAC4 encodes peptides with weak agonist activity and demonstrating that a single gene can produce both agonists and antagonists at tachykinin receptors.\",\n      \"evidence\": \"Intrathecal peptide and analog administration, behavioral assays, c-Fos immunohistochemistry, leucine-to-methionine substitution mutagenesis\",\n      \"pmids\": [\"17655832\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of EKC/D antagonism (competitive vs. allosteric) at the receptor level was not determined\", \"Relevance of EKC/D antagonism outside the spinal cord was untested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Characterization of the TAC4 promoter revealed a TATA-less architecture with NFκB-dependent transcriptional induction, explaining the immune-cell-enriched expression pattern that distinguishes TAC4 from neuronal tachykinin genes.\",\n      \"evidence\": \"5' RACE, promoter-reporter assays, NFκB pathway manipulation in a T cell line\",\n      \"pmids\": [\"19081134\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Additional transcription factors regulating tissue-specific TAC4 expression were not identified\", \"Chromatin-level regulation and epigenetic control were not examined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"TAC4 knockout mice revealed that HK-1 has an unexpected inhibitory role in pro-B cell expansion — TAC4-/- mice accumulated fraction B pro-B cells — refining the initial model from pure survival factor to a dual role as a survival signal for pre-B cells and a negative regulator of pro-B cell proliferation.\",\n      \"evidence\": \"TAC4-/- mice, flow cytometric bone marrow analysis, in vitro bone marrow and sorted pro-B cell cultures with exogenous HK-1 addition\",\n      \"pmids\": [\"20660792\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor and signaling pathway mediating pro-B cell inhibition were not identified\", \"Whether the knockout phenotype has functional immunological consequences (e.g., antibody responses) was not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Systematic truncation and structure-activity studies demonstrated that the N-terminal domain of human HK-1 confers biased agonism at NK1 receptors, preferentially engaging Gs/cAMP over Gq/Ca2+ signaling and modulating ERK1/2 phosphorylation and NF-κB activation, providing a molecular basis for functional selectivity among TAC4-derived peptides.\",\n      \"evidence\": \"Peptide truncation series, cAMP and intracellular Ca2+ assays, ERK1/2 phosphorylation, NF-κB reporter assay, receptor internalization assay in NK1-expressing cells\",\n      \"pmids\": [\"21168392\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of biased agonism (receptor conformation) was not resolved\", \"In vivo consequences of biased signaling in immune or vascular contexts were not tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"HK-1 was shown to drive Th17 cell differentiation through an indirect monocyte-dependent mechanism involving NK1 receptor–triggered IL-1β, IL-23, and TNF-like 1A production, establishing TAC4-derived peptides as regulators of adaptive immune polarization beyond their known innate/B cell roles.\",\n      \"evidence\": \"Human PBMC co-culture, cytokine neutralization experiments, monocyte depletion, flow cytometry and ELISA across multiple donors\",\n      \"pmids\": [\"21368235\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HK-1-driven Th17 polarization operates in vivo in autoimmune or inflammatory contexts was not demonstrated\", \"Relative contribution of HK-1 versus substance P to Th17 generation in tissues was not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"TAC4-encoded hemokinins were established as pro-angiogenic factors acting through NK1 receptors to activate ERK1/2, stimulate eNOS/NO production, and upregulate VEGF expression — extending the functional scope of TAC4 products from immune regulation to vascular biology.\",\n      \"evidence\": \"HUVEC proliferation, migration, adhesion, tube formation assays; chick embryo CAM model; NK1/NK2 antagonists; ERK1/2, eNOS, and VEGF Western blotting\",\n      \"pmids\": [\"22554585\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo angiogenic role in mammalian pathological contexts (tumor angiogenesis, wound healing) was not tested\", \"Whether this pathway operates independently of VEGF receptor signaling was unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The functional repertoire of TAC4 products was further extended to include promotion of melanoma cell migration via NK1 receptor–dependent upregulation of MMP-2 and MT1-MMP through PKC/PKA and MAPK cascades, and regulation of human ovarian granulosa cell function through reciprocal signaling with the kisspeptin system.\",\n      \"evidence\": \"Melanoma cell migration assays with NK1 antagonist and kinase inhibitors; primary human granulosa cell RT-PCR, Western blot, and Ca2+ assays with kisspeptin/SP co-treatment\",\n      \"pmids\": [\"27458061\", \"27146034\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of HK-1-driven melanoma metastasis was not established\", \"Molecular mechanism linking TAC4 and kisspeptin signaling was not fully resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Circulating HK-1 was found to desensitize MRGPRX2 on mast cells without inducing receptor internalization, providing a mechanism by which TAC4 products modulate innate immune responses by cross-regulating a non-tachykinin receptor.\",\n      \"evidence\": \"Serum HK-1 ELISA, primary skin mast cell histamine release assays, MRGPRX2 shRNA knockdown, receptor internalization assay\",\n      \"pmids\": [\"34090787\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of MRGPRX2 desensitization (competitive binding vs. allosteric modulation) was not resolved\", \"In vivo relevance of this HK-1/MRGPRX2 axis in allergic or inflammatory disease was not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major unresolved questions include the structural basis for HK-1's biased agonism at NK1 receptors, the in vivo significance of TAC4 products in adaptive immunity and cancer metastasis, the precise mechanism of EKC/D antagonism, and whether TAC4-derived peptides have dedicated non-NK receptors.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal structure or cryo-EM structure of HK-1 bound to NK1R\", \"In vivo role of TAC4 in tumor angiogenesis and metastasis lacks genetic evidence\", \"Potential non-NK receptor targets for EKC/D remain unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 12, 13, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [0, 2, 3, 14, 17, 19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0162582\", \"supporting_discovery_ids\": [1, 2, 4, 12, 14, 16]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 10, 13, 17]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [10, 15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"TACR1\",\n      \"TACR2\",\n      \"TACR3\",\n      \"MRGPRX2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}