{"gene":"VIPR1","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":2000,"finding":"Alanine scanning of VIP identified key residues (His1, Val5, Arg14, Lys15, Lys21, Leu23, Ile26) that directly interact with the VPAC1 receptor, and combining mutations at positions 11, 22, and 28 yielded the first highly selective (>1000-fold) VPAC1 agonist [Ala(11,22,28)]VIP.","method":"Solid-phase synthesis of VIP alanine-scan analogs, radioligand binding (125I-VIP), adenylyl cyclase activity assay, ab initio molecular modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic alanine scan with binding and functional assays, molecular modeling, multiple orthogonal methods in one rigorous study","pmids":["10801840"],"is_preprint":false},{"year":2001,"finding":"Two basic residues in the second transmembrane helix of VPAC1 (Arg188 and Lys195) are essential for receptor activation: the negatively charged Asp3 of VIP must penetrate into the transmembrane domain and interact with these residues to activate the receptor. This interaction is required for activation but not for antagonist recognition.","method":"Site-directed mutagenesis of VPAC1 receptor, radioligand binding, adenylyl cyclase activity assay in transfected CHO cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis combined with binding and functional assays; multiple mutants with orthogonal pharmacological probes","pmids":["11013258"],"is_preprint":false},{"year":2000,"finding":"Three-dimensional model of the VPAC1 receptor N-terminal domain was constructed by homology modeling; site-directed mutagenesis confirmed that Pro74, Pro87, Phe90, and Trp110 (in addition to previously identified Glu36, Trp67, Asp68, Trp73, Gly109) are important for VIP binding and adenylyl cyclase activation, defining a negatively charged binding groove with a tryptophan shell.","method":"Homology modeling, site-directed mutagenesis, stable transfection in CHO cells, 125I-VIP binding, adenylyl cyclase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — combined structure modeling and mutagenesis with binding and functional readouts, multiple residues validated","pmids":["11124960"],"is_preprint":false},{"year":2003,"finding":"Photoaffinity labeling with [Bpa22-VIP] demonstrated a direct physical contact between Tyr22 of VIP and the 109-120 fragment (GWTHLEPGPYPI) of the N-terminal ectodomain of hVPAC1 receptor, providing the first direct evidence for VIP–VPAC1 N-terminal ectodomain contact.","method":"Photoaffinity labeling, cyanogen bromide cleavage, V8 endoproteinase cleavage, creation of CNBr-cleavage-site receptor mutants, SDS-PAGE, Edman sequencing; 125I-labeled probe","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct photoaffinity crosslinking with fragment mapping and mutant confirmation, rigorous controls","pmids":["12807902"],"is_preprint":false},{"year":2006,"finding":"NMR structure of VIP (central alpha-helix, disordered N-terminal His1-Phe6, 310 helix at Ser25-Asn28) combined with photoaffinity labeling showed Phe6 contacts Asp107, Tyr22 contacts Gly116, and Asn24 contacts Cys122 in the N-terminal ectodomain of hVPAC1. A 3D model of the N-ted (short consensus repeat/Sushi domain with two antiparallel beta-sheets, three disulfide bonds) was built; docking showed the VIP 6-28 fragment occupies the C-terminal part of the N-ted while the N-terminus is free to interact with the transmembrane region.","method":"Photoaffinity labeling with Bpa at positions 6, 22, 24 of VIP, Edman sequencing of labeled fragments, NMR structure determination of VIP, homology modeling of N-ted using CRF receptor 2beta NMR structure as template, molecular docking","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (NMR, photoaffinity, modeling) in one rigorous study with structural validation","pmids":["16520374"],"is_preprint":false},{"year":2004,"finding":"Photoaffinity labeling with [Bpa6]-VIP demonstrated that position 6 of VIP contacts the 104-108 fragment of the hVPAC1 N-terminal ectodomain, adjacent to the fragment contacted by position 22 (Tyr22), showing that the central part of VIP (at least Phe6 to Tyr22) interacts with the N-terminal ectodomain.","method":"Photoaffinity labeling, sequential enzymatic and chemical cleavage (CNBr, PNGase F, Glu-C, trypsin), SDS-PAGE, receptor mutant with new CNBr cleavage site","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct photoaffinity crosslinking, multi-enzyme mapping, confirmed by mutant receptor","pmids":["15247290"],"is_preprint":false},{"year":2008,"finding":"The N-terminal part of VIP (residues 1-5/0) physically contacts the 130-137 region of the hVPAC1 N-terminal ectodomain (via [Bpa0]-VIP probe), while the N-terminal part of antagonist PG97-269 contacts a different region of the N-ted (residues 43-66), demonstrating distinct binding sites for the agonist and antagonist N-termini.","method":"Photoaffinity labeling with Bpa0-VIP and Bpa0-PG97-269, CNBr cleavage, NuPAGE analysis","journal":"Journal of molecular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct photoaffinity labeling, single lab, two probes with fragment identification","pmids":["18597186"],"is_preprint":false},{"year":2012,"finding":"Photoaffinity labeling established that the N-terminus of VIP ([Bpa0]-VIP probe) contacts VPAC1 residue Q135, while the N-terminus of antagonist PG97-269 ([Bpa0]-PG97-269) contacts G62 of the N-ted—distinct from the VIP contact site. Additionally, residues K143, T144, and T147 in the first transmembrane domain are critical for interaction with the His1 N-terminus of VIP, as shown by alanine substitution.","method":"Photoaffinity labeling, alanine mutagenesis of transmembrane domain residues, binding affinity measurements, pharmacological assays with VIP2-28","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — photoaffinity labeling plus mutagenesis with multiple orthogonal approaches in one rigorous study","pmids":["22291440"],"is_preprint":false},{"year":2010,"finding":"A network of conserved residues Arg188 (TM2), Asn229 (TM3), and Gln380 (TM7) governs ligand binding and receptor activation of VPAC1; double mutants of reciprocal residue exchanges showed strong cooperative or anticooperative effects confirming spatial proximity. Arg188 interacts with Asp3 of VIP and this is altered upon VIP binding to trigger activation.","method":"Structural modeling of TM domain, site-directed mutagenesis, cAMP production assay, binding assays, double mutant cooperativity analysis in CHO cells","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1 / Strong — combined structural model with systematic mutagenesis and double-mutant epistasis analysis, multiple orthogonal methods","pmids":["20573782"],"is_preprint":false},{"year":2005,"finding":"The carboxyl terminus of VPAC1 contains Ser/Thr residues (including Thr429, Ser435, Ser448/449, Ser455 in the distal C-terminus and Ser250 in the second intracellular loop) that mediate VIP-stimulated receptor phosphorylation and internalization; truncation removing all C-terminal Ser/Thr residues abolished phosphorylation and internalization, and also enabled receptor recycling (reversed within 2 h), an effect blocked by monensin.","method":"Site-directed mutagenesis and truncation of VPAC1, flow cytometry, confocal microscopy with monoclonal antibody against receptor, phosphorylation assays in CHO cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic truncations and mutations combined with multiple readouts (phosphorylation, internalization, recycling) and flow cytometry/confocal imaging","pmids":["15932876"],"is_preprint":false},{"year":2003,"finding":"Ser447 in the C-terminal tail of VPAC1 is crucial for VIP-induced receptor phosphorylation and rapid desensitization (10-fold right-shift of ED50 for adenylyl cyclase) but is not required for receptor internalization or down-regulation; Ser447 is a likely GRK target.","method":"Site-directed mutagenesis to Ala, adenylyl cyclase activity assay, phosphorylation assay, internalization assay using fluorescein-tagged VIP and Flag/GFP-tagged receptor in CHO cells","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 1 / Strong — specific single-residue mutagenesis with multiple orthogonal functional readouts (desensitization, phosphorylation, internalization)","pmids":["14645688"],"is_preprint":false},{"year":2005,"finding":"Mutations in the distal part of the third intracellular loop (R338, L339, R341) of VPAC1 markedly reduce VIP-stimulated calcium increase and Galphai coupling but only weakly affect adenylyl cyclase activity, whereas mutations in the proximal domain (K322) reduce adenylyl cyclase activity without changing the calcium response, demonstrating separate receptor sub-domains for Gs and Gi/Ca2+ coupling.","method":"Site-directed mutagenesis, adenylyl cyclase activity assay, calcium response assay in CHO cells","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic mutagenesis with two distinct functional readouts identifying separate coupling domains","pmids":["15451021"],"is_preprint":false},{"year":2002,"finding":"A small sequence in the third intracellular loop (IC3) of VPAC1, residues 328-331 (IRKS), is responsible for efficient agonist-stimulated [Ca2+]i increase, likely through coupling to Galphai/Galphaq proteins; swapping this sequence with the VPAC2 counterpart (VGGN) interconverted the calcium signaling phenotypes of the two receptors.","method":"VPAC1/VPAC2 chimeric receptors, point mutations in IC3, aequorin reporter gene calcium assay in transfected CHO cells","journal":"Molecular endocrinology","confidence":"High","confidence_rationale":"Tier 1 / Strong — chimeric receptor approach with reciprocal sequence swaps and functional validation","pmids":["11981043"],"is_preprint":false},{"year":1999,"finding":"Specific mutations of Thr343 (to Lys, Pro, or Ala) in human VPAC1 receptor produced constitutive activation with ~3.5-fold increase in cAMP; constitutive activation required integrity of the N-terminal extracellular VIP-binding domain (abolished by E36A or D68A double mutations), establishing Thr343 at the IC2/TM4 junction as a key constraint for receptor activation state.","method":"Site-directed mutagenesis, transient transfection in COS cells, cAMP production assay, double-mutant analysis","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple specific mutations including double mutants with functional dissection of activation mechanism","pmids":["9920725"],"is_preprint":false},{"year":2006,"finding":"Asn229 in TM3 of VPAC1 is essential for receptor activation (G protein coupling) but not for VIP/antagonist binding, not for agonist-stimulated phosphorylation and internalization; the N229A mutant could still internalize but re-expressed more rapidly than wild-type after agonist washout, dissociating G protein activation from receptor trafficking.","method":"Site-directed mutagenesis (Ala, Asp, Gln mutations), adenylyl cyclase assay, calcium assay, GTP sensitivity assay, receptor phosphorylation and internalization assays in CHO cells","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic mutagenesis with multiple orthogonal readouts dissecting activation, phosphorylation, and trafficking","pmids":["16650965"],"is_preprint":false},{"year":2001,"finding":"The conserved 'YL' motif (Tyr239, Leu240) in VPAC1 is functionally equivalent to the 'DRY' motif of rhodopsin-family GPCRs; Y239A caused moderate and L240A caused pronounced impairment of VIP-induced cAMP production, primarily by perturbing the G protein-binding site rather than the intrinsic low-to-high affinity equilibrium.","method":"Site-directed mutagenesis, GTP-gamma-S binding shift assay, VIP-induced cAMP production in whole cells","journal":"Journal of molecular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional and binding readouts, single lab","pmids":["11859928"],"is_preprint":false},{"year":2001,"finding":"Three conserved transmembrane prolines (P266, P300, P348) of VPAC1 are important for receptor expression, G protein coupling, and receptor activity; P266A decreased cAMP stimulation, while P300A and P348A increased potency and GTP sensitivity, suggesting these prolines constrain receptor activation. Adjacent leucines L346A and L349A also reduced receptor expression and G protein coupling.","method":"Alanine substitution mutagenesis, radioligand binding, cAMP production assay, GTP sensitivity in transfected cells","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with binding and functional readouts, single lab","pmids":["11513868"],"is_preprint":false},{"year":2002,"finding":"Immunoaffinity chromatography using an anti-VPAC1 first-extracellular-loop antibody showed that human VPAC1 in HEK293 cells couples to Gs but not Gi3, Gi1/2, or Gq; rat VPAC1 in brain couples to Gs and Gi3; rat VPAC1 in lung couples to Gs, Gi3, and Gq. VIP pretreatment increased G protein co-purification. Pre-coupling of VPAC1 to G protein occurred basally (without agonist), confirmed by covalent cross-linking.","method":"Immunoaffinity chromatography, covalent cross-linking in native membranes, Western blotting with G-protein-specific antibodies, 125I-VIP binding","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal pull-down strategy, cross-linking confirmation, single lab with multiple tissues/cell types","pmids":["11812005"],"is_preprint":false},{"year":2004,"finding":"VPAC1 receptor contains a functional N-terminal signal peptide (residues 1-30); deletion of the signal peptide abolished cell-surface expression and 125I-VIP binding, while the signal peptide is cleaved during translocation to the plasma membrane, probably in the endoplasmic reticulum.","method":"Deletion and Flag-insertion constructs, stable transfection in CHO cells, 125I-VIP binding, cAMP production, GFP fluorescence, indirect immunofluorescence on non-permeabilized cells","journal":"Regulatory peptides","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple constructs with binding, functional, and localization readouts confirming signal peptide function","pmids":["15518910"],"is_preprint":false},{"year":1999,"finding":"VPAC1/VPAC2 chimeric receptor analysis showed the N-terminal extracellular domain determines selectivity for the VIP1 antagonist, while VPAC1-selective agonist recognition additionally requires the first extracellular loop and distal receptor domains; replacement of EC1 in VPAC1 with the VPAC2 counterpart markedly reduced maximal cAMP response.","method":"Chimeric VPAC1/VPAC2 receptors, radioligand binding, adenylyl cyclase activity assay","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chimeric receptor approach with binding and functional readouts, single lab","pmids":["10491203"],"is_preprint":false},{"year":2004,"finding":"VPAC1 receptor activation by VIP and PACAP-27 on the basolateral surface of human bronchial epithelial Calu-3 cells induces CFTR-dependent chloride secretion; this requires both PKA and PKC activity. IB3-1 cystic fibrosis cells expressing VPAC1 but lacking functional CFTR showed no chloride transport in response to VIP/PACAP-27.","method":"Radioligand binding (125I-VIP, 125I-PACAP-27), iodide efflux assay, Ussing chamber short-circuit current measurements, PKA inhibitor (H-89) and PKC inhibitor (chelerythrine) pharmacology","journal":"British journal of pharmacology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (efflux, Ussing chamber, pharmacological inhibitors, CFTR-null control) in one study","pmids":["14744818"],"is_preprint":false},{"year":2014,"finding":"VIP stimulation of VPAC1 in airway Calu-3 cells increases CFTR membrane stability by promoting CFTR interaction with NHERF1 and phosphorylated ERM (via PKCε), while reducing CFTR interaction with CFTR-associated ligand (CAL); knockdown of NHERF1 or ERM by siRNA prevented the VIP effect on CFTR membrane stability and sustained CFTR activity.","method":"Immunocytochemistry, in situ proximity ligation assay, siRNA knockdown of NHERF1 and ERM, iodide efflux assays, Western blotting","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — proximity ligation, siRNA knockdown, and functional efflux assays with multiple protein partners identified","pmids":["24788249"],"is_preprint":false},{"year":2007,"finding":"VPAC1 signaling negatively regulates megakaryopoiesis: PACAP/VIP activation of VPAC1 on megakaryocytes inhibits megakaryocyte differentiation and reduces platelet counts; blocking VPAC1 with neutralizing antibody (23A11) or anti-PACAP (PP1A4) inhibited cAMP formation, stimulated megakaryopoiesis independently of thrombopoietin, and elevated platelet counts in mice and in models of myelosuppression and GATA1 deficiency.","method":"In vitro CD34+ cell differentiation assays, anti-VPAC1 and anti-PACAP neutralizing antibodies, transgenic mice overexpressing PACAP in megakaryocytes, VPAC1 KO mouse studies, cAMP measurement, histology","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — patient-derived cells, transgenic and KO mice, neutralizing antibodies, in vitro and in vivo experiments with multiple disease models","pmids":["18000164"],"is_preprint":false},{"year":2011,"finding":"VPAC1 receptor mediates the pro-inflammatory enhancement of DSS-induced colitis by VIP: VPAC1-KO mice showed milder colitis than wild-type mice, with reduced tissue myeloperoxidase, IL-6, IL-1β, and MMP-9; suppression of VPAC1 signals by PKA inhibitors in VPAC2-KO mice reduced colitis severity. Thus VIP enhancement of colitis is exclusively mediated by VPAC1.","method":"VPAC1-KO and VPAC2-KO mice, DSS-induced colitis model, myeloperoxidase assay, cytokine measurement, histopathology, pharmacological PKA inhibitors","journal":"Cellular immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO models for both receptor subtypes with multiple inflammatory readouts and pharmacological confirmation","pmids":["21295288"],"is_preprint":false},{"year":2016,"finding":"VPAC1 deficiency ameliorates experimental autoimmune encephalomyelitis (EAE) by impairing the effector phase: VPAC1 KO mice showed reduced CNS histopathology, reduced chemokine mRNAs, and impaired inflammatory cell infiltration. WT cells fully induced EAE in WT but not VPAC1-KO recipients (bone marrow chimeras). The resistance was minimally dependent on VPAC1 expression in the hematopoietic compartment, implicating non-hematopoietic VPAC1 in CNS chemokine induction.","method":"VPAC1 KO mice, MOG35-55 EAE induction, histology, real-time PCR, immunofluorescence, adoptive transfer, bone marrow chimeras, antigen-recall assays, pharmacological VPAC1 antagonist","journal":"Journal of neuroinflammation","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic approaches (KO, adoptive transfer, chimeras) with pharmacological validation","pmids":["27357191"],"is_preprint":false},{"year":2011,"finding":"VPAC1-null mice die perinatally with intestinal obstruction, disorganized hyperproliferative intestinal epithelium, small dysmorphic pancreatic islets, hypoglycemia, and impaired glucose homeostasis, demonstrating a required role for VPAC1 in embryonic and neonatal development of intestine and endocrine pancreas; VPAC1 promoter-driven transgene was expressed in E12.5/E14.5 intestinal epithelial and pancreatic endocrine cells.","method":"Homozygous VPAC1-null mutant mice, VPAC1 promoter-driven β-galactosidase transgenic mice, histology, glucose homeostasis assays (oral glucose tolerance, insulin challenge), blood glucose measurement","journal":"Pancreas","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic null mouse with multiple developmental and metabolic phenotypes and reporter gene localization","pmids":["21697765"],"is_preprint":false},{"year":2002,"finding":"VIP inhibits bone marrow progenitor proliferation (CFU-GM and erythroid progenitors) through VPAC1 (type 1 receptor), as shown by reversal of inhibition with VPAC1 antagonist but not VPAC2 agonist; direct effects on CD34+ cells were shown, with additional indirect effects via stromal TGF-β and TNF-α induction. Chemical cross-linking confirmed VPAC1 on stromal membranes.","method":"Clonogenic assays with unfractionated and CD34+ bone marrow cells, VPAC1/VPAC2 selective agonists and antagonists, semi-quantitative RT-PCR, chemical cross-linking","journal":"Experimental hematology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — selective receptor pharmacology with clonogenic assays and cross-linking, single lab","pmids":["12225791"],"is_preprint":false},{"year":2014,"finding":"VPAC1 receptor on cholinergic submucosal (NPY+) secretomotor neurons mediates VIP-induced chloride secretion: VIP-evoked secretion was depressed by VPAC1 antagonist PG97-269 and by hyoscine (muscarinic antagonist) but not eliminated by both together, indicating a direct epithelial VPAC1R component and an indirect VPAC1R-on-cholinergic-neuron component. VIP stimulates ACh-mediated longitudinal muscle contraction via VPAC1R on calretinin+ cholinergic motor neurons (inhibited by TTX, PG97-269, and hyoscine).","method":"Immunohistochemistry for VPAC1R and neurochemical markers, RT-PCR, Ussing chamber short-circuit current, isotonic muscle contraction with pharmacological dissection (TTX, VPAC1 antagonist PG97-269, hexamethonium, hyoscine)","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — immunohistochemical localization combined with functional Ussing chamber and muscle contraction assays with specific pharmacological agents","pmids":["24578344"],"is_preprint":false},{"year":2009,"finding":"VPAC1 receptor activation directs postnatal dentate gyrus neural stem/progenitor cells toward granule cell neurogenesis without a trophic effect, in contrast to VPAC2 which promotes symmetric division and nestin-positive cell pool expansion. This differential fate modulation was established by selective receptor agonists on postnatal hippocampal cultures.","method":"Selective VPAC1 and VPAC2 agonists applied to postnatal hippocampal cultures, cell fate analysis, BrdU incorporation, immunostaining for nestin and neuronal markers; in vivo Vipr2-/- mice for VPAC2 validation","journal":"Stem cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — selective pharmacology with cell fate readouts in cultures; in vivo validation for VPAC2 only","pmids":["19650041"],"is_preprint":false},{"year":2014,"finding":"Microglial VPAC1R mediates VIP-induced enhancement of neural stem/progenitor cell proliferation and pro-neurogenic effects in hippocampal cultures via IL-4 release from microglia; conditioned media from VIP-stimulated microglia was trophic for NSPCs, and this was dependent on VPAC1 receptor signaling leading to IL-4 secretion.","method":"Hippocampal mixed cultures with microglial depletion and readdition, conditioned media experiments, VIP stimulation with VPAC1 receptor activation, IL-4 measurement, NSPC proliferation and survival assays","journal":"Glia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditioned media and depletion-readdition experiments with cytokine identification, single lab","pmids":["24801739"],"is_preprint":false},{"year":2005,"finding":"VPAC1 receptor activation by VIP attenuates acute pancreatitis through inhibition of proinflammatory cytokine (IL-6, TNF-α) production from monocytes; selective VPAC1-R agonist decreased serum amylase, IL-6, and TNF-α and attenuated histological severity, while VPAC2-R agonist worsened outcomes. VPAC1-R and VPAC2-R mRNA were expressed in splenic monocytes.","method":"Cerulein/LPS mouse pancreatitis model, selective VPAC1-R and VPAC2-R agonists, serum amylase and cytokine ELISA, histology, in vitro monocyte cytokine production assay with LPS stimulation, RT-PCR","journal":"Pancreas","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — selective receptor pharmacology in vivo and in vitro with multiple readouts, single lab","pmids":["15632701"],"is_preprint":false},{"year":2003,"finding":"VIP inhibits intestinal dipeptide (Gly-Sar) transport via VPAC1 receptor in Caco-2 cells (VPAC2 mRNA not expressed) by a PKA-dependent mechanism; the inhibition is Na+-dependent and involves reduction of NHE3-dependent intracellular pH recovery after dipeptide-induced acidification, indicating modulation of hPepT1 activity indirectly through NHE3 inhibition.","method":"14C-Gly-Sar uptake assay, RT-PCR for receptor expression, BCECF pH measurement, PKA inhibitor H-89, NHE3 inhibitor S1611, Western blotting for NHERF1, selective VPAC1 agonist","journal":"British journal of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — selective agonist pharmacology, pH assay, transport assay, pharmacological dissection, single lab","pmids":["12598410"],"is_preprint":false},{"year":2002,"finding":"VPAC1 receptor actively facilitates productive HIV-1 infection: VPAC1 signal-blocking antibody inhibited ~80% of productive HIV-1 infection; VPAC1 antisense transfection reduced productive infection by ~50%; sense VPAC1 transfection increased productive infection >15-fold and increased syncytium formation. VPAC1 does not affect viral entry but is required for steps post-entry (absence of 2-LTR circles in VPAC1-negative cells). HIV-1 gp120 has sequence similarity to VIP, suggesting potential direct receptor activation.","method":"VPAC1 signal-blocking antibody, sense/antisense transfection of VPAC1 cDNA, HIV-1 p24 ELISA, luciferase pseudovirus assay, HIV-1 gag DNA PCR, 2-LTR circle analysis, syncytium formation assay","journal":"AIDS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic and antibody approaches with mechanistic post-entry analysis, single lab","pmids":["11834941"],"is_preprint":false},{"year":2002,"finding":"Ikaros transcription factors (IK-1 and IK-2) suppress endogenous VPAC1 expression: Ikaros binds high-affinity consensus sequences in the VPAC1 5'-flanking region (confirmed by EMSA with supershifts), overexpression of IK-1 or IK-2 in NIH-3T3 clones reduced VPAC1 mRNA and protein by 50-93%, and VPAC1 luciferase reporter activity was decreased up to 41% with two major Ikaros binding domains at -1076 to -623 bp and -222 to -35 bp.","method":"EMSA with native T cell nuclear extracts and recombinant IK-1/IK-2, antibody supershift, stable NIH-3T3 clones overexpressing Ikaros isoforms, RT-PCR and fluorometric kinetic RT-PCR, 125I-VIP binding, luciferase reporter with nested deletions","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — EMSA/supershift, stable overexpression, multiple reporter deletion constructs, protein and mRNA readouts in one comprehensive study","pmids":["11812772"],"is_preprint":false},{"year":2005,"finding":"VPAC1 receptor expression in gallbladder epithelial cells is transcriptionally regulated by FXR (farnesoid X receptor): the FXR agonist GW4064 upregulated VPAC1 mRNA and protein in primary human gallbladder epithelial cells dose-dependently; this effect was antagonized by 9-cis retinoic acid (RXRα ligand). Chenodeoxycholate activated endogenous FXR (confirmed by EMSA) and also increased VPAC1 expression.","method":"Quantitative RT-PCR, Western blot, primary human gallbladder epithelial cell cultures, pharmacological FXR agonist GW4064, EMSA for FXR binding, RXRα antagonism","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA plus RT-PCR/Western blot in primary human cells, single lab","pmids":["16037943"],"is_preprint":false},{"year":2017,"finding":"VPAC1 is localized to the apical membrane of intestinal epithelial cells (colocalizing with villin but not basolateral Na+/K+-ATPase) in both mouse and human colon, with highest expression in the colon compared to ileum and jejunum; this apical localization was determined by immunofluorescence and suggests potential for luminal peptide recognition.","method":"Quantitative RT-PCR, Western blotting, immunofluorescence with apical (villin) and basolateral (Na+/K+-ATPase) markers in mouse and human intestinal tissue","journal":"American journal of physiology. Gastrointestinal and liver physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct immunofluorescence colocalization with polarization markers in mouse and human tissues, single lab","pmids":["28385693"],"is_preprint":false},{"year":2017,"finding":"Palmitoylation of Cys37 in the N-terminal extracellular domain of VPAC1 mediates VIP-induced nuclear translocation of the receptor, which contributes to its anti-apoptotic activity; the C37A mutant failed to undergo nuclear translocation upon VIP stimulation and showed reduced anti-apoptotic activity, while exhibiting higher proliferative activity. Palmitoylation was confirmed by acyl-biotin exchange assay and click chemistry-based palmitoylation assay; the palmitoylation inhibitor 2-bromopalmitate blocked both nuclear translocation and anti-apoptotic activity.","method":"Site-directed mutagenesis (C37A), stable transfection with VPAC1-EYFP fusion proteins in CHO cells, confocal microscopy, Western blotting, fluorescence quantification, acyl-biotin exchange assay, click chemistry palmitoylation assay, 2-bromopalmitate treatment, apoptosis assay with camptothecin","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct palmitoylation detection by two biochemical methods, mutagenesis, pharmacological inhibition with functional readouts (nuclear translocation, apoptosis) in one study","pmids":["28473666"],"is_preprint":false},{"year":2019,"finding":"VPAC1 activation by VIP in gastric cancer cells induces TRPV4-mediated Ca2+ entry and promotes gastric cancer progression in a Ca2+-dependent manner; this VPAC1/TRPV4/Ca2+ signaling axis enhances VIP expression and secretion, establishing a positive autocrine feedback loop. Inhibition of VPAC1 blocked progressive responses.","method":"In vitro gastric cancer cell signaling assays, Ca2+ imaging, VPAC1 and TRPV4 inhibition, VIP/VPAC1 expression analysis in human cancer specimens and cell lines","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Ca2+ imaging with pharmacological inhibitors defining signaling pathway, in vitro and clinical correlation, single lab","pmids":["30692637"],"is_preprint":false},{"year":2022,"finding":"VIPR1 activation by VIP in HCC cells inhibits growth and metastasis through regulation of arginine biosynthesis: VIP treatment partially restored argininosuccinate synthase (ASS1) expression and inhibited de novo pyrimidine synthesis by suppressing CAD phosphorylation via mTOR/p70S6K signaling. Human HCC samples showed downregulation of ASS1 and upregulation of phospho-CAD correlating with VIPR1 loss.","method":"In vitro and in vivo HCC cell studies with VIP treatment, transcriptome sequencing, ASS1 expression analysis, CAD phosphorylation assay, mTOR/p70S6K pathway analysis, human HCC clinical sample analysis","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptomics plus mechanistic pathway analysis in vitro/in vivo with clinical correlation, single lab","pmids":["35864952"],"is_preprint":false},{"year":2005,"finding":"VPAC1 and VPAC2 receptors are both required for pressure-induced vasodilatation (PIV): blockade of VPAC1/VPAC2, or selectively of VPAC1 alone, eliminated the PIV response in anesthetized rodents, while PAC1 blockade had no effect. Vascular smooth muscle and endothelial vasodilator capacity were unaffected by VPAC1/2 antagonism.","method":"In vivo pharmacological blockade with selective antagonists (PG97-269 for VPAC1, PACAP6-38 for VPAC2/PAC1, Max.d.4 for PAC1, D-p-Cl-Phe6,Leu17-VIP for VPAC1/2) in anesthetized rodents, laser Doppler flowmetry","journal":"The Journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic pharmacological receptor dissection in vivo with multiple selective antagonists","pmids":["14578481"],"is_preprint":false},{"year":2006,"finding":"In porcine basilar arteries, VPAC1 receptor is localized specifically on the endothelium and mediates vasodilation via nitric oxide (NO) generation (inhibited by L-NAME, abolished by endothelial denudation), while VPAC2 on outer smooth muscle layers mediates NO-independent vasodilation.","method":"Immunocytochemistry, RT-PCR, pharmacological vasodilator responses to selective receptor agonists, L-NAME inhibition, endothelial denudation, electrical stimulation with VPAC2 antagonist","journal":"Journal of cerebral blood flow and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — immunocytochemical localization with functional pharmacological dissection in tissue preparations, single lab","pmids":["15959462"],"is_preprint":false},{"year":2011,"finding":"VPAC1 receptor on hippocampal nerve terminals inhibits exocytotic VGCC-dependent GABA release through Gi/o protein- and PKC-dependent mechanisms; this opposes the VPAC2 receptor effect (which enhances GABA release via Gs/PKA/PKC).","method":"Isolated rat hippocampal nerve terminal preparations, 3H-GABA release assay with selective VPAC1/VPAC2 agonists and antagonists, pertussis toxin, PKA inhibitor, PKC inhibitor, voltage-gated calcium channel blockers","journal":"British journal of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — nerve terminal preparation with selective pharmacology dissecting receptor subtypes and G protein/kinase coupling, single lab","pmids":["28945273"],"is_preprint":false},{"year":2005,"finding":"VPAC1 receptor activation by VIP and PACAP enhances hippocampal CA1 pyramidal cell synaptic transmission; VPAC1-mediated enhancement is dependent on PKC but not PKA activity (in contrast to VPAC2-mediated enhancement which requires PKA), demonstrating distinct intracellular signaling downstream of each receptor subtype in hippocampal synaptic modulation.","method":"Selective VPAC1 agonist [K15,R16,L27]VIP(1-7)/GRF(8-27) and VPAC2 agonist RO 25-1553, selective antagonists PG97-269 and PG99-465, PKA inhibitor H-89, PKC inhibitor GF109203X, field potential recording in hippocampal slices","journal":"Brain research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — selective receptor pharmacology with kinase inhibitor dissection in electrophysiology preparation, single lab","pmids":["15935995"],"is_preprint":false},{"year":2008,"finding":"In CD4 T cells, VPAC1 mRNA is upregulated by the vascular environment but downregulated by TCR signaling (anti-CD3); JNK kinases downstream of Zap70 mediate the suppressive regulation of VPAC1 after TCR activation, while inhibitors of PKC, ERK, p38, Zap70, and Rac1 show stimulatory influence on VPAC1 expression in the absence of TCR signaling.","method":"Primary murine splenic CD4 T cell isolation, pharmacological kinase inhibitors (10 inhibitors), anti-CD3 stimulation, qPCR for VPAC1 mRNA, blood vs. spleen comparison","journal":"Brain, behavior, and immunity","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pharmacological kinase inhibitor panel with qPCR, single lab, no direct pathway confirmation","pmids":["18534815","18555660"],"is_preprint":false},{"year":1999,"finding":"The mouse Vipr1 gene is encoded on chromosome 9 (syntenic with human chromosome 3p21.3) in a single-copy gene of >16 kb with 13 exons; the 5'-flanking region lacks a TATA box but contains a CCAAT box, Sp1- and AP-2-binding sites, and has functional promoter activity in luciferase reporter assays.","method":"Genomic cloning and sequencing, exon-intron mapping, chromosomal mapping by genetic crosses, luciferase reporter assay","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct genomic characterization and functional promoter assay, single lab","pmids":["10331949"],"is_preprint":false}],"current_model":"VPAC1 (VIPR1) is a class B G protein-coupled receptor that binds VIP and PACAP-27 with equal high affinity at its N-terminal ectodomain (structured as a Sushi/short consensus repeat domain with three disulfide bonds), where the central-to-C-terminal portion of VIP (residues 6-28) docks via contacts mapped by photoaffinity labeling (Phe6↔N-ted 104-108, Tyr22↔N-ted 109-120), while the VIP N-terminus (His1) interacts with TM1 residues K143/T144/T147; receptor activation requires Asp3 of VIP to penetrate the TM bundle and interact with Arg188/Lys195 in TM2, engaging a conserved network with Asn229 (TM3) and Gln380 (TM7); upon activation the receptor couples primarily to Gs/adenylyl cyclase (via a YL motif equivalent to the DRY motif) and also to Gi/o (via IC3 residues including IRKS 328-331) and Galphaq for Ca2+ signaling, with the third IC loop's distal basic residues (R338, L339, R341) selectively mediating Ca2+ and Gi coupling; rapid desensitization is mediated by GRK phosphorylation of Ser447 in the C-terminal tail, and sustained internalization requires multiple C-terminal Ser/Thr residues; Cys37 in the N-terminal ectodomain undergoes palmitoylation that mediates VIP-induced nuclear translocation contributing to anti-apoptotic signaling; a functional N-terminal signal peptide (residues 1-30) is required for cell-surface expression; physiologically, VPAC1 suppresses megakaryopoiesis (raising cAMP in megakaryocytes to inhibit differentiation), regulates intestinal secretion and motility via cholinergic neurons and epithelial CFTR (on the apical membrane of colonocytes), modulates hippocampal synaptic transmission via PKC-dependent GABA release inhibition, suppresses bone marrow progenitor proliferation, and mediates pro-inflammatory CNS chemokine induction in EAE; its transcription is regulated by Ikaros (suppression) and FXR agonists (activation in biliary epithelium), and by promoter methylation and H3K27 deacetylation in HCC."},"narrative":{"mechanistic_narrative":"VIPR1 (VPAC1) is a class B G protein-coupled receptor for the neuropeptides VIP and PACAP-27 that transduces these signals into cyclic AMP and calcium responses governing intestinal physiology, hematopoiesis, neural function, and inflammation [PMID:10801840, PMID:18000164]. Ligand recognition follows a two-domain mechanism: the central-to-C-terminal portion of VIP (residues 6–22) docks against a negatively charged, tryptophan-lined groove in the N-terminal ectodomain, a Sushi/short-consensus-repeat fold stabilized by three disulfide bonds, while the VIP N-terminus engages the transmembrane bundle [PMID:11124960, PMID:16520374, PMID:15247290]. Direct photoaffinity crosslinking mapped Phe6 to ectodomain residues 104–108 and Tyr22 to 109–120, and showed that the VIP His1 N-terminus contacts TM1 residues K143/T144/T147, contacts that are distinct from those used by antagonists [PMID:12807902, PMID:22291440]. Receptor activation requires the acidic Asp3 of VIP to penetrate the transmembrane core and engage Arg188/Lys195 in TM2 within a conserved network linking Arg188 (TM2), Asn229 (TM3), and Gln380 (TM7) [PMID:11013258, PMID:20573782, PMID:16650965]. Activated VPAC1 couples primarily to Gs/adenylyl cyclase via a DRY-equivalent 'YL' motif, and separately to Gi/Gq for calcium signaling through distinct third-intracellular-loop subdomains (IRKS 328–331 and distal basic residues R338/L339/R341) [PMID:15451021, PMID:11981043, PMID:11859928]. Agonist-induced phosphorylation of C-terminal Ser/Thr residues drives desensitization (Ser447 as a GRK target) and internalization [PMID:15932876, PMID:14645688]. Beyond canonical signaling, palmitoylation of Cys37 in the ectodomain directs VIP-induced nuclear translocation that supports anti-apoptotic activity, and a cleaved N-terminal signal peptide (residues 1–30) is required for surface expression [PMID:15518910, PMID:28473666]. Physiologically, VPAC1 raises cAMP in megakaryocytes to suppress megakaryopoiesis and platelet production [PMID:18000164], is required for intestinal and pancreatic islet development [PMID:21697765], drives CFTR-dependent chloride secretion in epithelia by stabilizing CFTR through NHERF1/ERM [PMID:14744818, PMID:24788249], acts on cholinergic secretomotor and motor neurons to regulate intestinal secretion and motility [PMID:24578344], and mediates pro-inflammatory responses in colitis and EAE [PMID:21295288, PMID:27357191]. Its transcription is suppressed by Ikaros and induced by FXR agonists in distinct epithelia [PMID:11812772, PMID:16037943].","teleology":[{"year":2000,"claim":"Establishing which VIP residues drive VPAC1 engagement defined the pharmacophore and yielded the first selective VPAC1 agonist, enabling all subsequent receptor-subtype dissection.","evidence":"Alanine-scan VIP analogs with radioligand binding, adenylyl cyclase assays, and molecular modeling","pmids":["10801840"],"confidence":"High","gaps":["Does not map the reciprocal receptor contact residues","Selectivity mechanism at the structural level not resolved"]},{"year":2001,"claim":"Identifying the transmembrane acidic-residue trigger answered how the VIP N-terminus converts binding into activation, separating activation from antagonist recognition.","evidence":"Site-directed mutagenesis of TM2 (Arg188, Lys195) with binding and cAMP assays in CHO cells","pmids":["11013258"],"confidence":"High","gaps":["Conformational changes accompanying Asp3 insertion not directly observed","No structural snapshot of the activated state"]},{"year":2000,"claim":"Building a 3D model of the N-terminal ectodomain and validating its residues defined the peptide-binding groove architecture of the receptor.","evidence":"Homology modeling plus mutagenesis (Pro74, Pro87, Phe90, Trp110 etc.) with binding and cyclase readouts","pmids":["11124960"],"confidence":"High","gaps":["Model lacks experimental high-resolution structure","Dynamics of ligand capture not addressed"]},{"year":2006,"claim":"Combining NMR of VIP with photoaffinity mapping resolved the two-domain binding mode, showing VIP 6–28 occupies the ectodomain while the N-terminus is free to reach the transmembrane region.","evidence":"Bpa photoaffinity probes at VIP positions 6/22/24, Edman sequencing, VIP NMR, and N-ted homology modeling/docking","pmids":["16520374","12807902","15247290"],"confidence":"High","gaps":["Full receptor–ligand complex not crystallized","Stoichiometry and order of binding events not defined"]},{"year":2012,"claim":"Distinguishing agonist versus antagonist N-terminal contact sites and pinpointing TM1 His1-interacting residues clarified why agonists and antagonists are recognized differently.","evidence":"Bpa0 photoaffinity probes for VIP and PG97-269 plus alanine mutagenesis of TM1 (K143/T144/T147)","pmids":["22291440","18597186"],"confidence":"High","gaps":["Functional consequence of distinct antagonist site for drug design not tested","Single-lab photoaffinity mapping"]},{"year":2010,"claim":"Demonstrating cooperativity among Arg188/Asn229/Gln380 established a conserved transmembrane activation network bridging ligand sensing and G-protein coupling.","evidence":"Structural modeling with double-mutant cooperativity analysis and cAMP/binding assays in CHO cells","pmids":["20573782","16650965"],"confidence":"High","gaps":["Activation network not visualized structurally","Allosteric pathway to G protein not fully traced"]},{"year":2002,"claim":"Mapping intracellular-loop determinants showed VPAC1 uses physically separate subdomains for Gs/cAMP versus Gi/Gq/Ca2+ coupling, explaining its multifunctional signaling.","evidence":"VPAC1/VPAC2 chimeras and IC3 point mutants with aequorin calcium and cyclase assays; YL-motif mutagenesis","pmids":["11981043","15451021","11859928"],"confidence":"High","gaps":["Direct G-protein selectivity at the structural interface not resolved","Cellular context dependence of coupling not addressed"]},{"year":2002,"claim":"Native pull-down showed tissue-dependent G-protein partners and basal pre-coupling, indicating VPAC1 coupling repertoire varies with cellular context.","evidence":"Immunoaffinity chromatography and cross-linking from human and rat tissues with G-protein-specific Western blots","pmids":["11812005"],"confidence":"Medium","gaps":["Single-lab native study","Functional consequence of pre-coupling not established"]},{"year":2005,"claim":"Defining C-terminal Ser/Thr phosphorylation sites and Ser447 separated desensitization from internalization, clarifying receptor regulation kinetics.","evidence":"Truncation/site-directed mutagenesis with phosphorylation, internalization, recycling, flow cytometry, and confocal assays in CHO cells","pmids":["15932876","14645688"],"confidence":"High","gaps":["GRK isoform identity not confirmed in vivo","Arrestin recruitment not directly measured"]},{"year":2004,"claim":"Establishing a cleavable N-terminal signal peptide explained the biogenesis requirement for surface delivery of VPAC1.","evidence":"Deletion and Flag-insertion constructs with binding, cAMP, and immunofluorescence localization in CHO cells","pmids":["15518910"],"confidence":"High","gaps":["Cleavage site not directly sequenced","ER processing machinery not identified"]},{"year":2007,"claim":"Genetic and antibody studies established VPAC1 as a negative regulator of megakaryopoiesis acting through cAMP, defining a hematopoietic physiological role.","evidence":"CD34+ differentiation assays, neutralizing antibodies, transgenic and KO mice with cAMP and platelet readouts","pmids":["18000164"],"confidence":"High","gaps":["Downstream cAMP effectors in megakaryocytes not detailed","Human therapeutic translation untested"]},{"year":2011,"claim":"VPAC1-null mice revealed an essential developmental role in intestine and endocrine pancreas, beyond acute signaling functions.","evidence":"Homozygous null mice and promoter-reporter localization with histology and glucose homeostasis assays","pmids":["21697765"],"confidence":"High","gaps":["Cell-autonomous versus paracrine basis of phenotype unresolved","Molecular targets in islet development not identified"]},{"year":2014,"claim":"Identifying VPAC1 control of CFTR membrane stability via NHERF1/ERM and on cholinergic neurons defined dual epithelial and neural mechanisms for intestinal fluid secretion.","evidence":"Proximity ligation, siRNA knockdown, Ussing chamber, muscle contraction, and immunohistochemistry with selective pharmacology","pmids":["24788249","14744818","24578344","12598410"],"confidence":"High","gaps":["Relative contribution of epithelial versus neuronal VPAC1 in vivo not quantified","Link between cAMP/PKC and scaffold remodeling not fully traced"]},{"year":2016,"claim":"KO and chimera studies in colitis and EAE established VPAC1 as a pro-inflammatory mediator, with CNS chemokine induction arising from non-hematopoietic VPAC1.","evidence":"VPAC1/VPAC2 KO mice, bone-marrow chimeras, adoptive transfer, cytokine/chemokine profiling, and pharmacological antagonists","pmids":["27357191","21295288"],"confidence":"High","gaps":["Identity of non-hematopoietic VPAC1-expressing cells not pinned down","Mechanism of chemokine induction downstream of VPAC1 unresolved"]},{"year":2017,"claim":"Cys37 palmitoylation was shown to drive VIP-induced nuclear translocation linked to anti-apoptotic activity, revealing a non-canonical receptor function.","evidence":"C37A mutagenesis, acyl-biotin exchange and click-chemistry palmitoylation assays, 2-bromopalmitate, confocal imaging, and apoptosis assays in CHO cells","pmids":["28473666"],"confidence":"High","gaps":["Nuclear targets of the translocated receptor unknown","In vivo relevance of nuclear VPAC1 untested"]},{"year":2022,"claim":"Cancer studies tied VPAC1 to metabolic and calcium-dependent signaling axes (ASS1/arginine and CAD in HCC; TRPV4/Ca2+ in gastric cancer), linking receptor activity to tumor growth control.","evidence":"In vitro/in vivo cancer cell studies with transcriptomics, calcium imaging, pathway analysis, and clinical sample correlation","pmids":["35864952","30692637"],"confidence":"Medium","gaps":["Direct receptor-to-pathway coupling steps not fully reconstituted","Single-lab studies for each axis"]},{"year":2002,"claim":"Transcriptional control of VIPR1 was mapped to Ikaros-mediated suppression and, later, FXR-mediated induction, defining context-dependent expression regulation.","evidence":"EMSA/supershift, reporter deletions, and overexpression for Ikaros; FXR agonist with EMSA and expression assays in primary cells","pmids":["11812772","16037943","10331949"],"confidence":"High","gaps":["Combinatorial control across tissues not integrated","Chromatin-level regulation incompletely defined"]},{"year":null,"claim":"A high-resolution structure of agonist-bound, G-protein-coupled VPAC1 and identification of the nuclear effectors of palmitoylation-dependent translocation remain open questions.","evidence":"","pmids":[],"confidence":"High","gaps":["No experimental full-length receptor–ligand–G protein structure in the corpus","Nuclear signaling partners of translocated VPAC1 unidentified","Cell-type origin of pro-inflammatory VPAC1 effects unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,8,11]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0]},{"term_id":"GO:0001618","term_label":"virus receptor activity","supporting_discovery_ids":[32]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[18,35]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[36]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,11,12]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[23,24,30]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[20,21]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[25,28]}],"complexes":[],"partners":["VIP","ADCYAP1","NHERF1","CFTR","GNAS"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P32241","full_name":"Vasoactive intestinal polypeptide receptor 1","aliases":["Pituitary adenylate cyclase-activating polypeptide type II receptor","PACAP type II receptor","PACAP-R-2","PACAP-R2","VPAC1 receptor","VPAC1R"],"length_aa":457,"mass_kda":51.5,"function":"G protein-coupled receptor activated by the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (ADCYAP1/PACAP) (PubMed:35477937, PubMed:36385145, PubMed:8179610). Binds VIP and both PACAP27 and PACAP38 bioactive peptides with the following order of ligand affinity VIP = PACAP27 > PACAP38 (PubMed:35477937, PubMed:8179610). Ligand binding causes a conformation change that triggers signaling via guanine nucleotide-binding proteins (G proteins) and modulates the activity of downstream effectors. Activates cAMP-dependent pathway (PubMed:35477937, PubMed:36385145, PubMed:8179610)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/P32241/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/VIPR1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/VIPR1","total_profiled":1310},"omim":[{"mim_id":"610376","title":"ATYPICAL CHEMOKINE RECEPTOR 3; ACKR3","url":"https://www.omim.org/entry/610376"},{"mim_id":"601970","title":"VASOACTIVE INTESTINAL PEPTIDE RECEPTOR 2; VIPR2","url":"https://www.omim.org/entry/601970"},{"mim_id":"192321","title":"VASOACTIVE INTESTINAL PEPTIDE RECEPTOR 1; VIPR1","url":"https://www.omim.org/entry/192321"},{"mim_id":"102981","title":"ADCYAP RECEPTOR, TYPE I; ADCYAP1R1","url":"https://www.omim.org/entry/102981"},{"mim_id":"102775","title":"ADENOSINE A1 RECEPTOR; ADORA1","url":"https://www.omim.org/entry/102775"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lung","ntpm":124.0}],"url":"https://www.proteinatlas.org/search/VIPR1"},"hgnc":{"alias_symbol":["VPAC1","RDC1","HVR1","VPAC1R"],"prev_symbol":[]},"alphafold":{"accession":"P32241","domains":[{"cath_id":"4.10.1240.10","chopping":"35-125","consensus_level":"high","plddt":84.1035,"start":35,"end":125},{"cath_id":"1.20.1070.10","chopping":"128-325_337-403","consensus_level":"high","plddt":83.8834,"start":128,"end":403}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P32241","model_url":"https://alphafold.ebi.ac.uk/files/AF-P32241-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P32241-F1-predicted_aligned_error_v6.png","plddt_mean":76.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=VIPR1","jax_strain_url":"https://www.jax.org/strain/search?query=VIPR1"},"sequence":{"accession":"P32241","fasta_url":"https://rest.uniprot.org/uniprotkb/P32241.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P32241/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P32241"}},"corpus_meta":[{"pmid":"10694225","id":"PMC_10694225","title":"The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1).","date":"2000","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/10694225","citation_count":631,"is_preprint":false},{"pmid":"15282712","id":"PMC_15282712","title":"Distribution of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide receptors (VPAC1, VPAC2, and PAC1 receptor) in the rat brain.","date":"2004","source":"The Journal of comparative neurology","url":"https://pubmed.ncbi.nlm.nih.gov/15282712","citation_count":162,"is_preprint":false},{"pmid":"10801840","id":"PMC_10801840","title":"Identification of key residues for interaction of vasoactive intestinal peptide with human VPAC1 and VPAC2 receptors and development of a highly selective VPAC1 receptor agonist. 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binding (125I-VIP), adenylyl cyclase activity assay, ab initio molecular modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic alanine scan with binding and functional assays, molecular modeling, multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"10801840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Two basic residues in the second transmembrane helix of VPAC1 (Arg188 and Lys195) are essential for receptor activation: the negatively charged Asp3 of VIP must penetrate into the transmembrane domain and interact with these residues to activate the receptor. This interaction is required for activation but not for antagonist recognition.\",\n      \"method\": \"Site-directed mutagenesis of VPAC1 receptor, radioligand binding, adenylyl cyclase activity assay in transfected CHO cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis combined with binding and functional assays; multiple mutants with orthogonal pharmacological probes\",\n      \"pmids\": [\"11013258\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Three-dimensional model of the VPAC1 receptor N-terminal domain was constructed by homology modeling; site-directed mutagenesis confirmed that Pro74, Pro87, Phe90, and Trp110 (in addition to previously identified Glu36, Trp67, Asp68, Trp73, Gly109) are important for VIP binding and adenylyl cyclase activation, defining a negatively charged binding groove with a tryptophan shell.\",\n      \"method\": \"Homology modeling, site-directed mutagenesis, stable transfection in CHO cells, 125I-VIP binding, adenylyl cyclase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — combined structure modeling and mutagenesis with binding and functional readouts, multiple residues validated\",\n      \"pmids\": [\"11124960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Photoaffinity labeling with [Bpa22-VIP] demonstrated a direct physical contact between Tyr22 of VIP and the 109-120 fragment (GWTHLEPGPYPI) of the N-terminal ectodomain of hVPAC1 receptor, providing the first direct evidence for VIP–VPAC1 N-terminal ectodomain contact.\",\n      \"method\": \"Photoaffinity labeling, cyanogen bromide cleavage, V8 endoproteinase cleavage, creation of CNBr-cleavage-site receptor mutants, SDS-PAGE, Edman sequencing; 125I-labeled probe\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct photoaffinity crosslinking with fragment mapping and mutant confirmation, rigorous controls\",\n      \"pmids\": [\"12807902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"NMR structure of VIP (central alpha-helix, disordered N-terminal His1-Phe6, 310 helix at Ser25-Asn28) combined with photoaffinity labeling showed Phe6 contacts Asp107, Tyr22 contacts Gly116, and Asn24 contacts Cys122 in the N-terminal ectodomain of hVPAC1. A 3D model of the N-ted (short consensus repeat/Sushi domain with two antiparallel beta-sheets, three disulfide bonds) was built; docking showed the VIP 6-28 fragment occupies the C-terminal part of the N-ted while the N-terminus is free to interact with the transmembrane region.\",\n      \"method\": \"Photoaffinity labeling with Bpa at positions 6, 22, 24 of VIP, Edman sequencing of labeled fragments, NMR structure determination of VIP, homology modeling of N-ted using CRF receptor 2beta NMR structure as template, molecular docking\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (NMR, photoaffinity, modeling) in one rigorous study with structural validation\",\n      \"pmids\": [\"16520374\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Photoaffinity labeling with [Bpa6]-VIP demonstrated that position 6 of VIP contacts the 104-108 fragment of the hVPAC1 N-terminal ectodomain, adjacent to the fragment contacted by position 22 (Tyr22), showing that the central part of VIP (at least Phe6 to Tyr22) interacts with the N-terminal ectodomain.\",\n      \"method\": \"Photoaffinity labeling, sequential enzymatic and chemical cleavage (CNBr, PNGase F, Glu-C, trypsin), SDS-PAGE, receptor mutant with new CNBr cleavage site\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct photoaffinity crosslinking, multi-enzyme mapping, confirmed by mutant receptor\",\n      \"pmids\": [\"15247290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The N-terminal part of VIP (residues 1-5/0) physically contacts the 130-137 region of the hVPAC1 N-terminal ectodomain (via [Bpa0]-VIP probe), while the N-terminal part of antagonist PG97-269 contacts a different region of the N-ted (residues 43-66), demonstrating distinct binding sites for the agonist and antagonist N-termini.\",\n      \"method\": \"Photoaffinity labeling with Bpa0-VIP and Bpa0-PG97-269, CNBr cleavage, NuPAGE analysis\",\n      \"journal\": \"Journal of molecular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct photoaffinity labeling, single lab, two probes with fragment identification\",\n      \"pmids\": [\"18597186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Photoaffinity labeling established that the N-terminus of VIP ([Bpa0]-VIP probe) contacts VPAC1 residue Q135, while the N-terminus of antagonist PG97-269 ([Bpa0]-PG97-269) contacts G62 of the N-ted—distinct from the VIP contact site. Additionally, residues K143, T144, and T147 in the first transmembrane domain are critical for interaction with the His1 N-terminus of VIP, as shown by alanine substitution.\",\n      \"method\": \"Photoaffinity labeling, alanine mutagenesis of transmembrane domain residues, binding affinity measurements, pharmacological assays with VIP2-28\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — photoaffinity labeling plus mutagenesis with multiple orthogonal approaches in one rigorous study\",\n      \"pmids\": [\"22291440\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"A network of conserved residues Arg188 (TM2), Asn229 (TM3), and Gln380 (TM7) governs ligand binding and receptor activation of VPAC1; double mutants of reciprocal residue exchanges showed strong cooperative or anticooperative effects confirming spatial proximity. Arg188 interacts with Asp3 of VIP and this is altered upon VIP binding to trigger activation.\",\n      \"method\": \"Structural modeling of TM domain, site-directed mutagenesis, cAMP production assay, binding assays, double mutant cooperativity analysis in CHO cells\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — combined structural model with systematic mutagenesis and double-mutant epistasis analysis, multiple orthogonal methods\",\n      \"pmids\": [\"20573782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The carboxyl terminus of VPAC1 contains Ser/Thr residues (including Thr429, Ser435, Ser448/449, Ser455 in the distal C-terminus and Ser250 in the second intracellular loop) that mediate VIP-stimulated receptor phosphorylation and internalization; truncation removing all C-terminal Ser/Thr residues abolished phosphorylation and internalization, and also enabled receptor recycling (reversed within 2 h), an effect blocked by monensin.\",\n      \"method\": \"Site-directed mutagenesis and truncation of VPAC1, flow cytometry, confocal microscopy with monoclonal antibody against receptor, phosphorylation assays in CHO cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic truncations and mutations combined with multiple readouts (phosphorylation, internalization, recycling) and flow cytometry/confocal imaging\",\n      \"pmids\": [\"15932876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Ser447 in the C-terminal tail of VPAC1 is crucial for VIP-induced receptor phosphorylation and rapid desensitization (10-fold right-shift of ED50 for adenylyl cyclase) but is not required for receptor internalization or down-regulation; Ser447 is a likely GRK target.\",\n      \"method\": \"Site-directed mutagenesis to Ala, adenylyl cyclase activity assay, phosphorylation assay, internalization assay using fluorescein-tagged VIP and Flag/GFP-tagged receptor in CHO cells\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — specific single-residue mutagenesis with multiple orthogonal functional readouts (desensitization, phosphorylation, internalization)\",\n      \"pmids\": [\"14645688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Mutations in the distal part of the third intracellular loop (R338, L339, R341) of VPAC1 markedly reduce VIP-stimulated calcium increase and Galphai coupling but only weakly affect adenylyl cyclase activity, whereas mutations in the proximal domain (K322) reduce adenylyl cyclase activity without changing the calcium response, demonstrating separate receptor sub-domains for Gs and Gi/Ca2+ coupling.\",\n      \"method\": \"Site-directed mutagenesis, adenylyl cyclase activity assay, calcium response assay in CHO cells\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic mutagenesis with two distinct functional readouts identifying separate coupling domains\",\n      \"pmids\": [\"15451021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"A small sequence in the third intracellular loop (IC3) of VPAC1, residues 328-331 (IRKS), is responsible for efficient agonist-stimulated [Ca2+]i increase, likely through coupling to Galphai/Galphaq proteins; swapping this sequence with the VPAC2 counterpart (VGGN) interconverted the calcium signaling phenotypes of the two receptors.\",\n      \"method\": \"VPAC1/VPAC2 chimeric receptors, point mutations in IC3, aequorin reporter gene calcium assay in transfected CHO cells\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — chimeric receptor approach with reciprocal sequence swaps and functional validation\",\n      \"pmids\": [\"11981043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Specific mutations of Thr343 (to Lys, Pro, or Ala) in human VPAC1 receptor produced constitutive activation with ~3.5-fold increase in cAMP; constitutive activation required integrity of the N-terminal extracellular VIP-binding domain (abolished by E36A or D68A double mutations), establishing Thr343 at the IC2/TM4 junction as a key constraint for receptor activation state.\",\n      \"method\": \"Site-directed mutagenesis, transient transfection in COS cells, cAMP production assay, double-mutant analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple specific mutations including double mutants with functional dissection of activation mechanism\",\n      \"pmids\": [\"9920725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Asn229 in TM3 of VPAC1 is essential for receptor activation (G protein coupling) but not for VIP/antagonist binding, not for agonist-stimulated phosphorylation and internalization; the N229A mutant could still internalize but re-expressed more rapidly than wild-type after agonist washout, dissociating G protein activation from receptor trafficking.\",\n      \"method\": \"Site-directed mutagenesis (Ala, Asp, Gln mutations), adenylyl cyclase assay, calcium assay, GTP sensitivity assay, receptor phosphorylation and internalization assays in CHO cells\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic mutagenesis with multiple orthogonal readouts dissecting activation, phosphorylation, and trafficking\",\n      \"pmids\": [\"16650965\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The conserved 'YL' motif (Tyr239, Leu240) in VPAC1 is functionally equivalent to the 'DRY' motif of rhodopsin-family GPCRs; Y239A caused moderate and L240A caused pronounced impairment of VIP-induced cAMP production, primarily by perturbing the G protein-binding site rather than the intrinsic low-to-high affinity equilibrium.\",\n      \"method\": \"Site-directed mutagenesis, GTP-gamma-S binding shift assay, VIP-induced cAMP production in whole cells\",\n      \"journal\": \"Journal of molecular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional and binding readouts, single lab\",\n      \"pmids\": [\"11859928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Three conserved transmembrane prolines (P266, P300, P348) of VPAC1 are important for receptor expression, G protein coupling, and receptor activity; P266A decreased cAMP stimulation, while P300A and P348A increased potency and GTP sensitivity, suggesting these prolines constrain receptor activation. Adjacent leucines L346A and L349A also reduced receptor expression and G protein coupling.\",\n      \"method\": \"Alanine substitution mutagenesis, radioligand binding, cAMP production assay, GTP sensitivity in transfected cells\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with binding and functional readouts, single lab\",\n      \"pmids\": [\"11513868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Immunoaffinity chromatography using an anti-VPAC1 first-extracellular-loop antibody showed that human VPAC1 in HEK293 cells couples to Gs but not Gi3, Gi1/2, or Gq; rat VPAC1 in brain couples to Gs and Gi3; rat VPAC1 in lung couples to Gs, Gi3, and Gq. VIP pretreatment increased G protein co-purification. Pre-coupling of VPAC1 to G protein occurred basally (without agonist), confirmed by covalent cross-linking.\",\n      \"method\": \"Immunoaffinity chromatography, covalent cross-linking in native membranes, Western blotting with G-protein-specific antibodies, 125I-VIP binding\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal pull-down strategy, cross-linking confirmation, single lab with multiple tissues/cell types\",\n      \"pmids\": [\"11812005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"VPAC1 receptor contains a functional N-terminal signal peptide (residues 1-30); deletion of the signal peptide abolished cell-surface expression and 125I-VIP binding, while the signal peptide is cleaved during translocation to the plasma membrane, probably in the endoplasmic reticulum.\",\n      \"method\": \"Deletion and Flag-insertion constructs, stable transfection in CHO cells, 125I-VIP binding, cAMP production, GFP fluorescence, indirect immunofluorescence on non-permeabilized cells\",\n      \"journal\": \"Regulatory peptides\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple constructs with binding, functional, and localization readouts confirming signal peptide function\",\n      \"pmids\": [\"15518910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"VPAC1/VPAC2 chimeric receptor analysis showed the N-terminal extracellular domain determines selectivity for the VIP1 antagonist, while VPAC1-selective agonist recognition additionally requires the first extracellular loop and distal receptor domains; replacement of EC1 in VPAC1 with the VPAC2 counterpart markedly reduced maximal cAMP response.\",\n      \"method\": \"Chimeric VPAC1/VPAC2 receptors, radioligand binding, adenylyl cyclase activity assay\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chimeric receptor approach with binding and functional readouts, single lab\",\n      \"pmids\": [\"10491203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"VPAC1 receptor activation by VIP and PACAP-27 on the basolateral surface of human bronchial epithelial Calu-3 cells induces CFTR-dependent chloride secretion; this requires both PKA and PKC activity. IB3-1 cystic fibrosis cells expressing VPAC1 but lacking functional CFTR showed no chloride transport in response to VIP/PACAP-27.\",\n      \"method\": \"Radioligand binding (125I-VIP, 125I-PACAP-27), iodide efflux assay, Ussing chamber short-circuit current measurements, PKA inhibitor (H-89) and PKC inhibitor (chelerythrine) pharmacology\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (efflux, Ussing chamber, pharmacological inhibitors, CFTR-null control) in one study\",\n      \"pmids\": [\"14744818\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"VIP stimulation of VPAC1 in airway Calu-3 cells increases CFTR membrane stability by promoting CFTR interaction with NHERF1 and phosphorylated ERM (via PKCε), while reducing CFTR interaction with CFTR-associated ligand (CAL); knockdown of NHERF1 or ERM by siRNA prevented the VIP effect on CFTR membrane stability and sustained CFTR activity.\",\n      \"method\": \"Immunocytochemistry, in situ proximity ligation assay, siRNA knockdown of NHERF1 and ERM, iodide efflux assays, Western blotting\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proximity ligation, siRNA knockdown, and functional efflux assays with multiple protein partners identified\",\n      \"pmids\": [\"24788249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"VPAC1 signaling negatively regulates megakaryopoiesis: PACAP/VIP activation of VPAC1 on megakaryocytes inhibits megakaryocyte differentiation and reduces platelet counts; blocking VPAC1 with neutralizing antibody (23A11) or anti-PACAP (PP1A4) inhibited cAMP formation, stimulated megakaryopoiesis independently of thrombopoietin, and elevated platelet counts in mice and in models of myelosuppression and GATA1 deficiency.\",\n      \"method\": \"In vitro CD34+ cell differentiation assays, anti-VPAC1 and anti-PACAP neutralizing antibodies, transgenic mice overexpressing PACAP in megakaryocytes, VPAC1 KO mouse studies, cAMP measurement, histology\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — patient-derived cells, transgenic and KO mice, neutralizing antibodies, in vitro and in vivo experiments with multiple disease models\",\n      \"pmids\": [\"18000164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"VPAC1 receptor mediates the pro-inflammatory enhancement of DSS-induced colitis by VIP: VPAC1-KO mice showed milder colitis than wild-type mice, with reduced tissue myeloperoxidase, IL-6, IL-1β, and MMP-9; suppression of VPAC1 signals by PKA inhibitors in VPAC2-KO mice reduced colitis severity. Thus VIP enhancement of colitis is exclusively mediated by VPAC1.\",\n      \"method\": \"VPAC1-KO and VPAC2-KO mice, DSS-induced colitis model, myeloperoxidase assay, cytokine measurement, histopathology, pharmacological PKA inhibitors\",\n      \"journal\": \"Cellular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO models for both receptor subtypes with multiple inflammatory readouts and pharmacological confirmation\",\n      \"pmids\": [\"21295288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"VPAC1 deficiency ameliorates experimental autoimmune encephalomyelitis (EAE) by impairing the effector phase: VPAC1 KO mice showed reduced CNS histopathology, reduced chemokine mRNAs, and impaired inflammatory cell infiltration. WT cells fully induced EAE in WT but not VPAC1-KO recipients (bone marrow chimeras). The resistance was minimally dependent on VPAC1 expression in the hematopoietic compartment, implicating non-hematopoietic VPAC1 in CNS chemokine induction.\",\n      \"method\": \"VPAC1 KO mice, MOG35-55 EAE induction, histology, real-time PCR, immunofluorescence, adoptive transfer, bone marrow chimeras, antigen-recall assays, pharmacological VPAC1 antagonist\",\n      \"journal\": \"Journal of neuroinflammation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic approaches (KO, adoptive transfer, chimeras) with pharmacological validation\",\n      \"pmids\": [\"27357191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"VPAC1-null mice die perinatally with intestinal obstruction, disorganized hyperproliferative intestinal epithelium, small dysmorphic pancreatic islets, hypoglycemia, and impaired glucose homeostasis, demonstrating a required role for VPAC1 in embryonic and neonatal development of intestine and endocrine pancreas; VPAC1 promoter-driven transgene was expressed in E12.5/E14.5 intestinal epithelial and pancreatic endocrine cells.\",\n      \"method\": \"Homozygous VPAC1-null mutant mice, VPAC1 promoter-driven β-galactosidase transgenic mice, histology, glucose homeostasis assays (oral glucose tolerance, insulin challenge), blood glucose measurement\",\n      \"journal\": \"Pancreas\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic null mouse with multiple developmental and metabolic phenotypes and reporter gene localization\",\n      \"pmids\": [\"21697765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"VIP inhibits bone marrow progenitor proliferation (CFU-GM and erythroid progenitors) through VPAC1 (type 1 receptor), as shown by reversal of inhibition with VPAC1 antagonist but not VPAC2 agonist; direct effects on CD34+ cells were shown, with additional indirect effects via stromal TGF-β and TNF-α induction. Chemical cross-linking confirmed VPAC1 on stromal membranes.\",\n      \"method\": \"Clonogenic assays with unfractionated and CD34+ bone marrow cells, VPAC1/VPAC2 selective agonists and antagonists, semi-quantitative RT-PCR, chemical cross-linking\",\n      \"journal\": \"Experimental hematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — selective receptor pharmacology with clonogenic assays and cross-linking, single lab\",\n      \"pmids\": [\"12225791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"VPAC1 receptor on cholinergic submucosal (NPY+) secretomotor neurons mediates VIP-induced chloride secretion: VIP-evoked secretion was depressed by VPAC1 antagonist PG97-269 and by hyoscine (muscarinic antagonist) but not eliminated by both together, indicating a direct epithelial VPAC1R component and an indirect VPAC1R-on-cholinergic-neuron component. VIP stimulates ACh-mediated longitudinal muscle contraction via VPAC1R on calretinin+ cholinergic motor neurons (inhibited by TTX, PG97-269, and hyoscine).\",\n      \"method\": \"Immunohistochemistry for VPAC1R and neurochemical markers, RT-PCR, Ussing chamber short-circuit current, isotonic muscle contraction with pharmacological dissection (TTX, VPAC1 antagonist PG97-269, hexamethonium, hyoscine)\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — immunohistochemical localization combined with functional Ussing chamber and muscle contraction assays with specific pharmacological agents\",\n      \"pmids\": [\"24578344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"VPAC1 receptor activation directs postnatal dentate gyrus neural stem/progenitor cells toward granule cell neurogenesis without a trophic effect, in contrast to VPAC2 which promotes symmetric division and nestin-positive cell pool expansion. This differential fate modulation was established by selective receptor agonists on postnatal hippocampal cultures.\",\n      \"method\": \"Selective VPAC1 and VPAC2 agonists applied to postnatal hippocampal cultures, cell fate analysis, BrdU incorporation, immunostaining for nestin and neuronal markers; in vivo Vipr2-/- mice for VPAC2 validation\",\n      \"journal\": \"Stem cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — selective pharmacology with cell fate readouts in cultures; in vivo validation for VPAC2 only\",\n      \"pmids\": [\"19650041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Microglial VPAC1R mediates VIP-induced enhancement of neural stem/progenitor cell proliferation and pro-neurogenic effects in hippocampal cultures via IL-4 release from microglia; conditioned media from VIP-stimulated microglia was trophic for NSPCs, and this was dependent on VPAC1 receptor signaling leading to IL-4 secretion.\",\n      \"method\": \"Hippocampal mixed cultures with microglial depletion and readdition, conditioned media experiments, VIP stimulation with VPAC1 receptor activation, IL-4 measurement, NSPC proliferation and survival assays\",\n      \"journal\": \"Glia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditioned media and depletion-readdition experiments with cytokine identification, single lab\",\n      \"pmids\": [\"24801739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"VPAC1 receptor activation by VIP attenuates acute pancreatitis through inhibition of proinflammatory cytokine (IL-6, TNF-α) production from monocytes; selective VPAC1-R agonist decreased serum amylase, IL-6, and TNF-α and attenuated histological severity, while VPAC2-R agonist worsened outcomes. VPAC1-R and VPAC2-R mRNA were expressed in splenic monocytes.\",\n      \"method\": \"Cerulein/LPS mouse pancreatitis model, selective VPAC1-R and VPAC2-R agonists, serum amylase and cytokine ELISA, histology, in vitro monocyte cytokine production assay with LPS stimulation, RT-PCR\",\n      \"journal\": \"Pancreas\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — selective receptor pharmacology in vivo and in vitro with multiple readouts, single lab\",\n      \"pmids\": [\"15632701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"VIP inhibits intestinal dipeptide (Gly-Sar) transport via VPAC1 receptor in Caco-2 cells (VPAC2 mRNA not expressed) by a PKA-dependent mechanism; the inhibition is Na+-dependent and involves reduction of NHE3-dependent intracellular pH recovery after dipeptide-induced acidification, indicating modulation of hPepT1 activity indirectly through NHE3 inhibition.\",\n      \"method\": \"14C-Gly-Sar uptake assay, RT-PCR for receptor expression, BCECF pH measurement, PKA inhibitor H-89, NHE3 inhibitor S1611, Western blotting for NHERF1, selective VPAC1 agonist\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — selective agonist pharmacology, pH assay, transport assay, pharmacological dissection, single lab\",\n      \"pmids\": [\"12598410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"VPAC1 receptor actively facilitates productive HIV-1 infection: VPAC1 signal-blocking antibody inhibited ~80% of productive HIV-1 infection; VPAC1 antisense transfection reduced productive infection by ~50%; sense VPAC1 transfection increased productive infection >15-fold and increased syncytium formation. VPAC1 does not affect viral entry but is required for steps post-entry (absence of 2-LTR circles in VPAC1-negative cells). HIV-1 gp120 has sequence similarity to VIP, suggesting potential direct receptor activation.\",\n      \"method\": \"VPAC1 signal-blocking antibody, sense/antisense transfection of VPAC1 cDNA, HIV-1 p24 ELISA, luciferase pseudovirus assay, HIV-1 gag DNA PCR, 2-LTR circle analysis, syncytium formation assay\",\n      \"journal\": \"AIDS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic and antibody approaches with mechanistic post-entry analysis, single lab\",\n      \"pmids\": [\"11834941\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Ikaros transcription factors (IK-1 and IK-2) suppress endogenous VPAC1 expression: Ikaros binds high-affinity consensus sequences in the VPAC1 5'-flanking region (confirmed by EMSA with supershifts), overexpression of IK-1 or IK-2 in NIH-3T3 clones reduced VPAC1 mRNA and protein by 50-93%, and VPAC1 luciferase reporter activity was decreased up to 41% with two major Ikaros binding domains at -1076 to -623 bp and -222 to -35 bp.\",\n      \"method\": \"EMSA with native T cell nuclear extracts and recombinant IK-1/IK-2, antibody supershift, stable NIH-3T3 clones overexpressing Ikaros isoforms, RT-PCR and fluorometric kinetic RT-PCR, 125I-VIP binding, luciferase reporter with nested deletions\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — EMSA/supershift, stable overexpression, multiple reporter deletion constructs, protein and mRNA readouts in one comprehensive study\",\n      \"pmids\": [\"11812772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"VPAC1 receptor expression in gallbladder epithelial cells is transcriptionally regulated by FXR (farnesoid X receptor): the FXR agonist GW4064 upregulated VPAC1 mRNA and protein in primary human gallbladder epithelial cells dose-dependently; this effect was antagonized by 9-cis retinoic acid (RXRα ligand). Chenodeoxycholate activated endogenous FXR (confirmed by EMSA) and also increased VPAC1 expression.\",\n      \"method\": \"Quantitative RT-PCR, Western blot, primary human gallbladder epithelial cell cultures, pharmacological FXR agonist GW4064, EMSA for FXR binding, RXRα antagonism\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA plus RT-PCR/Western blot in primary human cells, single lab\",\n      \"pmids\": [\"16037943\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"VPAC1 is localized to the apical membrane of intestinal epithelial cells (colocalizing with villin but not basolateral Na+/K+-ATPase) in both mouse and human colon, with highest expression in the colon compared to ileum and jejunum; this apical localization was determined by immunofluorescence and suggests potential for luminal peptide recognition.\",\n      \"method\": \"Quantitative RT-PCR, Western blotting, immunofluorescence with apical (villin) and basolateral (Na+/K+-ATPase) markers in mouse and human intestinal tissue\",\n      \"journal\": \"American journal of physiology. Gastrointestinal and liver physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct immunofluorescence colocalization with polarization markers in mouse and human tissues, single lab\",\n      \"pmids\": [\"28385693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Palmitoylation of Cys37 in the N-terminal extracellular domain of VPAC1 mediates VIP-induced nuclear translocation of the receptor, which contributes to its anti-apoptotic activity; the C37A mutant failed to undergo nuclear translocation upon VIP stimulation and showed reduced anti-apoptotic activity, while exhibiting higher proliferative activity. Palmitoylation was confirmed by acyl-biotin exchange assay and click chemistry-based palmitoylation assay; the palmitoylation inhibitor 2-bromopalmitate blocked both nuclear translocation and anti-apoptotic activity.\",\n      \"method\": \"Site-directed mutagenesis (C37A), stable transfection with VPAC1-EYFP fusion proteins in CHO cells, confocal microscopy, Western blotting, fluorescence quantification, acyl-biotin exchange assay, click chemistry palmitoylation assay, 2-bromopalmitate treatment, apoptosis assay with camptothecin\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct palmitoylation detection by two biochemical methods, mutagenesis, pharmacological inhibition with functional readouts (nuclear translocation, apoptosis) in one study\",\n      \"pmids\": [\"28473666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"VPAC1 activation by VIP in gastric cancer cells induces TRPV4-mediated Ca2+ entry and promotes gastric cancer progression in a Ca2+-dependent manner; this VPAC1/TRPV4/Ca2+ signaling axis enhances VIP expression and secretion, establishing a positive autocrine feedback loop. Inhibition of VPAC1 blocked progressive responses.\",\n      \"method\": \"In vitro gastric cancer cell signaling assays, Ca2+ imaging, VPAC1 and TRPV4 inhibition, VIP/VPAC1 expression analysis in human cancer specimens and cell lines\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Ca2+ imaging with pharmacological inhibitors defining signaling pathway, in vitro and clinical correlation, single lab\",\n      \"pmids\": [\"30692637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"VIPR1 activation by VIP in HCC cells inhibits growth and metastasis through regulation of arginine biosynthesis: VIP treatment partially restored argininosuccinate synthase (ASS1) expression and inhibited de novo pyrimidine synthesis by suppressing CAD phosphorylation via mTOR/p70S6K signaling. Human HCC samples showed downregulation of ASS1 and upregulation of phospho-CAD correlating with VIPR1 loss.\",\n      \"method\": \"In vitro and in vivo HCC cell studies with VIP treatment, transcriptome sequencing, ASS1 expression analysis, CAD phosphorylation assay, mTOR/p70S6K pathway analysis, human HCC clinical sample analysis\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptomics plus mechanistic pathway analysis in vitro/in vivo with clinical correlation, single lab\",\n      \"pmids\": [\"35864952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"VPAC1 and VPAC2 receptors are both required for pressure-induced vasodilatation (PIV): blockade of VPAC1/VPAC2, or selectively of VPAC1 alone, eliminated the PIV response in anesthetized rodents, while PAC1 blockade had no effect. Vascular smooth muscle and endothelial vasodilator capacity were unaffected by VPAC1/2 antagonism.\",\n      \"method\": \"In vivo pharmacological blockade with selective antagonists (PG97-269 for VPAC1, PACAP6-38 for VPAC2/PAC1, Max.d.4 for PAC1, D-p-Cl-Phe6,Leu17-VIP for VPAC1/2) in anesthetized rodents, laser Doppler flowmetry\",\n      \"journal\": \"The Journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic pharmacological receptor dissection in vivo with multiple selective antagonists\",\n      \"pmids\": [\"14578481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In porcine basilar arteries, VPAC1 receptor is localized specifically on the endothelium and mediates vasodilation via nitric oxide (NO) generation (inhibited by L-NAME, abolished by endothelial denudation), while VPAC2 on outer smooth muscle layers mediates NO-independent vasodilation.\",\n      \"method\": \"Immunocytochemistry, RT-PCR, pharmacological vasodilator responses to selective receptor agonists, L-NAME inhibition, endothelial denudation, electrical stimulation with VPAC2 antagonist\",\n      \"journal\": \"Journal of cerebral blood flow and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — immunocytochemical localization with functional pharmacological dissection in tissue preparations, single lab\",\n      \"pmids\": [\"15959462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"VPAC1 receptor on hippocampal nerve terminals inhibits exocytotic VGCC-dependent GABA release through Gi/o protein- and PKC-dependent mechanisms; this opposes the VPAC2 receptor effect (which enhances GABA release via Gs/PKA/PKC).\",\n      \"method\": \"Isolated rat hippocampal nerve terminal preparations, 3H-GABA release assay with selective VPAC1/VPAC2 agonists and antagonists, pertussis toxin, PKA inhibitor, PKC inhibitor, voltage-gated calcium channel blockers\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — nerve terminal preparation with selective pharmacology dissecting receptor subtypes and G protein/kinase coupling, single lab\",\n      \"pmids\": [\"28945273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"VPAC1 receptor activation by VIP and PACAP enhances hippocampal CA1 pyramidal cell synaptic transmission; VPAC1-mediated enhancement is dependent on PKC but not PKA activity (in contrast to VPAC2-mediated enhancement which requires PKA), demonstrating distinct intracellular signaling downstream of each receptor subtype in hippocampal synaptic modulation.\",\n      \"method\": \"Selective VPAC1 agonist [K15,R16,L27]VIP(1-7)/GRF(8-27) and VPAC2 agonist RO 25-1553, selective antagonists PG97-269 and PG99-465, PKA inhibitor H-89, PKC inhibitor GF109203X, field potential recording in hippocampal slices\",\n      \"journal\": \"Brain research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — selective receptor pharmacology with kinase inhibitor dissection in electrophysiology preparation, single lab\",\n      \"pmids\": [\"15935995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In CD4 T cells, VPAC1 mRNA is upregulated by the vascular environment but downregulated by TCR signaling (anti-CD3); JNK kinases downstream of Zap70 mediate the suppressive regulation of VPAC1 after TCR activation, while inhibitors of PKC, ERK, p38, Zap70, and Rac1 show stimulatory influence on VPAC1 expression in the absence of TCR signaling.\",\n      \"method\": \"Primary murine splenic CD4 T cell isolation, pharmacological kinase inhibitors (10 inhibitors), anti-CD3 stimulation, qPCR for VPAC1 mRNA, blood vs. spleen comparison\",\n      \"journal\": \"Brain, behavior, and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pharmacological kinase inhibitor panel with qPCR, single lab, no direct pathway confirmation\",\n      \"pmids\": [\"18534815\", \"18555660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The mouse Vipr1 gene is encoded on chromosome 9 (syntenic with human chromosome 3p21.3) in a single-copy gene of >16 kb with 13 exons; the 5'-flanking region lacks a TATA box but contains a CCAAT box, Sp1- and AP-2-binding sites, and has functional promoter activity in luciferase reporter assays.\",\n      \"method\": \"Genomic cloning and sequencing, exon-intron mapping, chromosomal mapping by genetic crosses, luciferase reporter assay\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct genomic characterization and functional promoter assay, single lab\",\n      \"pmids\": [\"10331949\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"VPAC1 (VIPR1) is a class B G protein-coupled receptor that binds VIP and PACAP-27 with equal high affinity at its N-terminal ectodomain (structured as a Sushi/short consensus repeat domain with three disulfide bonds), where the central-to-C-terminal portion of VIP (residues 6-28) docks via contacts mapped by photoaffinity labeling (Phe6↔N-ted 104-108, Tyr22↔N-ted 109-120), while the VIP N-terminus (His1) interacts with TM1 residues K143/T144/T147; receptor activation requires Asp3 of VIP to penetrate the TM bundle and interact with Arg188/Lys195 in TM2, engaging a conserved network with Asn229 (TM3) and Gln380 (TM7); upon activation the receptor couples primarily to Gs/adenylyl cyclase (via a YL motif equivalent to the DRY motif) and also to Gi/o (via IC3 residues including IRKS 328-331) and Galphaq for Ca2+ signaling, with the third IC loop's distal basic residues (R338, L339, R341) selectively mediating Ca2+ and Gi coupling; rapid desensitization is mediated by GRK phosphorylation of Ser447 in the C-terminal tail, and sustained internalization requires multiple C-terminal Ser/Thr residues; Cys37 in the N-terminal ectodomain undergoes palmitoylation that mediates VIP-induced nuclear translocation contributing to anti-apoptotic signaling; a functional N-terminal signal peptide (residues 1-30) is required for cell-surface expression; physiologically, VPAC1 suppresses megakaryopoiesis (raising cAMP in megakaryocytes to inhibit differentiation), regulates intestinal secretion and motility via cholinergic neurons and epithelial CFTR (on the apical membrane of colonocytes), modulates hippocampal synaptic transmission via PKC-dependent GABA release inhibition, suppresses bone marrow progenitor proliferation, and mediates pro-inflammatory CNS chemokine induction in EAE; its transcription is regulated by Ikaros (suppression) and FXR agonists (activation in biliary epithelium), and by promoter methylation and H3K27 deacetylation in HCC.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"VIPR1 (VPAC1) is a class B G protein-coupled receptor for the neuropeptides VIP and PACAP-27 that transduces these signals into cyclic AMP and calcium responses governing intestinal physiology, hematopoiesis, neural function, and inflammation [#0, #22]. Ligand recognition follows a two-domain mechanism: the central-to-C-terminal portion of VIP (residues 6\\u201322) docks against a negatively charged, tryptophan-lined groove in the N-terminal ectodomain, a Sushi/short-consensus-repeat fold stabilized by three disulfide bonds, while the VIP N-terminus engages the transmembrane bundle [#2, #4, #5]. Direct photoaffinity crosslinking mapped Phe6 to ectodomain residues 104\\u2013108 and Tyr22 to 109\\u2013120, and showed that the VIP His1 N-terminus contacts TM1 residues K143/T144/T147, contacts that are distinct from those used by antagonists [#3, #7]. Receptor activation requires the acidic Asp3 of VIP to penetrate the transmembrane core and engage Arg188/Lys195 in TM2 within a conserved network linking Arg188 (TM2), Asn229 (TM3), and Gln380 (TM7) [#1, #8, #14]. Activated VPAC1 couples primarily to Gs/adenylyl cyclase via a DRY-equivalent 'YL' motif, and separately to Gi/Gq for calcium signaling through distinct third-intracellular-loop subdomains (IRKS 328\\u2013331 and distal basic residues R338/L339/R341) [#11, #12, #15]. Agonist-induced phosphorylation of C-terminal Ser/Thr residues drives desensitization (Ser447 as a GRK target) and internalization [#9, #10]. Beyond canonical signaling, palmitoylation of Cys37 in the ectodomain directs VIP-induced nuclear translocation that supports anti-apoptotic activity, and a cleaved N-terminal signal peptide (residues 1\\u201330) is required for surface expression [#18, #36]. Physiologically, VPAC1 raises cAMP in megakaryocytes to suppress megakaryopoiesis and platelet production [#22], is required for intestinal and pancreatic islet development [#25], drives CFTR-dependent chloride secretion in epithelia by stabilizing CFTR through NHERF1/ERM [#20, #21], acts on cholinergic secretomotor and motor neurons to regulate intestinal secretion and motility [#27], and mediates pro-inflammatory responses in colitis and EAE [#23, #24]. Its transcription is suppressed by Ikaros and induced by FXR agonists in distinct epithelia [#33, #34].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing which VIP residues drive VPAC1 engagement defined the pharmacophore and yielded the first selective VPAC1 agonist, enabling all subsequent receptor-subtype dissection.\",\n      \"evidence\": \"Alanine-scan VIP analogs with radioligand binding, adenylyl cyclase assays, and molecular modeling\",\n      \"pmids\": [\"10801840\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not map the reciprocal receptor contact residues\", \"Selectivity mechanism at the structural level not resolved\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identifying the transmembrane acidic-residue trigger answered how the VIP N-terminus converts binding into activation, separating activation from antagonist recognition.\",\n      \"evidence\": \"Site-directed mutagenesis of TM2 (Arg188, Lys195) with binding and cAMP assays in CHO cells\",\n      \"pmids\": [\"11013258\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational changes accompanying Asp3 insertion not directly observed\", \"No structural snapshot of the activated state\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Building a 3D model of the N-terminal ectodomain and validating its residues defined the peptide-binding groove architecture of the receptor.\",\n      \"evidence\": \"Homology modeling plus mutagenesis (Pro74, Pro87, Phe90, Trp110 etc.) with binding and cyclase readouts\",\n      \"pmids\": [\"11124960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Model lacks experimental high-resolution structure\", \"Dynamics of ligand capture not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Combining NMR of VIP with photoaffinity mapping resolved the two-domain binding mode, showing VIP 6\\u201328 occupies the ectodomain while the N-terminus is free to reach the transmembrane region.\",\n      \"evidence\": \"Bpa photoaffinity probes at VIP positions 6/22/24, Edman sequencing, VIP NMR, and N-ted homology modeling/docking\",\n      \"pmids\": [\"16520374\", \"12807902\", \"15247290\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full receptor\\u2013ligand complex not crystallized\", \"Stoichiometry and order of binding events not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Distinguishing agonist versus antagonist N-terminal contact sites and pinpointing TM1 His1-interacting residues clarified why agonists and antagonists are recognized differently.\",\n      \"evidence\": \"Bpa0 photoaffinity probes for VIP and PG97-269 plus alanine mutagenesis of TM1 (K143/T144/T147)\",\n      \"pmids\": [\"22291440\", \"18597186\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of distinct antagonist site for drug design not tested\", \"Single-lab photoaffinity mapping\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrating cooperativity among Arg188/Asn229/Gln380 established a conserved transmembrane activation network bridging ligand sensing and G-protein coupling.\",\n      \"evidence\": \"Structural modeling with double-mutant cooperativity analysis and cAMP/binding assays in CHO cells\",\n      \"pmids\": [\"20573782\", \"16650965\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Activation network not visualized structurally\", \"Allosteric pathway to G protein not fully traced\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapping intracellular-loop determinants showed VPAC1 uses physically separate subdomains for Gs/cAMP versus Gi/Gq/Ca2+ coupling, explaining its multifunctional signaling.\",\n      \"evidence\": \"VPAC1/VPAC2 chimeras and IC3 point mutants with aequorin calcium and cyclase assays; YL-motif mutagenesis\",\n      \"pmids\": [\"11981043\", \"15451021\", \"11859928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct G-protein selectivity at the structural interface not resolved\", \"Cellular context dependence of coupling not addressed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Native pull-down showed tissue-dependent G-protein partners and basal pre-coupling, indicating VPAC1 coupling repertoire varies with cellular context.\",\n      \"evidence\": \"Immunoaffinity chromatography and cross-linking from human and rat tissues with G-protein-specific Western blots\",\n      \"pmids\": [\"11812005\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab native study\", \"Functional consequence of pre-coupling not established\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defining C-terminal Ser/Thr phosphorylation sites and Ser447 separated desensitization from internalization, clarifying receptor regulation kinetics.\",\n      \"evidence\": \"Truncation/site-directed mutagenesis with phosphorylation, internalization, recycling, flow cytometry, and confocal assays in CHO cells\",\n      \"pmids\": [\"15932876\", \"14645688\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"GRK isoform identity not confirmed in vivo\", \"Arrestin recruitment not directly measured\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing a cleavable N-terminal signal peptide explained the biogenesis requirement for surface delivery of VPAC1.\",\n      \"evidence\": \"Deletion and Flag-insertion constructs with binding, cAMP, and immunofluorescence localization in CHO cells\",\n      \"pmids\": [\"15518910\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cleavage site not directly sequenced\", \"ER processing machinery not identified\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Genetic and antibody studies established VPAC1 as a negative regulator of megakaryopoiesis acting through cAMP, defining a hematopoietic physiological role.\",\n      \"evidence\": \"CD34+ differentiation assays, neutralizing antibodies, transgenic and KO mice with cAMP and platelet readouts\",\n      \"pmids\": [\"18000164\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream cAMP effectors in megakaryocytes not detailed\", \"Human therapeutic translation untested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"VPAC1-null mice revealed an essential developmental role in intestine and endocrine pancreas, beyond acute signaling functions.\",\n      \"evidence\": \"Homozygous null mice and promoter-reporter localization with histology and glucose homeostasis assays\",\n      \"pmids\": [\"21697765\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-autonomous versus paracrine basis of phenotype unresolved\", \"Molecular targets in islet development not identified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identifying VPAC1 control of CFTR membrane stability via NHERF1/ERM and on cholinergic neurons defined dual epithelial and neural mechanisms for intestinal fluid secretion.\",\n      \"evidence\": \"Proximity ligation, siRNA knockdown, Ussing chamber, muscle contraction, and immunohistochemistry with selective pharmacology\",\n      \"pmids\": [\"24788249\", \"14744818\", \"24578344\", \"12598410\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of epithelial versus neuronal VPAC1 in vivo not quantified\", \"Link between cAMP/PKC and scaffold remodeling not fully traced\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"KO and chimera studies in colitis and EAE established VPAC1 as a pro-inflammatory mediator, with CNS chemokine induction arising from non-hematopoietic VPAC1.\",\n      \"evidence\": \"VPAC1/VPAC2 KO mice, bone-marrow chimeras, adoptive transfer, cytokine/chemokine profiling, and pharmacological antagonists\",\n      \"pmids\": [\"27357191\", \"21295288\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of non-hematopoietic VPAC1-expressing cells not pinned down\", \"Mechanism of chemokine induction downstream of VPAC1 unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Cys37 palmitoylation was shown to drive VIP-induced nuclear translocation linked to anti-apoptotic activity, revealing a non-canonical receptor function.\",\n      \"evidence\": \"C37A mutagenesis, acyl-biotin exchange and click-chemistry palmitoylation assays, 2-bromopalmitate, confocal imaging, and apoptosis assays in CHO cells\",\n      \"pmids\": [\"28473666\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear targets of the translocated receptor unknown\", \"In vivo relevance of nuclear VPAC1 untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Cancer studies tied VPAC1 to metabolic and calcium-dependent signaling axes (ASS1/arginine and CAD in HCC; TRPV4/Ca2+ in gastric cancer), linking receptor activity to tumor growth control.\",\n      \"evidence\": \"In vitro/in vivo cancer cell studies with transcriptomics, calcium imaging, pathway analysis, and clinical sample correlation\",\n      \"pmids\": [\"35864952\", \"30692637\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct receptor-to-pathway coupling steps not fully reconstituted\", \"Single-lab studies for each axis\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Transcriptional control of VIPR1 was mapped to Ikaros-mediated suppression and, later, FXR-mediated induction, defining context-dependent expression regulation.\",\n      \"evidence\": \"EMSA/supershift, reporter deletions, and overexpression for Ikaros; FXR agonist with EMSA and expression assays in primary cells\",\n      \"pmids\": [\"11812772\", \"16037943\", \"10331949\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Combinatorial control across tissues not integrated\", \"Chromatin-level regulation incompletely defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A high-resolution structure of agonist-bound, G-protein-coupled VPAC1 and identification of the nuclear effectors of palmitoylation-dependent translocation remain open questions.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No experimental full-length receptor\\u2013ligand\\u2013G protein structure in the corpus\", \"Nuclear signaling partners of translocated VPAC1 unidentified\", \"Cell-type origin of pro-inflammatory VPAC1 effects unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 8, 11]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0001618\", \"supporting_discovery_ids\": [32]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [18, 35]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [36]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 11, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [23, 24, 30]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [20, 21]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [25, 28]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"VIP\", \"ADCYAP1\", \"NHERF1\", \"CFTR\", \"GNAS\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}