{"gene":"ADCYAP1R1","run_date":"2026-06-09T22:02:41","timeline":{"discoveries":[{"year":2011,"finding":"Crystal structure of the PAC1R extracellular domain (ECD) at 1.9 Å resolution reveals it adopts the same consensus fold as other Class B GPCRs. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor confirm that PAC1R uses this conserved ECD fold for PACAP binding, unifying the hormone-recognition mechanism for this receptor family.","method":"X-ray crystallography (1.9 Å), alanine-scan peptide binding assays, cell-based cAMP assays, mutant receptor functional assays","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure combined with mutagenesis and functional cell-based assays in a single rigorous study","pmids":["21625560"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structures of VIP-bound, Gs-coupled PAC1R short variant (PAC1sR) and null variant (PAC1nR) reveal that the alternatively spliced null loop in PAC1nR transiently engages residues in extracellular loop 2 (ECL2) and the N-terminal helix of the ECD, resulting in differential engagement of VIP with these domains and the top of TM2/ECL1. This structural difference underlies the selectively enhanced VIP (but not PACAP) functional activity across multiple signaling endpoints at PAC1sR compared to PAC1nR. MD simulations further predict differential Gs protein interactions with the two variants when VIP-bound, with greater allosteric influence of Gs on VIP affinity at PAC1sR.","method":"Cryo-EM structure determination, molecular dynamics (MD) simulations, pharmacological signaling assays across multiple functional endpoints","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structures of both splice variants with MD simulations and comprehensive pharmacological profiling in one rigorous study","pmids":["41264251"],"is_preprint":false},{"year":1998,"finding":"PAC1R isoform expression determines second-messenger coupling and mitogenic outcome: sympathetic neuroblasts expressing the 'hop' receptor isoform show PACAP-induced increases in both cAMP and PI (phosphoinositide) signaling and are mitogenically stimulated, whereas cerebral cortical precursors expressing primarily the 'short' isoform show only cAMP increases and are mitogenically inhibited. Thus, opposing mitogenic effects of PACAP correlate with differential receptor isoform expression and distinct intracellular signaling.","method":"RT-PCR isoform identification, cAMP and PI second-messenger assays, cell proliferation assays in primary neuronal cultures","journal":"Journal of neuroscience research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (RT-PCR, second-messenger assays, proliferation assays) in primary cultures, single lab","pmids":["9753193"],"is_preprint":false},{"year":2001,"finding":"A novel alternatively spliced exon (exon 3a, encoding 24 amino acids) inserted into the extracellular domain of PAC1R during spermatogenesis generates PAC1R(3a). In HEK293 cells stably expressing PAC1R(3a), ligand binding affinity for PACAP-38 is increased 6-fold and coupling to both cAMP (Gs) and inositol phosphate (Gq) signaling pathways is altered relative to wild-type PAC1R. PAC1R(3a) mRNA is preferentially expressed in seminiferous tubules, round spermatids, and Sertoli cells.","method":"Cloning and sequencing of novel splice variant, stable transfection of HEK293 cells, radioligand binding assays, cAMP and inositol phosphate signaling assays, RT-PCR expression analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with stably transfected cells, binding assays, and two second-messenger pathway assays, rigorous controls, single lab","pmids":["11278585"],"is_preprint":false},{"year":2009,"finding":"PACAP acts via selective high-affinity PAC1Rs to enhance nicotinic synaptic transmission at parasympathetic ciliary ganglion neurons by increasing spontaneous EPSC frequency and amplitude. The mechanism requires adenylate cyclase (AC)/cAMP/PKA signaling (not PLC), and acts presynaptically to increase vesicular ACh release (quantal content), not postsynaptic nAChR function. Nitric oxide (NO) signaling downstream of AC is critical: PACAP-induced increases in EPSC frequency, amplitude, and quantal content are mimicked by NO donor and abolished by NOS inhibition.","method":"Electrophysiology (whole-cell patch clamp, spontaneous and evoked EPSC recordings), pharmacological inhibitors of AC, PKA, PLC, NOS; 8-Bromo-cAMP mimicry; quantal content analysis","journal":"Molecular and cellular neurosciences","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal electrophysiology and pharmacology approaches, rigorous presynaptic mechanism dissection, single lab","pmids":["19958833"],"is_preprint":false},{"year":2016,"finding":"Estradiol (E2) regulates ADCYAP1R1 expression through estrogen receptor alpha (ERα) binding to an estrogen response element (ERE) in the ADCYAP1R1 gene that contains the PTSD-risk SNP rs2267735. In vitro, E2/ERα binds less efficiently to the ERE containing the risk C allele. In vivo in mice, fear conditioning and E2 additively increase ADCYAP1R1 expression. In women with low serum E2, the CC genotype associates with lower ADCYAP1R1 expression.","method":"In vitro ERE binding assays (E2/ERα), cell culture E2 treatment with ADCYAP1R1 expression measurement, mouse fear conditioning with E2 treatment, human genotype-expression correlation","journal":"Translational psychiatry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (in vitro binding, cell culture, mouse in vivo, human expression), single lab","pmids":["27959335"],"is_preprint":false},{"year":1999,"finding":"The tumor suppressor p53 and zinc finger protein Zac induce expression of the PAC1-R gene, establishing a transcriptional regulatory link between apoptosis/cell-cycle arrest pathways and PAC1-R expression.","method":"Gene reporter assays, overexpression of p53 and Zac in cell culture with PAC1-R gene expression readout","journal":"Brain research. Molecular brain research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, gene expression readout only, no direct mechanistic dissection of promoter binding","pmids":["10366751"],"is_preprint":false},{"year":2011,"finding":"PAC1R stimulates prolactin promoter activity and mRNA synthesis in pituitary GH3 cells through Gs/cAMP and ERK/PKC signaling pathways; transfection of PAC1R expression vector dramatically potentiates the prolactin promoter response to PACAP proportional to receptor amount, and PAC1R itself is induced by PACAP (auto-upregulation).","method":"Transfection of PAC1R expression vector into GH3 cells, prolactin promoter-reporter assay, ERK phosphorylation assay (Western blot), RT-PCR for prolactin and GH mRNA","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter reporter assay, Western blot ERK activation, and mRNA measurement; multiple orthogonal methods, single lab","pmids":["21539889"],"is_preprint":false},{"year":2016,"finding":"In human adipose-derived stem cells (hADSCs), PAC1R activation by the selective agonist maxadilan promotes cell proliferation, migration, and anti-apoptotic effects, and enhances neural differentiation. Two distinct PAC1R-coupled pathways are identified: ligand-dependent PKA signaling enhances viability and neural differentiation (blocked by H-89); and dimer-dependent Wnt/β-catenin signaling also promotes viability (blocked by XAV939).","method":"Cell Counting Kit-8 proliferation assay, cell cycle analysis, wound-healing migration assay, Annexin V apoptosis assay, mitochondrial membrane potential assay, Western blot, qPCR, whole-cell patch clamp, pharmacological inhibition of PKA and Wnt pathways","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal assays identifying two mechanistically distinct PAC1R signaling branches, single lab","pmids":["26798992"],"is_preprint":false},{"year":2022,"finding":"PAC1R undergoes nuclear translocation when treated with positive allosteric modulators PACAP(28-38) or TAT peptide. Nuclear PAC1R binds the promoter regions of PAC1R and PACAP genes (demonstrated by ChIP-PCR), upregulating their expression. Nuclear translocation requires palmitoylation at Cys25 (abolished by mutation C25A or palmitoylation inhibitor 2-bromopalmitate). ChIP-seq analysis identifies transcription factors SP1, Zic2, GATA1, REST, and YY1 as potentially recruited by nuclear PAC1R.","method":"Confocal fluorescence microscopy (nuclear translocation), ChIP-PCR (promoter binding), site-directed mutagenesis (C25A), palmitoylation inhibitor treatment, ChIP-sequencing, promoter-reporter assays","journal":"Acta biochimica et biophysica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-PCR promoter binding and mutagenesis with fluorescence imaging, multiple methods, single lab","pmids":["35593471"],"is_preprint":false},{"year":2021,"finding":"Blue light and hydrogen peroxide (H2O2) trigger nuclear translocation of PAC1R (observed in PAC1R-CHO cells expressing PAC1R-eGFP and in retinal ganglion cells RGC-5 with native PAC1R). Nuclear translocation is blocked by ROS scavenger NAC and palmitoylation inhibitor 2-bromopalmitate, and is markedly reduced by the C25A mutation in PAC1R. Nuclear translocation positively correlates with upregulation of PAC1R expression and promoter activity.","method":"Fluorescence microscopy (nuclear translocation of PAC1R-eGFP), pharmacological inhibition (NAC, 2-bromopalmitate), site-directed mutagenesis (C25A), Western blot, promoter-reporter assay","journal":"Biochimica et biophysica acta. General subjects","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (imaging, pharmacology, mutagenesis, reporter), single lab, corroborates prior findings","pmids":["33647387"],"is_preprint":false},{"year":2022,"finding":"PAC1R positive allosteric modulator SPAM1 triggers nuclear translocation of PAC1R. The nuclear 35 kDa (but not 15 kDa) fragment of PAC1R interacts with SP1 to upregulate Huntingtin (Htt) expression, which then attenuates NRSF (neuron-restrictive silencer factor) binding to NRSE, leading to upregulated expression of NRSF-target neuropeptides (PACAP, BDNF, TH, SYN1).","method":"Fluorescence microscopy (nuclear translocation), co-immunoprecipitation (nuclear PAC1R-SP1 interaction), Western blot, in vivo D-gal aging mouse model, cell viability assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for nuclear PAC1R-SP1 interaction combined with in vivo mouse model and imaging, single lab","pmids":["36555637"],"is_preprint":false},{"year":2016,"finding":"PACAP auto-regulates its own expression through PAC1R activation in human neuroblastoma NB-1 cells. PAC1R-mediated PACAP gene induction requires parallel, independent activation of PKA, novel (but not conventional) PKC isoforms, and ERK. EGR1 induction by PACAP requires PKC and ERK (not PKA), but siRNA knockdown of EGR1 did not affect PACAP auto-regulation, excluding EGR1 from the PACAP auto-regulatory pathway.","method":"Pharmacological kinase inhibitors (PKA, PKC, MEK), RT-PCR for PACAP mRNA, siRNA knockdown of EGR1, PAC1R-selective agonist Maxadilan","journal":"Neuropeptides","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic pharmacological pathway dissection with siRNA validation, single lab, multiple orthogonal approaches","pmids":["27745706"],"is_preprint":false},{"year":2019,"finding":"The human ADCYAP1R1 gene promoter (cloned 2,526 bp region from -2500 to +26) shows a hormetic response to H2O2 in SH-SY5Y cells: low concentrations (<10 nM) activate the promoter in a dose-dependent manner via SP1 binding sites (blocked by mithramycin A and SP1 site deletion mutants), whereas higher concentrations (>10 nM) inhibit promoter activity.","method":"Promoter cloning, reporter assay, SP1 inhibitor (mithramycin A), deletion mutagenesis of SP1 binding sites","journal":"Journal of food biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assay combined with pharmacological inhibition and deletion mutagenesis, single lab","pmids":["31353704"],"is_preprint":false},{"year":2019,"finding":"Doxycycline and minocycline function as positive allosteric modulators (PAMs) of PAC1-R by binding the N-terminal extracellular domain (PAC1-EC1). Isothermal titration calorimetry confirms direct binding. Site-directed mutagenesis identifies ASN60 as the most important residue for PAM binding, with ASP116 playing a sensitive marginal role. Tetracycline/tigecycline derivatives show lower affinity and lack PAM activity.","method":"Computational molecular docking, isothermal titration calorimetry, cAMP assay with PACAP27, site-directed mutagenesis of PAC1-EC1","journal":"Acta biochimica et biophysica Sinica","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro binding assay (ITC), mutagenesis, and functional cAMP assay, single lab","pmids":["31056648"],"is_preprint":false},{"year":2021,"finding":"A co-crystal structure of a PAC1R antagonist peptide (des(24-42) Maxadilan analog 18) bound to the PAC1R extracellular domain was determined, representing the first structural data for a PAC1R antagonist. SAR studies identified peptide modifications that increased PAC1R inhibitory activity to the pM IC90 range. Analog 17 and 18 showed in vivo efficacy in a rat Maxadilan-induced blood flow pharmacodynamic model at 0.3 mg/kg subcutaneous dosing.","method":"X-ray co-crystallography of PAC1R ECD with antagonist peptide, in vitro PAC1R binding/inhibition assays, in vivo rat pharmacodynamic model","journal":"Journal of medicinal chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure of receptor-antagonist complex combined with in vitro SAR and in vivo pharmacodynamic validation","pmids":["33715378"],"is_preprint":false},{"year":2022,"finding":"Peptide OM-LV20 protects astrocytes from oxidative stress via a PAC1R/JNK/TPH1 signaling axis: OM-LV20 increases TPH1 expression in astrocytes through PAC1R and the JNK signaling pathway, protecting against H2O2-induced decreases in cell viability and catalase levels.","method":"Primary astrocyte culture, quantitative RT-PCR, double immunofluorescence, lentiviral infection (loss-of-function), cell viability analysis, Western blotting","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — lentiviral knockdown plus pharmacological pathway inhibition with multiple readouts, single lab","pmids":["36037970"],"is_preprint":false},{"year":2003,"finding":"PAC1-R is expressed in reactive astrocytes but not resting astrocytes. Immunostaining of transgenic mice (GFAP-EGFP) following cortical stab wound revealed PAC1-R immunoreactivity specifically in EGFP/GFAP-positive reactive astrocytes (and vimentin-positive cells) around the wound at 5 days post-surgery, but not in the contralateral hemisphere.","method":"Transgenic GFAP-EGFP mouse model, cortical stab wound surgery, immunohistochemistry for PAC1-R, GFAP, vimentin, CD11b, NeuN","journal":"Brain research. Molecular brain research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vivo localization using transgenic reporter mice with injury model, single lab","pmids":["12824050"],"is_preprint":false},{"year":2008,"finding":"PAC1-R heterozygous (-/+) mice fail to use geomagnetic orientation or fear signals to guide memory retrieval (no errors when maze is rotated 180° relative to geomagnetic field or upon fear signal exposure), whereas wild-type mice do make errors in these conditions. This implicates PAC1-R in gating passage of geomagnetic and fear sensory signals for memory retrieval.","method":"Behavioral testing in PAC1-R heterozygous transgenic mutant mice vs. wild-type littermates in multiple-maze memory tasks with rotation and fear signal challenges","journal":"Journal of molecular neuroscience : MN","confidence":"Low","confidence_rationale":"Tier 3 / Weak — behavioral phenotype in heterozygous KO mice, single lab, no molecular mechanism dissection","pmids":["18781286"],"is_preprint":false},{"year":2024,"finding":"PACAP/PAC1R signaling in the locus coeruleus (LC) from anterior insular cortex (aIC) PACAP neurons regulates anxiety-related behaviors. PAC1R mRNA is highly expressed in LC noradrenergic neurons. Targeted deletion of PAC1R from noradrenergic cells produces a hyperactive/low anxiety phenotype. Selective activation of PACAP neurons in the aIC increases anxiety. Retrograde tracing confirmed aIC PACAP neurons project to the LC. shRNA knockdown of PACAP in the aIC decreases anxiety.","method":"Histochemistry (PAC1R mRNA in situ hybridization), conditional cell-type-specific PAC1R deletion, retrograde neurocircuit tracing, chemogenetic activation of aIC PACAP neurons, shRNA knockdown of PACAP in aIC, open field and elevated-plus maze behavioral tests","journal":"Neurochemistry international","confidence":"High","confidence_rationale":"Tier 2 / Moderate — conditional KO, circuit tracing, chemogenetics, and shRNA knockdown in multiple orthogonal experiments establishing a defined aIC→LC→anxiety circuit requiring PAC1R","pmids":["39396708"],"is_preprint":false},{"year":2025,"finding":"In a rat nucleus accumbens core (NAcc) model, endogenous PAC1R (ADCYAP1R1) is expressed in NAcc and PACAP mRNA is present in medial prefrontal cortical projections to NAcc. Intra-NAcc PACAP infusion blocked cocaine-primed reinstatement and blocked reinstatement driven by D1 receptor agonist co-infusion, but did not block reinstatement driven by D2 receptor agonist, demonstrating PAC1R signaling in NAcc selectively interferes with D1-mediated (not D2-mediated) reinstatement mechanisms.","method":"In situ hybridization/immunofluorescence for PAC1R expression, retrograde tracing for mPFC→NAcc PACAP projections, intra-NAcc pharmacological infusion, cocaine-primed and receptor agonist-driven reinstatement behavioral assays","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, pharmacological infusion with behavioral readout only, no molecular mechanism dissection beyond receptor selectivity","pmids":["bio_10.1101_2025.03.17.643720"],"is_preprint":true},{"year":2023,"finding":"Celastrol promotes cAMP production after spinal cord injury (SCI) by upregulating the VIP-ADCYAP1R1-GNAS signaling pathway, thereby inhibiting JNK phosphorylation and preventing apoptosis in injured spinal cord tissue.","method":"In vivo SCI mouse model, cAMP measurement, Western blot (JNK phosphorylation, apoptotic markers), gene expression analysis","journal":"Biomedicine & pharmacotherapy","confidence":"Low","confidence_rationale":"Tier 3 / Weak — in vivo pharmacology with pathway readouts, but ADCYAP1R1-specific mechanistic evidence is indirect (upregulation of pathway), single lab","pmids":["37531781"],"is_preprint":false},{"year":2025,"finding":"PACAP/PAC1R signaling promotes ILC2 (group 2 innate lymphoid cell) proliferation and IL-5/IL-13 cytokine production via the ERK pathway. In OVA-induced allergic rhinitis mice, PAC1R knockdown or antagonist (PA-8) markedly suppressed ILC2 proliferation and cytokine production. ERK inhibitor PD98059 reversed PAC1R-mediated ILC2 activation, and PAC1R knockdown decreased p-ERK and ILC2 numbers.","method":"OVA-induced AR mouse model, PAC1R siRNA knockdown, PAC1R antagonist PA-8, ERK inhibitor PD98059, Western blot (p-ERK), flow cytometry (ILC2s, IL-5/IL-13)","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PAC1R knockdown and antagonist combined with ERK inhibition and in vivo model, multiple orthogonal methods, single lab","pmids":["40713003"],"is_preprint":false},{"year":2005,"finding":"PAC1R is expressed on tuberoinfundibular dopamine (TIDA) neurons in the arcuate nucleus of the ewe hypothalamus; dual immunofluorescence shows 21.2% of arcuate tyrosine hydroxylase-positive dopaminergic neurons co-express PAC1R, while other hypothalamic dopaminergic cell groups show little (<3%) co-localization. This positions PAC1R to mediate PACAP's inhibition of prolactin secretion via direct action on TIDA neurons.","method":"RT-PCR, in situ hybridization, immunohistochemistry, dual immunofluorescence for PAC1R and tyrosine hydroxylase","journal":"Journal of neuroendocrinology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — localization study establishing cell-type specificity; functional consequence is inferred from prior in vivo studies, not directly tested here","pmids":["15869565"],"is_preprint":false}],"current_model":"ADCYAP1R1 (PAC1R) is a class B G protein-coupled receptor that binds PACAP (and, with splice variant-dependent selectivity, VIP) through its extracellular domain; alternatively spliced isoforms of the receptor (short, null, hop, hip, 3a, etc.) differ in PACAP/VIP affinity and in coupling to Gs/cAMP versus Gq/inositol phosphate pathways, with structural basis established by cryo-EM and X-ray crystallography; upon activation, PAC1R signals canonically via cAMP/PKA and PKC/ERK to regulate synaptic vesicle release, neuronal proliferation, prolactin synthesis, and ILC2 activation, and non-canonically undergoes palmitoylation-dependent nuclear translocation where it binds gene promoters (including its own and PACAP) to upregulate their expression; estrogen receptor alpha regulates ADCYAP1R1 transcription via an ERE overlapping the PTSD-risk SNP rs2267735; and in specific circuits (e.g., anterior insular cortex → locus coeruleus noradrenergic neurons; nucleus accumbens), PAC1R signaling is required for anxiety-related behaviors and modulates dopamine-dependent reinstatement of cocaine seeking."},"narrative":{"mechanistic_narrative":"ADCYAP1R1 (PAC1R) is a class B G protein-coupled receptor that recognizes the neuropeptide PACAP through a conserved extracellular-domain fold and transduces this signal into diverse neuronal, endocrine, and immune outcomes [PMID:21625560]. Receptor function is diversified by alternative splicing: variants differ in ligand affinity and in coupling to Gs/cAMP versus Gq/inositol phosphate pathways, and structural work on the VIP-bound short and null variants shows that the spliced null loop engages ECL2 and the ECD N-terminal helix to set variant-selective VIP responsiveness [PMID:41264251, PMID:11278585]. Isoform identity also determines biological output—'hop'-expressing neuroblasts couple to both cAMP and phosphoinositide signaling and are mitogenically stimulated, whereas 'short'-isoform cortical precursors raise only cAMP and are mitogenically inhibited [PMID:9753193]. Downstream, PAC1R signals canonically via AC/cAMP/PKA and PKC/ERK: it enhances presynaptic vesicular acetylcholine release through an AC/cAMP/PKA/nitric oxide cascade [PMID:19958833], drives prolactin promoter activity and mRNA synthesis in pituitary cells with self-induction of the receptor [PMID:21539889], auto-regulates PACAP expression through parallel PKA, novel-PKC, and ERK activation [PMID:27745706], and promotes ILC2 proliferation and IL-5/IL-13 production via ERK [PMID:40713003]. Non-canonically, PAC1R undergoes palmitoylation-dependent (Cys25) nuclear translocation triggered by allosteric modulators or by oxidative/blue-light stress, where it binds the promoters of its own and the PACAP gene to upregulate them and interacts with SP1 to drive a Huntingtin-NRSF neuropeptide program [PMID:35593471, PMID:33647387, PMID:36555637]. Its transcription is controlled by estrogen receptor alpha acting at an ERE that contains the PTSD-risk SNP rs2267735, providing a hormonal link to fear-related expression [PMID:27959335]. At the circuit level, PAC1R in locus coeruleus noradrenergic neurons is required for anxiety-related behavior within an anterior-insula→LC pathway [PMID:39396708].","teleology":[{"year":1998,"claim":"Established that PAC1R splice-isoform identity, not ligand alone, dictates second-messenger coupling and opposing mitogenic outcomes, explaining context-dependent PACAP biology.","evidence":"RT-PCR isoform typing with cAMP/PI assays and proliferation assays in primary neuronal cultures","pmids":["9753193"],"confidence":"Medium","gaps":["Molecular basis of isoform-specific coupling not structurally resolved","Single lab, primary culture only"]},{"year":1999,"claim":"Linked PAC1R transcription to apoptosis/cell-cycle regulators by showing p53 and Zac induce the gene, but only at the expression-readout level.","evidence":"Reporter assays and p53/Zac overexpression in cell culture","pmids":["10366751"],"confidence":"Low","gaps":["No direct promoter-binding dissection","Single lab, expression readout only"]},{"year":2001,"claim":"Demonstrated that tissue-specific splicing (exon 3a in spermatogenic cells) reshapes PACAP affinity and Gs/Gq coupling, extending the splicing-diversification principle to the reproductive tract.","evidence":"Cloning of PAC1R(3a), stable HEK293 expression, radioligand binding, cAMP and IP assays, RT-PCR","pmids":["11278585"],"confidence":"High","gaps":["Functional role in spermatogenesis not tested in vivo","Structural basis of altered coupling not determined"]},{"year":2005,"claim":"Positioned PAC1R to inhibit prolactin secretion by localizing it to a subset of tuberoinfundibular dopamine neurons in the arcuate nucleus.","evidence":"RT-PCR, in situ hybridization, dual immunofluorescence for PAC1R and tyrosine hydroxylase in ewe hypothalamus","pmids":["15869565"],"confidence":"Low","gaps":["Functional consequence inferred, not directly tested","Restricted to one species and cell group"]},{"year":2009,"claim":"Defined a presynaptic AC/cAMP/PKA/NO mechanism by which PAC1R enhances cholinergic synaptic transmission, distinguishing it from postsynaptic and PLC routes.","evidence":"Whole-cell patch-clamp EPSC recordings with AC/PKA/PLC/NOS inhibitors and cAMP mimicry in ciliary ganglion neurons","pmids":["19958833"],"confidence":"High","gaps":["NO source and downstream presynaptic targets not identified","Receptor isoform not specified"]},{"year":2011,"claim":"Resolved the conserved class B ECD fold of PAC1R and confirmed it as the PACAP-recognition module, unifying hormone binding across the family.","evidence":"1.9 Å X-ray crystallography with alanine-scan peptide binding and cell-based cAMP/mutant assays","pmids":["21625560"],"confidence":"High","gaps":["Full-length receptor and transmembrane activation not captured","Isoform-specific ECD differences not addressed"]},{"year":2011,"claim":"Showed PAC1R drives prolactin gene expression via Gs/cAMP and ERK/PKC and is itself upregulated by PACAP, introducing receptor auto-induction.","evidence":"PAC1R transfection, prolactin promoter-reporter, ERK Western blot, and mRNA assays in GH3 pituitary cells","pmids":["21539889"],"confidence":"Medium","gaps":["Transcription factors mediating prolactin induction not identified","Single cell line"]},{"year":2016,"claim":"Established hormonal control of PAC1R transcription through ERα at an ERE overlapping the PTSD-risk SNP rs2267735, mechanistically connecting estrogen, the risk allele, and receptor expression.","evidence":"In vitro ERE binding, cell-culture E2 treatment, mouse fear conditioning, and human genotype-expression correlation","pmids":["27959335"],"confidence":"Medium","gaps":["Causal link to PTSD behavior not demonstrated","Allele-specific binding effect size modest"]},{"year":2016,"claim":"Dissected two parallel PAC1R signaling branches—PKA and dimer-dependent Wnt/β-catenin—governing stem-cell viability and neural differentiation.","evidence":"Proliferation, migration, apoptosis, patch-clamp assays with PKA (H-89) and Wnt (XAV939) inhibition in human ADSCs","pmids":["26798992"],"confidence":"Medium","gaps":["Mechanism of Wnt engagement by receptor dimers unresolved","Single lab, ex vivo cells"]},{"year":2016,"claim":"Defined the PAC1R-driven PACAP auto-regulatory circuit requiring parallel PKA, novel PKC, and ERK, and ruled out EGR1 as the mediator.","evidence":"Kinase inhibitors, RT-PCR, and EGR1 siRNA in NB-1 neuroblastoma cells with PAC1R agonist maxadilan","pmids":["27745706"],"confidence":"Medium","gaps":["Transcription factor mediating PACAP induction unidentified","Single cell line"]},{"year":2019,"claim":"Identified doxycycline/minocycline as direct ECD-binding positive allosteric modulators of PAC1R, mapping key residues ASN60 and ASP116.","evidence":"Molecular docking, ITC binding, cAMP assay, and ECD site-directed mutagenesis","pmids":["31056648"],"confidence":"Medium","gaps":["In vivo relevance of PAM activity untested","Effect on Gq coupling not examined"]},{"year":2019,"claim":"Showed the ADCYAP1R1 promoter has a hormetic, SP1-dependent response to H2O2, linking oxidative state to receptor transcription.","evidence":"Promoter cloning, reporter assay, SP1 inhibitor mithramycin A, and SP1-site deletion in SH-SY5Y cells","pmids":["31353704"],"confidence":"Medium","gaps":["In vivo oxidative regulation not tested","Mechanism of biphasic switch unclear"]},{"year":2021,"claim":"Determined the first PAC1R ECD–antagonist co-structure and developed picomolar antagonist peptides with in vivo efficacy.","evidence":"X-ray co-crystallography of ECD-antagonist, SAR binding/inhibition, and rat maxadilan-induced blood-flow model","pmids":["33715378"],"confidence":"High","gaps":["Antagonism in CNS circuits not assessed","Full-receptor binding mode not captured"]},{"year":2021,"claim":"Showed oxidative and blue-light stress trigger palmitoylation-dependent (Cys25) nuclear translocation of PAC1R coupled to its own promoter upregulation, extending the non-canonical nuclear mechanism to stress signaling.","evidence":"PAC1R-eGFP imaging, NAC/2-bromopalmitate inhibition, C25A mutagenesis, Western blot, reporter assay in CHO and RGC-5 cells","pmids":["33647387"],"confidence":"Medium","gaps":["How a membrane GPCR reaches the nucleus mechanistically unresolved","Physiological trigger in vivo unclear"]},{"year":2022,"claim":"Established that allosterically induced nuclear PAC1R binds the PAC1R and PACAP promoters and recruits transcription factors, defining a receptor-as-transcriptional-regulator mode requiring Cys25 palmitoylation.","evidence":"Confocal imaging, ChIP-PCR, ChIP-seq, C25A mutagenesis, and promoter-reporter assays","pmids":["35593471"],"confidence":"Medium","gaps":["Direct DNA binding vs. cofactor recruitment not separated","Functional importance relative to membrane signaling unknown"]},{"year":2022,"claim":"Connected the nuclear PAC1R fragment to a transcriptional program via SP1 interaction, Huntingtin upregulation, and NRSF derepression of neuropeptide genes.","evidence":"Nuclear translocation imaging, PAC1R-SP1 Co-IP, Western blot, and D-gal aging mouse model","pmids":["36555637"],"confidence":"Medium","gaps":["Single Co-IP for the PAC1R-SP1 interaction without reciprocal validation","Origin of the 35 kDa nuclear fragment not defined"]},{"year":2022,"claim":"Defined a PAC1R/JNK/TPH1 axis by which a PACAP-family peptide protects astrocytes from oxidative stress.","evidence":"Primary astrocyte culture, qRT-PCR, immunofluorescence, lentiviral knockdown, viability and Western blot assays","pmids":["36037970"],"confidence":"Medium","gaps":["Receptor-proximal coupling to JNK not mapped","In vivo relevance untested"]},{"year":2024,"claim":"Demonstrated that PAC1R in locus coeruleus noradrenergic neurons is required for anxiety behavior within a defined anterior-insula→LC PACAP circuit.","evidence":"In situ hybridization, conditional cell-type PAC1R deletion, retrograde tracing, chemogenetics, aIC PACAP shRNA, and behavioral tests","pmids":["39396708"],"confidence":"High","gaps":["Downstream signaling in LC neurons not dissected","Sex-specific and hormonal modulation not addressed"]},{"year":2025,"claim":"Resolved the structural basis of splice-variant-selective VIP responsiveness, showing the null loop reshapes ECD/ECL2 engagement and Gs allostery.","evidence":"Cryo-EM of VIP-bound Gs-coupled PAC1sR and PAC1nR, MD simulations, and multi-endpoint pharmacology","pmids":["41264251"],"confidence":"High","gaps":["PACAP-bound complexes of the variants not compared","Functional consequence in native neurons untested"]},{"year":2025,"claim":"Indicated PAC1R signaling in the nucleus accumbens selectively blocks D1-mediated, not D2-mediated, cocaine reinstatement.","evidence":"PAC1R/PACAP expression mapping, retrograde tracing, and intra-NAcc pharmacological infusion in cocaine reinstatement assays (preprint)","pmids":["bio_10.1101_2025.03.17.643720"],"confidence":"Low","gaps":["Preprint, not peer reviewed","No molecular mechanism beyond receptor selectivity","Single lab"]},{"year":null,"claim":"How membrane-resident PAC1R is processed and trafficked to the nucleus, and how this transcriptional mode is balanced against canonical Gs/Gq signaling in vivo, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Mechanism of receptor entry into the nucleus unknown","Relative physiological weight of nuclear vs. membrane signaling unquantified","No in vivo validation of the nuclear transcriptional program"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0140110","term_label":"transcription regulator 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polypeptide (ADCYAP1/PACAP) (PubMed:32047270, PubMed:33715378, PubMed:35477937, PubMed:36385145). Binds both PACAP27 and PACAP38 bioactive peptides (PubMed:32047270, PubMed:35477937, PubMed:36385145). 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:32047270, PubMed:33715378, PubMed:35477937, PubMed:36385145). May regulate the release of adrenocorticotropin, luteinizing hormone, growth hormone, prolactin, epinephrine, and catecholamine. May play a role in spermatogenesis and sperm motility. Causes smooth muscle relaxation and secretion in the gastrointestinal tract (PubMed:32047270, PubMed:33715378)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/P41586/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ADCYAP1R1","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/ADCYAP1R1","total_profiled":1310},"omim":[{"mim_id":"607834","title":"ANXIETY","url":"https://www.omim.org/entry/607834"},{"mim_id":"102981","title":"ADCYAP RECEPTOR, TYPE I; ADCYAP1R1","url":"https://www.omim.org/entry/102981"},{"mim_id":"102980","title":"ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE 1; ADCYAP1","url":"https://www.omim.org/entry/102980"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Vesicles","reliability":"Enhanced"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":76.2},{"tissue":"fallopian tube","ntpm":28.5}],"url":"https://www.proteinatlas.org/search/ADCYAP1R1"},"hgnc":{"alias_symbol":["PAC1","PACAPR","PAC1R"],"prev_symbol":[]},"alphafold":{"accession":"P41586","domains":[{"cath_id":"4.10.1240.10","chopping":"22-82_109-133","consensus_level":"high","plddt":78.6313,"start":22,"end":133},{"cath_id":"1.20.1070.10","chopping":"145-410","consensus_level":"high","plddt":84.9831,"start":145,"end":410}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P41586","model_url":"https://alphafold.ebi.ac.uk/files/AF-P41586-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P41586-F1-predicted_aligned_error_v6.png","plddt_mean":75.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ADCYAP1R1","jax_strain_url":"https://www.jax.org/strain/search?query=ADCYAP1R1"},"sequence":{"accession":"P41586","fasta_url":"https://rest.uniprot.org/uniprotkb/P41586.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P41586/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P41586"}},"corpus_meta":[{"pmid":"23505260","id":"PMC_23505260","title":"ADCYAP1R1 genotype associates with post-traumatic stress symptoms in highly traumatized African-American females.","date":"2013","source":"American journal of medical genetics. 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Folia pharmacologica Japonica","url":"https://pubmed.ncbi.nlm.nih.gov/29887573","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.13.682090","title":"PERINATAL ORGANOPHOSPHATE FLAME RETARDANT EXPOSURE ALTERS ADULT STRESS AXIS AND AVOIDANCE BEHAVIOR IN MICE","date":"2025-10-14","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.13.682090","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.09.02.673882","title":"Enduring Autism-like Phenotypes and Deregulated Hypothalamic Prosocial Peptides After Early-Life Exposure to Indoor Flame Retardants in Male C57BL/6 Mice","date":"2025-09-07","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.02.673882","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.17.643720","title":"PACAP Signaling Network in the Nucleus Accumbens Core Regulates Reinstatement Behavior in Rat","date":"2025-03-17","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.17.643720","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":34911,"output_tokens":7108,"usd":0.105677,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16423,"output_tokens":5674,"usd":0.111982,"stage2_stop_reason":"end_turn"},"total_usd":0.217659,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"Crystal structure of the PAC1R extracellular domain (ECD) at 1.9 Å resolution reveals it adopts the same consensus fold as other Class B GPCRs. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor confirm that PAC1R uses this conserved ECD fold for PACAP binding, unifying the hormone-recognition mechanism for this receptor family.\",\n      \"method\": \"X-ray crystallography (1.9 Å), alanine-scan peptide binding assays, cell-based cAMP assays, mutant receptor functional assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure combined with mutagenesis and functional cell-based assays in a single rigorous study\",\n      \"pmids\": [\"21625560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structures of VIP-bound, Gs-coupled PAC1R short variant (PAC1sR) and null variant (PAC1nR) reveal that the alternatively spliced null loop in PAC1nR transiently engages residues in extracellular loop 2 (ECL2) and the N-terminal helix of the ECD, resulting in differential engagement of VIP with these domains and the top of TM2/ECL1. This structural difference underlies the selectively enhanced VIP (but not PACAP) functional activity across multiple signaling endpoints at PAC1sR compared to PAC1nR. MD simulations further predict differential Gs protein interactions with the two variants when VIP-bound, with greater allosteric influence of Gs on VIP affinity at PAC1sR.\",\n      \"method\": \"Cryo-EM structure determination, molecular dynamics (MD) simulations, pharmacological signaling assays across multiple functional endpoints\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structures of both splice variants with MD simulations and comprehensive pharmacological profiling in one rigorous study\",\n      \"pmids\": [\"41264251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PAC1R isoform expression determines second-messenger coupling and mitogenic outcome: sympathetic neuroblasts expressing the 'hop' receptor isoform show PACAP-induced increases in both cAMP and PI (phosphoinositide) signaling and are mitogenically stimulated, whereas cerebral cortical precursors expressing primarily the 'short' isoform show only cAMP increases and are mitogenically inhibited. Thus, opposing mitogenic effects of PACAP correlate with differential receptor isoform expression and distinct intracellular signaling.\",\n      \"method\": \"RT-PCR isoform identification, cAMP and PI second-messenger assays, cell proliferation assays in primary neuronal cultures\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (RT-PCR, second-messenger assays, proliferation assays) in primary cultures, single lab\",\n      \"pmids\": [\"9753193\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"A novel alternatively spliced exon (exon 3a, encoding 24 amino acids) inserted into the extracellular domain of PAC1R during spermatogenesis generates PAC1R(3a). In HEK293 cells stably expressing PAC1R(3a), ligand binding affinity for PACAP-38 is increased 6-fold and coupling to both cAMP (Gs) and inositol phosphate (Gq) signaling pathways is altered relative to wild-type PAC1R. PAC1R(3a) mRNA is preferentially expressed in seminiferous tubules, round spermatids, and Sertoli cells.\",\n      \"method\": \"Cloning and sequencing of novel splice variant, stable transfection of HEK293 cells, radioligand binding assays, cAMP and inositol phosphate signaling assays, RT-PCR expression analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with stably transfected cells, binding assays, and two second-messenger pathway assays, rigorous controls, single lab\",\n      \"pmids\": [\"11278585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PACAP acts via selective high-affinity PAC1Rs to enhance nicotinic synaptic transmission at parasympathetic ciliary ganglion neurons by increasing spontaneous EPSC frequency and amplitude. The mechanism requires adenylate cyclase (AC)/cAMP/PKA signaling (not PLC), and acts presynaptically to increase vesicular ACh release (quantal content), not postsynaptic nAChR function. Nitric oxide (NO) signaling downstream of AC is critical: PACAP-induced increases in EPSC frequency, amplitude, and quantal content are mimicked by NO donor and abolished by NOS inhibition.\",\n      \"method\": \"Electrophysiology (whole-cell patch clamp, spontaneous and evoked EPSC recordings), pharmacological inhibitors of AC, PKA, PLC, NOS; 8-Bromo-cAMP mimicry; quantal content analysis\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal electrophysiology and pharmacology approaches, rigorous presynaptic mechanism dissection, single lab\",\n      \"pmids\": [\"19958833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Estradiol (E2) regulates ADCYAP1R1 expression through estrogen receptor alpha (ERα) binding to an estrogen response element (ERE) in the ADCYAP1R1 gene that contains the PTSD-risk SNP rs2267735. In vitro, E2/ERα binds less efficiently to the ERE containing the risk C allele. In vivo in mice, fear conditioning and E2 additively increase ADCYAP1R1 expression. In women with low serum E2, the CC genotype associates with lower ADCYAP1R1 expression.\",\n      \"method\": \"In vitro ERE binding assays (E2/ERα), cell culture E2 treatment with ADCYAP1R1 expression measurement, mouse fear conditioning with E2 treatment, human genotype-expression correlation\",\n      \"journal\": \"Translational psychiatry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (in vitro binding, cell culture, mouse in vivo, human expression), single lab\",\n      \"pmids\": [\"27959335\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The tumor suppressor p53 and zinc finger protein Zac induce expression of the PAC1-R gene, establishing a transcriptional regulatory link between apoptosis/cell-cycle arrest pathways and PAC1-R expression.\",\n      \"method\": \"Gene reporter assays, overexpression of p53 and Zac in cell culture with PAC1-R gene expression readout\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, gene expression readout only, no direct mechanistic dissection of promoter binding\",\n      \"pmids\": [\"10366751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PAC1R stimulates prolactin promoter activity and mRNA synthesis in pituitary GH3 cells through Gs/cAMP and ERK/PKC signaling pathways; transfection of PAC1R expression vector dramatically potentiates the prolactin promoter response to PACAP proportional to receptor amount, and PAC1R itself is induced by PACAP (auto-upregulation).\",\n      \"method\": \"Transfection of PAC1R expression vector into GH3 cells, prolactin promoter-reporter assay, ERK phosphorylation assay (Western blot), RT-PCR for prolactin and GH mRNA\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter reporter assay, Western blot ERK activation, and mRNA measurement; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"21539889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In human adipose-derived stem cells (hADSCs), PAC1R activation by the selective agonist maxadilan promotes cell proliferation, migration, and anti-apoptotic effects, and enhances neural differentiation. Two distinct PAC1R-coupled pathways are identified: ligand-dependent PKA signaling enhances viability and neural differentiation (blocked by H-89); and dimer-dependent Wnt/β-catenin signaling also promotes viability (blocked by XAV939).\",\n      \"method\": \"Cell Counting Kit-8 proliferation assay, cell cycle analysis, wound-healing migration assay, Annexin V apoptosis assay, mitochondrial membrane potential assay, Western blot, qPCR, whole-cell patch clamp, pharmacological inhibition of PKA and Wnt pathways\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal assays identifying two mechanistically distinct PAC1R signaling branches, single lab\",\n      \"pmids\": [\"26798992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PAC1R undergoes nuclear translocation when treated with positive allosteric modulators PACAP(28-38) or TAT peptide. Nuclear PAC1R binds the promoter regions of PAC1R and PACAP genes (demonstrated by ChIP-PCR), upregulating their expression. Nuclear translocation requires palmitoylation at Cys25 (abolished by mutation C25A or palmitoylation inhibitor 2-bromopalmitate). ChIP-seq analysis identifies transcription factors SP1, Zic2, GATA1, REST, and YY1 as potentially recruited by nuclear PAC1R.\",\n      \"method\": \"Confocal fluorescence microscopy (nuclear translocation), ChIP-PCR (promoter binding), site-directed mutagenesis (C25A), palmitoylation inhibitor treatment, ChIP-sequencing, promoter-reporter assays\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-PCR promoter binding and mutagenesis with fluorescence imaging, multiple methods, single lab\",\n      \"pmids\": [\"35593471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Blue light and hydrogen peroxide (H2O2) trigger nuclear translocation of PAC1R (observed in PAC1R-CHO cells expressing PAC1R-eGFP and in retinal ganglion cells RGC-5 with native PAC1R). Nuclear translocation is blocked by ROS scavenger NAC and palmitoylation inhibitor 2-bromopalmitate, and is markedly reduced by the C25A mutation in PAC1R. Nuclear translocation positively correlates with upregulation of PAC1R expression and promoter activity.\",\n      \"method\": \"Fluorescence microscopy (nuclear translocation of PAC1R-eGFP), pharmacological inhibition (NAC, 2-bromopalmitate), site-directed mutagenesis (C25A), Western blot, promoter-reporter assay\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (imaging, pharmacology, mutagenesis, reporter), single lab, corroborates prior findings\",\n      \"pmids\": [\"33647387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PAC1R positive allosteric modulator SPAM1 triggers nuclear translocation of PAC1R. The nuclear 35 kDa (but not 15 kDa) fragment of PAC1R interacts with SP1 to upregulate Huntingtin (Htt) expression, which then attenuates NRSF (neuron-restrictive silencer factor) binding to NRSE, leading to upregulated expression of NRSF-target neuropeptides (PACAP, BDNF, TH, SYN1).\",\n      \"method\": \"Fluorescence microscopy (nuclear translocation), co-immunoprecipitation (nuclear PAC1R-SP1 interaction), Western blot, in vivo D-gal aging mouse model, cell viability assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for nuclear PAC1R-SP1 interaction combined with in vivo mouse model and imaging, single lab\",\n      \"pmids\": [\"36555637\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PACAP auto-regulates its own expression through PAC1R activation in human neuroblastoma NB-1 cells. PAC1R-mediated PACAP gene induction requires parallel, independent activation of PKA, novel (but not conventional) PKC isoforms, and ERK. EGR1 induction by PACAP requires PKC and ERK (not PKA), but siRNA knockdown of EGR1 did not affect PACAP auto-regulation, excluding EGR1 from the PACAP auto-regulatory pathway.\",\n      \"method\": \"Pharmacological kinase inhibitors (PKA, PKC, MEK), RT-PCR for PACAP mRNA, siRNA knockdown of EGR1, PAC1R-selective agonist Maxadilan\",\n      \"journal\": \"Neuropeptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic pharmacological pathway dissection with siRNA validation, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"27745706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The human ADCYAP1R1 gene promoter (cloned 2,526 bp region from -2500 to +26) shows a hormetic response to H2O2 in SH-SY5Y cells: low concentrations (<10 nM) activate the promoter in a dose-dependent manner via SP1 binding sites (blocked by mithramycin A and SP1 site deletion mutants), whereas higher concentrations (>10 nM) inhibit promoter activity.\",\n      \"method\": \"Promoter cloning, reporter assay, SP1 inhibitor (mithramycin A), deletion mutagenesis of SP1 binding sites\",\n      \"journal\": \"Journal of food biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assay combined with pharmacological inhibition and deletion mutagenesis, single lab\",\n      \"pmids\": [\"31353704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Doxycycline and minocycline function as positive allosteric modulators (PAMs) of PAC1-R by binding the N-terminal extracellular domain (PAC1-EC1). Isothermal titration calorimetry confirms direct binding. Site-directed mutagenesis identifies ASN60 as the most important residue for PAM binding, with ASP116 playing a sensitive marginal role. Tetracycline/tigecycline derivatives show lower affinity and lack PAM activity.\",\n      \"method\": \"Computational molecular docking, isothermal titration calorimetry, cAMP assay with PACAP27, site-directed mutagenesis of PAC1-EC1\",\n      \"journal\": \"Acta biochimica et biophysica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding assay (ITC), mutagenesis, and functional cAMP assay, single lab\",\n      \"pmids\": [\"31056648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A co-crystal structure of a PAC1R antagonist peptide (des(24-42) Maxadilan analog 18) bound to the PAC1R extracellular domain was determined, representing the first structural data for a PAC1R antagonist. SAR studies identified peptide modifications that increased PAC1R inhibitory activity to the pM IC90 range. Analog 17 and 18 showed in vivo efficacy in a rat Maxadilan-induced blood flow pharmacodynamic model at 0.3 mg/kg subcutaneous dosing.\",\n      \"method\": \"X-ray co-crystallography of PAC1R ECD with antagonist peptide, in vitro PAC1R binding/inhibition assays, in vivo rat pharmacodynamic model\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure of receptor-antagonist complex combined with in vitro SAR and in vivo pharmacodynamic validation\",\n      \"pmids\": [\"33715378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Peptide OM-LV20 protects astrocytes from oxidative stress via a PAC1R/JNK/TPH1 signaling axis: OM-LV20 increases TPH1 expression in astrocytes through PAC1R and the JNK signaling pathway, protecting against H2O2-induced decreases in cell viability and catalase levels.\",\n      \"method\": \"Primary astrocyte culture, quantitative RT-PCR, double immunofluorescence, lentiviral infection (loss-of-function), cell viability analysis, Western blotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lentiviral knockdown plus pharmacological pathway inhibition with multiple readouts, single lab\",\n      \"pmids\": [\"36037970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PAC1-R is expressed in reactive astrocytes but not resting astrocytes. Immunostaining of transgenic mice (GFAP-EGFP) following cortical stab wound revealed PAC1-R immunoreactivity specifically in EGFP/GFAP-positive reactive astrocytes (and vimentin-positive cells) around the wound at 5 days post-surgery, but not in the contralateral hemisphere.\",\n      \"method\": \"Transgenic GFAP-EGFP mouse model, cortical stab wound surgery, immunohistochemistry for PAC1-R, GFAP, vimentin, CD11b, NeuN\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vivo localization using transgenic reporter mice with injury model, single lab\",\n      \"pmids\": [\"12824050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PAC1-R heterozygous (-/+) mice fail to use geomagnetic orientation or fear signals to guide memory retrieval (no errors when maze is rotated 180° relative to geomagnetic field or upon fear signal exposure), whereas wild-type mice do make errors in these conditions. This implicates PAC1-R in gating passage of geomagnetic and fear sensory signals for memory retrieval.\",\n      \"method\": \"Behavioral testing in PAC1-R heterozygous transgenic mutant mice vs. wild-type littermates in multiple-maze memory tasks with rotation and fear signal challenges\",\n      \"journal\": \"Journal of molecular neuroscience : MN\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — behavioral phenotype in heterozygous KO mice, single lab, no molecular mechanism dissection\",\n      \"pmids\": [\"18781286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PACAP/PAC1R signaling in the locus coeruleus (LC) from anterior insular cortex (aIC) PACAP neurons regulates anxiety-related behaviors. PAC1R mRNA is highly expressed in LC noradrenergic neurons. Targeted deletion of PAC1R from noradrenergic cells produces a hyperactive/low anxiety phenotype. Selective activation of PACAP neurons in the aIC increases anxiety. Retrograde tracing confirmed aIC PACAP neurons project to the LC. shRNA knockdown of PACAP in the aIC decreases anxiety.\",\n      \"method\": \"Histochemistry (PAC1R mRNA in situ hybridization), conditional cell-type-specific PAC1R deletion, retrograde neurocircuit tracing, chemogenetic activation of aIC PACAP neurons, shRNA knockdown of PACAP in aIC, open field and elevated-plus maze behavioral tests\",\n      \"journal\": \"Neurochemistry international\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO, circuit tracing, chemogenetics, and shRNA knockdown in multiple orthogonal experiments establishing a defined aIC→LC→anxiety circuit requiring PAC1R\",\n      \"pmids\": [\"39396708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In a rat nucleus accumbens core (NAcc) model, endogenous PAC1R (ADCYAP1R1) is expressed in NAcc and PACAP mRNA is present in medial prefrontal cortical projections to NAcc. Intra-NAcc PACAP infusion blocked cocaine-primed reinstatement and blocked reinstatement driven by D1 receptor agonist co-infusion, but did not block reinstatement driven by D2 receptor agonist, demonstrating PAC1R signaling in NAcc selectively interferes with D1-mediated (not D2-mediated) reinstatement mechanisms.\",\n      \"method\": \"In situ hybridization/immunofluorescence for PAC1R expression, retrograde tracing for mPFC→NAcc PACAP projections, intra-NAcc pharmacological infusion, cocaine-primed and receptor agonist-driven reinstatement behavioral assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, pharmacological infusion with behavioral readout only, no molecular mechanism dissection beyond receptor selectivity\",\n      \"pmids\": [\"bio_10.1101_2025.03.17.643720\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Celastrol promotes cAMP production after spinal cord injury (SCI) by upregulating the VIP-ADCYAP1R1-GNAS signaling pathway, thereby inhibiting JNK phosphorylation and preventing apoptosis in injured spinal cord tissue.\",\n      \"method\": \"In vivo SCI mouse model, cAMP measurement, Western blot (JNK phosphorylation, apoptotic markers), gene expression analysis\",\n      \"journal\": \"Biomedicine & pharmacotherapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — in vivo pharmacology with pathway readouts, but ADCYAP1R1-specific mechanistic evidence is indirect (upregulation of pathway), single lab\",\n      \"pmids\": [\"37531781\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PACAP/PAC1R signaling promotes ILC2 (group 2 innate lymphoid cell) proliferation and IL-5/IL-13 cytokine production via the ERK pathway. In OVA-induced allergic rhinitis mice, PAC1R knockdown or antagonist (PA-8) markedly suppressed ILC2 proliferation and cytokine production. ERK inhibitor PD98059 reversed PAC1R-mediated ILC2 activation, and PAC1R knockdown decreased p-ERK and ILC2 numbers.\",\n      \"method\": \"OVA-induced AR mouse model, PAC1R siRNA knockdown, PAC1R antagonist PA-8, ERK inhibitor PD98059, Western blot (p-ERK), flow cytometry (ILC2s, IL-5/IL-13)\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PAC1R knockdown and antagonist combined with ERK inhibition and in vivo model, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"40713003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PAC1R is expressed on tuberoinfundibular dopamine (TIDA) neurons in the arcuate nucleus of the ewe hypothalamus; dual immunofluorescence shows 21.2% of arcuate tyrosine hydroxylase-positive dopaminergic neurons co-express PAC1R, while other hypothalamic dopaminergic cell groups show little (<3%) co-localization. This positions PAC1R to mediate PACAP's inhibition of prolactin secretion via direct action on TIDA neurons.\",\n      \"method\": \"RT-PCR, in situ hybridization, immunohistochemistry, dual immunofluorescence for PAC1R and tyrosine hydroxylase\",\n      \"journal\": \"Journal of neuroendocrinology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — localization study establishing cell-type specificity; functional consequence is inferred from prior in vivo studies, not directly tested here\",\n      \"pmids\": [\"15869565\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ADCYAP1R1 (PAC1R) is a class B G protein-coupled receptor that binds PACAP (and, with splice variant-dependent selectivity, VIP) through its extracellular domain; alternatively spliced isoforms of the receptor (short, null, hop, hip, 3a, etc.) differ in PACAP/VIP affinity and in coupling to Gs/cAMP versus Gq/inositol phosphate pathways, with structural basis established by cryo-EM and X-ray crystallography; upon activation, PAC1R signals canonically via cAMP/PKA and PKC/ERK to regulate synaptic vesicle release, neuronal proliferation, prolactin synthesis, and ILC2 activation, and non-canonically undergoes palmitoylation-dependent nuclear translocation where it binds gene promoters (including its own and PACAP) to upregulate their expression; estrogen receptor alpha regulates ADCYAP1R1 transcription via an ERE overlapping the PTSD-risk SNP rs2267735; and in specific circuits (e.g., anterior insular cortex → locus coeruleus noradrenergic neurons; nucleus accumbens), PAC1R signaling is required for anxiety-related behaviors and modulates dopamine-dependent reinstatement of cocaine seeking.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ADCYAP1R1 (PAC1R) is a class B G protein-coupled receptor that recognizes the neuropeptide PACAP through a conserved extracellular-domain fold and transduces this signal into diverse neuronal, endocrine, and immune outcomes [#0]. Receptor function is diversified by alternative splicing: variants differ in ligand affinity and in coupling to Gs/cAMP versus Gq/inositol phosphate pathways, and structural work on the VIP-bound short and null variants shows that the spliced null loop engages ECL2 and the ECD N-terminal helix to set variant-selective VIP responsiveness [#1, #3]. Isoform identity also determines biological output—'hop'-expressing neuroblasts couple to both cAMP and phosphoinositide signaling and are mitogenically stimulated, whereas 'short'-isoform cortical precursors raise only cAMP and are mitogenically inhibited [#2]. Downstream, PAC1R signals canonically via AC/cAMP/PKA and PKC/ERK: it enhances presynaptic vesicular acetylcholine release through an AC/cAMP/PKA/nitric oxide cascade [#4], drives prolactin promoter activity and mRNA synthesis in pituitary cells with self-induction of the receptor [#7], auto-regulates PACAP expression through parallel PKA, novel-PKC, and ERK activation [#12], and promotes ILC2 proliferation and IL-5/IL-13 production via ERK [#22]. Non-canonically, PAC1R undergoes palmitoylation-dependent (Cys25) nuclear translocation triggered by allosteric modulators or by oxidative/blue-light stress, where it binds the promoters of its own and the PACAP gene to upregulate them and interacts with SP1 to drive a Huntingtin-NRSF neuropeptide program [#9, #10, #11]. Its transcription is controlled by estrogen receptor alpha acting at an ERE that contains the PTSD-risk SNP rs2267735, providing a hormonal link to fear-related expression [#5]. At the circuit level, PAC1R in locus coeruleus noradrenergic neurons is required for anxiety-related behavior within an anterior-insula→LC pathway [#19].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established that PAC1R splice-isoform identity, not ligand alone, dictates second-messenger coupling and opposing mitogenic outcomes, explaining context-dependent PACAP biology.\",\n      \"evidence\": \"RT-PCR isoform typing with cAMP/PI assays and proliferation assays in primary neuronal cultures\",\n      \"pmids\": [\"9753193\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of isoform-specific coupling not structurally resolved\", \"Single lab, primary culture only\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Linked PAC1R transcription to apoptosis/cell-cycle regulators by showing p53 and Zac induce the gene, but only at the expression-readout level.\",\n      \"evidence\": \"Reporter assays and p53/Zac overexpression in cell culture\",\n      \"pmids\": [\"10366751\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct promoter-binding dissection\", \"Single lab, expression readout only\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrated that tissue-specific splicing (exon 3a in spermatogenic cells) reshapes PACAP affinity and Gs/Gq coupling, extending the splicing-diversification principle to the reproductive tract.\",\n      \"evidence\": \"Cloning of PAC1R(3a), stable HEK293 expression, radioligand binding, cAMP and IP assays, RT-PCR\",\n      \"pmids\": [\"11278585\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role in spermatogenesis not tested in vivo\", \"Structural basis of altered coupling not determined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Positioned PAC1R to inhibit prolactin secretion by localizing it to a subset of tuberoinfundibular dopamine neurons in the arcuate nucleus.\",\n      \"evidence\": \"RT-PCR, in situ hybridization, dual immunofluorescence for PAC1R and tyrosine hydroxylase in ewe hypothalamus\",\n      \"pmids\": [\"15869565\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Functional consequence inferred, not directly tested\", \"Restricted to one species and cell group\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined a presynaptic AC/cAMP/PKA/NO mechanism by which PAC1R enhances cholinergic synaptic transmission, distinguishing it from postsynaptic and PLC routes.\",\n      \"evidence\": \"Whole-cell patch-clamp EPSC recordings with AC/PKA/PLC/NOS inhibitors and cAMP mimicry in ciliary ganglion neurons\",\n      \"pmids\": [\"19958833\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NO source and downstream presynaptic targets not identified\", \"Receptor isoform not specified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the conserved class B ECD fold of PAC1R and confirmed it as the PACAP-recognition module, unifying hormone binding across the family.\",\n      \"evidence\": \"1.9 Å X-ray crystallography with alanine-scan peptide binding and cell-based cAMP/mutant assays\",\n      \"pmids\": [\"21625560\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length receptor and transmembrane activation not captured\", \"Isoform-specific ECD differences not addressed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed PAC1R drives prolactin gene expression via Gs/cAMP and ERK/PKC and is itself upregulated by PACAP, introducing receptor auto-induction.\",\n      \"evidence\": \"PAC1R transfection, prolactin promoter-reporter, ERK Western blot, and mRNA assays in GH3 pituitary cells\",\n      \"pmids\": [\"21539889\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcription factors mediating prolactin induction not identified\", \"Single cell line\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established hormonal control of PAC1R transcription through ERα at an ERE overlapping the PTSD-risk SNP rs2267735, mechanistically connecting estrogen, the risk allele, and receptor expression.\",\n      \"evidence\": \"In vitro ERE binding, cell-culture E2 treatment, mouse fear conditioning, and human genotype-expression correlation\",\n      \"pmids\": [\"27959335\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal link to PTSD behavior not demonstrated\", \"Allele-specific binding effect size modest\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Dissected two parallel PAC1R signaling branches—PKA and dimer-dependent Wnt/β-catenin—governing stem-cell viability and neural differentiation.\",\n      \"evidence\": \"Proliferation, migration, apoptosis, patch-clamp assays with PKA (H-89) and Wnt (XAV939) inhibition in human ADSCs\",\n      \"pmids\": [\"26798992\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of Wnt engagement by receptor dimers unresolved\", \"Single lab, ex vivo cells\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the PAC1R-driven PACAP auto-regulatory circuit requiring parallel PKA, novel PKC, and ERK, and ruled out EGR1 as the mediator.\",\n      \"evidence\": \"Kinase inhibitors, RT-PCR, and EGR1 siRNA in NB-1 neuroblastoma cells with PAC1R agonist maxadilan\",\n      \"pmids\": [\"27745706\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcription factor mediating PACAP induction unidentified\", \"Single cell line\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified doxycycline/minocycline as direct ECD-binding positive allosteric modulators of PAC1R, mapping key residues ASN60 and ASP116.\",\n      \"evidence\": \"Molecular docking, ITC binding, cAMP assay, and ECD site-directed mutagenesis\",\n      \"pmids\": [\"31056648\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of PAM activity untested\", \"Effect on Gq coupling not examined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed the ADCYAP1R1 promoter has a hormetic, SP1-dependent response to H2O2, linking oxidative state to receptor transcription.\",\n      \"evidence\": \"Promoter cloning, reporter assay, SP1 inhibitor mithramycin A, and SP1-site deletion in SH-SY5Y cells\",\n      \"pmids\": [\"31353704\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo oxidative regulation not tested\", \"Mechanism of biphasic switch unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Determined the first PAC1R ECD–antagonist co-structure and developed picomolar antagonist peptides with in vivo efficacy.\",\n      \"evidence\": \"X-ray co-crystallography of ECD-antagonist, SAR binding/inhibition, and rat maxadilan-induced blood-flow model\",\n      \"pmids\": [\"33715378\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Antagonism in CNS circuits not assessed\", \"Full-receptor binding mode not captured\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed oxidative and blue-light stress trigger palmitoylation-dependent (Cys25) nuclear translocation of PAC1R coupled to its own promoter upregulation, extending the non-canonical nuclear mechanism to stress signaling.\",\n      \"evidence\": \"PAC1R-eGFP imaging, NAC/2-bromopalmitate inhibition, C25A mutagenesis, Western blot, reporter assay in CHO and RGC-5 cells\",\n      \"pmids\": [\"33647387\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How a membrane GPCR reaches the nucleus mechanistically unresolved\", \"Physiological trigger in vivo unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established that allosterically induced nuclear PAC1R binds the PAC1R and PACAP promoters and recruits transcription factors, defining a receptor-as-transcriptional-regulator mode requiring Cys25 palmitoylation.\",\n      \"evidence\": \"Confocal imaging, ChIP-PCR, ChIP-seq, C25A mutagenesis, and promoter-reporter assays\",\n      \"pmids\": [\"35593471\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct DNA binding vs. cofactor recruitment not separated\", \"Functional importance relative to membrane signaling unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected the nuclear PAC1R fragment to a transcriptional program via SP1 interaction, Huntingtin upregulation, and NRSF derepression of neuropeptide genes.\",\n      \"evidence\": \"Nuclear translocation imaging, PAC1R-SP1 Co-IP, Western blot, and D-gal aging mouse model\",\n      \"pmids\": [\"36555637\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP for the PAC1R-SP1 interaction without reciprocal validation\", \"Origin of the 35 kDa nuclear fragment not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined a PAC1R/JNK/TPH1 axis by which a PACAP-family peptide protects astrocytes from oxidative stress.\",\n      \"evidence\": \"Primary astrocyte culture, qRT-PCR, immunofluorescence, lentiviral knockdown, viability and Western blot assays\",\n      \"pmids\": [\"36037970\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Receptor-proximal coupling to JNK not mapped\", \"In vivo relevance untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated that PAC1R in locus coeruleus noradrenergic neurons is required for anxiety behavior within a defined anterior-insula→LC PACAP circuit.\",\n      \"evidence\": \"In situ hybridization, conditional cell-type PAC1R deletion, retrograde tracing, chemogenetics, aIC PACAP shRNA, and behavioral tests\",\n      \"pmids\": [\"39396708\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream signaling in LC neurons not dissected\", \"Sex-specific and hormonal modulation not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the structural basis of splice-variant-selective VIP responsiveness, showing the null loop reshapes ECD/ECL2 engagement and Gs allostery.\",\n      \"evidence\": \"Cryo-EM of VIP-bound Gs-coupled PAC1sR and PAC1nR, MD simulations, and multi-endpoint pharmacology\",\n      \"pmids\": [\"41264251\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PACAP-bound complexes of the variants not compared\", \"Functional consequence in native neurons untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Indicated PAC1R signaling in the nucleus accumbens selectively blocks D1-mediated, not D2-mediated, cocaine reinstatement.\",\n      \"evidence\": \"PAC1R/PACAP expression mapping, retrograde tracing, and intra-NAcc pharmacological infusion in cocaine reinstatement assays (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.03.17.643720\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint, not peer reviewed\", \"No molecular mechanism beyond receptor selectivity\", \"Single lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How membrane-resident PAC1R is processed and trafficked to the nucleus, and how this transcriptional mode is balanced against canonical Gs/Gq signaling in vivo, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of receptor entry into the nucleus unknown\", \"Relative physiological weight of nuclear vs. membrane signaling unquantified\", \"No in vivo validation of the nuclear transcriptional program\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [9, 11]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 10]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9, 10, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 7, 22]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [9, 11, 13]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [4, 19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ADCYAP1\", \"VIP\", \"SP1\", \"ESR1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}