{"gene":"RAMP1","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":1999,"finding":"RAMP1 coexpression with the calcitonin receptor-like receptor (CRLR/CLR) converts it from an adrenomedullin receptor into a functional CGRP receptor; RAMP1 competes with RAMP2 for CRLR association, and RAMP1 expression inhibits RAMP2-evoked ADM receptor function while generating CGRP binding sites and CGRP-stimulated cAMP responses.","method":"Radioligand binding assays ([125I]hαCGRP, [125I]rADM) and CRE-luciferase cAMP reporter assays in transiently transfected COS-7 and UMR-106 cells with combinations of rCRLR, hRAMP1, and hRAMP2","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with binding and functional (cAMP) assays, multiple receptor combinations tested, replicated across two cell lines","pmids":["10342881"],"is_preprint":false},{"year":2001,"finding":"When expressed alone, RAMP1 is retained intracellularly in the ER and Golgi as a disulfide-linked homodimer; coexpression with CRLR redirects RAMP1 to the cell surface as a 1:1 heterodimer (without intermolecular disulfide bonds) and promotes intramolecular disulfide bond formation within RAMP1. CGRP activation leads to CRLR phosphorylation and dynamin- and β-arrestin-dependent endocytosis of a stable CRLR/RAMP1/β-arrestin ternary complex via clathrin-coated pits.","method":"Subcellular fractionation, co-immunoprecipitation, disulfide bond analysis, CGRP-stimulated internalization assays with dominant-negative dynamin and β-arrestin mutants in transfected cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (biochemical fractionation, co-IP, dominant-negative constructs, trafficking assays) in a single focused study","pmids":["11535606"],"is_preprint":false},{"year":2007,"finding":"CRLR and RAMP1 associate selectively to form heterodimers at the cell surface, as shown by BRET titration; both proteins can also form homodimers. RAMP1 is required for CRLR to engage G proteins and β-arrestin upon CGRP stimulation; a trafficking-deficient RAMP1 mutant still heterodimerizes with CRLR, indicating deficient surface targeting results from altered complex conformation rather than loss of heterodimerization.","method":"Bioluminescence resonance energy transfer (BRET) titration in living cells, radioligand binding, cAMP production assay, mutagenesis","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — BRET in living cells combined with binding and functional assays and mutagenesis, multiple orthogonal methods in one study","pmids":["17503773"],"is_preprint":false},{"year":2006,"finding":"The extracellular domain of RAMP1 adopts a three-helix structure with disulfide bonds at Cys27–Cys82, Cys40–Cys72, and Cys57–Cys104 (determined by site-directed mutagenesis); modeling identified Phe93, Tyr100, and Phe101 as a putative CLR-binding interface and Trp74/Phe92 as potential ligand interaction sites.","method":"Ab initio molecular modeling of RAMP1 extracellular domain, site-directed mutagenesis to map disulfide bonds","journal":"Biophysical journal","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — disulfide bond assignments validated by mutagenesis, but structural model is computational with no crystal structure; single lab","pmids":["16632510"],"is_preprint":false},{"year":2009,"finding":"Alanine mutagenesis of RAMP1 identified Tyr66 and His97 as critical for CLR trafficking to the cell surface (CLR recognition site), while Leu69 and Thr73 in helix 2 contribute to CGRP potency (CGRP recognition site); Met48 in helix 1 had only a modest effect on CLR surface expression.","method":"Alanine scanning mutagenesis of RAMP1 coexpressed with CLR in COS-7 cells; cell-surface ELISA and cAMP assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic alanine mutagenesis with functional readouts (trafficking and cAMP), multiple residues tested, single lab but comprehensive","pmids":["19072332"],"is_preprint":false},{"year":2009,"finding":"Non-peptide CGRP receptor antagonists BIBN4096BS and MK-0974 interact with the CGRP receptor via Met42 of CLR and Trp74 of RAMP1; Trp74Lys mutation reduces BIBN4096BS affinity >300-fold and similarly reduces MK-0974 affinity, and Met42Ala reduces BIBN4096BS affinity 48-fold and MK-0974 affinity ~900-fold, placing the antagonist binding site at the CLR/RAMP1 extracellular domain interface.","method":"Site-directed mutagenesis of CLR residues 23–63 and RAMP1 Trp74, radioligand binding and functional assays in transfected cells","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis with quantitative binding affinities for two independent antagonists, confirms and extends prior Trp74 finding, single lab but rigorous","pmids":["19914210"],"is_preprint":false},{"year":2010,"finding":"Tryptophan-84 of RAMP1 is critical for both high-affinity non-peptide antagonist (telcagepant, BIBN4096BS) binding and agonist (CGRP and CGRP8-37) potency; Arg67 is important for telcagepant but not compound 3 (BIBN4096BS analog) binding, indicating distinct contact points for different antagonists within the RAMP1 N-terminal domain.","method":"Alanine replacement mutagenesis of RAMP1 residues, radioligand binding and cAMP assays in cells coexpressing CLR","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis with quantitative binding and functional readouts for both agonists and antagonists, single lab","pmids":["20188075"],"is_preprint":false},{"year":2010,"finding":"RAMP1-RAMP3 chimera analysis showed that helix 1 of RAMP1 and residues 62–69 in helix 2 are required for CLR trafficking; residues 86–89 of RAMP1 at the helix 2–3 junction are important for hαCGRP and adrenomedullin potency but not hβCGRP potency, suggesting a peptide-binding role; residues 90–94 have dual roles in CLR recognition and CGRP binding.","method":"RAMP1–RAMP3 chimera expression with CLR in COS-7 cells; cell-surface ELISA and cAMP assays","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — chimeric protein approach with multiple segment exchanges and functional readouts, single lab","pmids":["20017504"],"is_preprint":false},{"year":2011,"finding":"Position 74 of RAMP1 (Trp74) vs RAMP3 (Glu74) differentially affects adrenomedullin potency at the respective CLR complexes; geometry and charge at this residue contribute to differential AM pharmacology at AM2 and CGRP receptors, while CGRP and AM2/intermedin potency are largely unaffected by substitutions at this position in RAMP1.","method":"Site-directed mutagenesis of RAMP1 and RAMP3 at position 74 with multiple amino acid substitutions, coexpressed with CLR in COS-7 cells; cAMP assays and ELISA for surface expression","journal":"Peptides","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis across both RAMPs with multiple substitutions and functional readouts, single lab","pmids":["21402116"],"is_preprint":false},{"year":2018,"finding":"Photoaffinity cross-linking using unnatural amino acid mutagenesis in CLR ECL2 identified key contacts for CGRP within the transmembrane bundle: I284 in ECL2 and L291 at the top of TM5 are primary contacts; M223 in TM3 and F349 in TM6 are minor contacts. Molecular modeling indicates CGRP sits at the top of the TM bundle with Thr6 contacting H295 (important for activation) and Leu12/Ala13 contacting I284.","method":"Genetic code expansion and unnatural amino acid (p-azido-Phe) mutagenesis in CLR ECL2, photoaffinity cross-linking with fluorescein-CGRP analog, functional validation with [Leu16Ala]-CGRP analogue","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — photoaffinity cross-linking plus unnatural amino acid mutagenesis plus functional peptide analogue validation; multiple orthogonal methods in one study","pmids":["30004692"],"is_preprint":false},{"year":2013,"finding":"NKX3.1 directly represses RAMP1 expression in prostate cells; multiple NKX3.1 binding sites were identified in the RAMP1 locus by ChIP-seq. RAMP1 knockdown reduces prostate cancer cell proliferation and tumorigenicity, associated with reduced MEK1 expression and decreased phospho-ERK1/2 levels, placing RAMP1 upstream of the MAPK signaling pathway in prostate cancer.","method":"ChIP-seq for NKX3.1 binding at RAMP1 locus, Nkx3.1 knockout mice analysis, shRNA knockdown of RAMP1 in prostate cancer cell lines with in vitro and in vivo tumorigenicity assays, gene expression profiling","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq plus KO mice plus functional knockdown with pathway readouts, multiple orthogonal methods, single lab","pmids":["23867798"],"is_preprint":false},{"year":2014,"finding":"RAMP1 deficiency in mice reduces antigen-induced airway hyperresponsiveness and lung inflammation in an ovalbumin asthma model; loss of CLR from smooth muscle cells specifically does NOT alter airway resistance, indicating CGRP/RAMP1 signaling drives asthma pathology via endothelial and inflammatory cells rather than smooth muscle cells.","method":"RAMP1 knockout mice (exon 3 excision) and smooth-muscle-specific CLR heterozygous mice subjected to ovalbumin sensitization/challenge; airway resistance (methacholine challenge) and inflammatory cell measurements","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined phenotypic readout, cell-type-specific CLR deletion identifies non-smooth muscle mechanism; single lab","pmids":["25010197"],"is_preprint":false},{"year":2012,"finding":"RAMP1 deficiency improves bacterial clearance in the early phase of polymicrobial septic peritonitis by reducing IL-10-mediated immunosuppression; CGRP directly increases IL-10 production in macrophages via RAMP1 signaling in response to LPS, and RAMP1 deficiency increases neutrophil recruitment and defensin-α1 release.","method":"RAMP1 KO mice in cecal ligation and puncture model; cytokine measurements in plasma and peritoneal lavage, in vitro CGRP stimulation of macrophages, neutrophil and defensin assays","journal":"Immunobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus in vitro macrophage CGRP stimulation with cytokine readouts; single lab, two complementary methods","pmids":["22656887"],"is_preprint":false},{"year":2019,"finding":"RAMP1 mediates amylin's anorectic signaling: RAMP1/3 double KO mice are insensitive to both amylin and salmon calcitonin (sCT) food intake suppression; RAMP1 single KO mice respond to amylin but not sCT, while RAMP3 KO mice respond to sCT but not amylin. RAMP1 is also required for normal fat utilization and meal pattern regulation.","method":"RAMP1, RAMP3, and RAMP1/3 global KO mice; acute intraperitoneal amylin/sCT injection food intake assays, metabolic measurements, meal pattern analysis","journal":"Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined pharmacological and feeding phenotype, multiple KO lines compared; single lab","pmids":["31881259"],"is_preprint":false},{"year":2019,"finding":"RAMP1 signaling in CD4+ T cells promotes inflammation-associated lymphangiogenesis by driving VEGF-C and VEGF-D expression; RAMP1-deficient mice show suppressed LPS-induced lymphangiogenesis, reduced CD4+ cell numbers, and a shift toward pro-inflammatory (M1) macrophage phenotype with increased CD11b+ cells.","method":"RAMP1 KO mice with LPS-induced peritoneal inflammation model; FITC-dextran lymphatic flow assay, immunostaining for CD4+/CD11b+ cells, VEGF-C/D expression analysis, CD4+ cell depletion experiments","journal":"Laboratory investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus cell depletion plus functional lymphatic flow assay; single lab","pmids":["31911634"],"is_preprint":false},{"year":2017,"finding":"RAMP1 signaling promotes lymphangiogenesis during secondary lymphedema by supporting VEGF-C/VEGFR3 expression and reparative macrophage polarization; RAMP1-deficient mice show sustained tail lymphedema, suppressed lymphangiogenesis, dilated/dysfunctional lymphatics, and elevated M1 macrophage gene expression (TNF-α, IL-1β) with decreased M2 markers (IL-10).","method":"RAMP1 KO mice in surgical tail lymphedema model; tail diameter measurements, VEGF-C/VEGFR3 expression, macrophage marker analysis","journal":"The Journal of surgical research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with functional lymphatic readouts and macrophage phenotyping; single lab","pmids":["29078910"],"is_preprint":false},{"year":2020,"finding":"RAMP1 deficiency severely impairs liver regeneration after partial hepatectomy and carbon tetrachloride injury; mechanistically, RAMP1 loss leads to hyperphosphorylation of YAP at Ser127 and Ser397, reducing YAP/TAZ protein levels and expression of YAP-controlled cell cycle regulators. In vitro, CGRP treatment of liver slice cultures and primary human hepatocytes increases YAP protein and reduces YAP phosphorylation.","method":"RAMP1 KO mice subjected to 70% partial hepatectomy and CCl4 model; liver mass recovery, hepatocyte proliferation (Ki67), YAP/TAZ protein levels and phosphorylation (Western blot), ex vivo CGRP treatment of liver slices and primary hepatocytes","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO in two injury models plus in vitro mechanistic validation in multiple systems (mouse and human tissue); multiple orthogonal methods","pmids":["32329113"],"is_preprint":false},{"year":2022,"finding":"RAMP1 is C-mannosylated at Trp56 (identified by mass spectrometry); C-mannosylation enhances RAMP1 protein stability and promotes cell migration activity but does not affect RAMP1 plasma membrane trafficking or its function in transporting CRLR to the cell surface.","method":"Mass spectrometry to identify C-mannosylation site, C-mannosylation-deficient RAMP1 mutants, protein stability assays, cell migration assays","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — MS identification of modification site plus functional mutagenesis for stability and migration; single lab, two orthogonal methods","pmids":["35942636"],"is_preprint":false},{"year":2022,"finding":"RAMP1 is expressed on intestinal goblet cells in mice and humans; nociceptor (Nav1.8+CGRP+) signaling via the CGRP-RAMP1 pathway induces rapid goblet cell emptying and mucus secretion. Loss of epithelial Ramp1 increases susceptibility to colitis; CGRP administration protects nociceptor-ablated mice against colitis.","method":"RAMP1 cell-specific KO (epithelial), nociceptor ablation, chemogenetic nociceptor activation, capsaicin treatment, mouse colitis model (DSS), CGRP administration rescue experiments; histology, mucus thickness measurement","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic cell-type-specific KO, nociceptor ablation and chemogenetic activation, pharmacological rescue, multiple complementary approaches in one study","pmids":["36243004"],"is_preprint":false},{"year":2024,"finding":"The CGRP-RAMP1 axis in skin mediates direct neuroimmune communication: commensal colonization upregulates RAMP1 on CD8+ T lymphocytes; CGRP-RAMP1 signaling in commensal-specific T cells constrains Type 17 responses and moderates the activation status of microbiota-reactive lymphocytes, shaping the overall skin epithelial activation state and wound healing responses.","method":"Intravital imaging of T cell proximity to nerve fibers, flow cytometry of RAMP1+ T cells after commensal colonization, RAMP1 conditional KO in T cells, wound healing assays, commensal-specific T cell transfer experiments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — intravital imaging plus conditional genetic KO plus functional immunological readouts; multiple orthogonal methods","pmids":["38451947"],"is_preprint":false},{"year":2024,"finding":"CGRP/RAMP1 signaling in macrophages drives endometriosis lesion growth and pain: CGRP stimulation changes macrophage phenotype toward a pro-endometriosis state with impaired efferocytosis and support of endometrial cell growth in a RAMP1-dependent manner. FDA-approved CGRP-RAMP1 blockers reduce mechanical hyperalgesia, spontaneous pain, and lesion size in mice.","method":"Nonsurgical mouse endometriosis model, nociceptor ablation, in vitro CGRP-stimulated macrophage co-culture with endometrial cells (RAMP1 KO macrophages as controls), efferocytosis assays, pharmacological CGRP blockade","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple in vitro and in vivo approaches, genetic KO macrophages, pharmacological rescue, and human tissue samples; strong mechanistic evidence","pmids":["39504351"],"is_preprint":false},{"year":2024,"finding":"CGRP-RAMP1 signaling promotes liver fibrosis via TGFβ1/Smad2 and YAP pathways: RAMP1 deficiency attenuates fibrogenesis and hepatic stellate cell (HSC) activity. In LX-2 HSC cells, CGRP induces TGFβ1 production and downstream Smad2 signaling, increases α-SMA expression and collagen synthesis, and promotes YAP activation and nuclear translocation downstream of TGFβ1/Smad2.","method":"RAMP1 KO mice in liver fibrosis model; in vitro CGRP stimulation of LX-2 cells; Western blot for Smad2 phosphorylation, α-SMA, collagen, YAP nuclear translocation","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo KO model plus in vitro mechanistic cell signaling with multiple pathway readouts; single lab","pmids":["39103072"],"is_preprint":false},{"year":2024,"finding":"RAMP1 protects against hepatic ischemia-reperfusion injury by inhibiting ERK/MAPK activation and YAP phosphorylation; RAMP1 KO mice show increased apoptosis and liver damage associated with elevated ERK and YAP phosphorylation, and ERK or YAP inhibitors reduce apoptosis in RAMP1 KO livers.","method":"RAMP1 KO mice in 70% liver ischemia-reperfusion model; histology, serum transaminase, ERK/YAP phosphorylation (Western blot), pharmacological rescue with SCH772984 (ERK inhibitor) and PY-60 (YAP inhibitor)","journal":"Journal of clinical and translational hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with pharmacological rescue experiments validating specific pathway; single lab","pmids":["38638379"],"is_preprint":false},{"year":2024,"finding":"In dental pulp, CGRP released from sensory nerves binds RAMP1 on dental pulp stem cells (DPSCs) to promote collective cell migration to injury sites; sensory denervation impairs DPSC recruitment and pulp repair. Single-cell RNA-seq identified the CGRP-Ramp1 axis as the communication mechanism; BIBN4096 (CGRP receptor antagonist) blocks the pro-migratory effect.","method":"Sensory denervation model, indirect co-culture of DPSCs with trigeminal neurons, CGRP recombinant protein and BIBN4096 antagonist treatment, single-cell RNA-seq, immunohistochemistry, migration assays","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo denervation plus in vitro co-culture with pharmacological antagonism plus scRNA-seq; single lab","pmids":["39196292"],"is_preprint":false},{"year":2018,"finding":"RAMP1 expressed on Kupffer cells (KCs) is a critical regulator of immune-mediated hepatitis: RAMP1 KO mice show severe ConA-induced liver injury with increased inflammatory cytokines; KC depletion ameliorates damage; co-culture of KCs with splenic T cells increases cytokine expression from both cell types in a RAMP1-dependent manner. Adoptive transfer of RAMP1-deficient splenic T cells causes a modest increase in liver injury.","method":"RAMP1 KO mice in ConA hepatitis model; KC depletion, splenectomy, T cell depletion, adoptive transfer of splenic T cells, KC/T cell co-culture with cytokine measurements","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus cell depletion plus adoptive transfer plus co-culture; multiple approaches, single lab","pmids":["30462657"],"is_preprint":false},{"year":2019,"finding":"CGRP/CRLR/RAMP1 signaling in hematopoietic progenitor cells is required for stress-induced hematopoiesis: Ramp1-deficient bone marrow cells have decreased repopulation capacity and increased ROS production and apoptosis under proliferative stress, while steady-state hematopoiesis is maintained. Continuous CGRP exposure reduces immature hematopoietic and differentiated myeloid cells in vivo.","method":"Ramp1 KO mice, BM transplantation/repopulation assays, ROS and apoptosis measurements in transplanted BM cells, continuous CGRP infusion in vivo","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with BM transplantation assay and ROS/apoptosis readouts plus CGRP infusion experiment; single lab","pmids":["30674976"],"is_preprint":false},{"year":2025,"finding":"Ramp1 expression in the trigeminal ganglion (TG) varies across the estrous cycle (peaking in proestrus) in inverse correlation with Calca (CGRPα); Ramp1 expression correlates with Esr2 (ERβ), and estrogen treatment upregulates Ramp1. In Ramp1 KO mice, the cyclical variation of Calca, Ramp2, and Ramp3 seen in WT is absent, and basal Calca is elevated in males, establishing RAMP1 as essential for hormonal regulation of the CGRP system in the TG.","method":"RT-qPCR across estrous cycle stages in WT and Ramp1 KO mice, exogenous estrogen/progesterone administration, sex-stratified analysis","journal":"The journal of headache and pain","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus hormone manipulation with quantitative gene expression across multiple timepoints and both sexes; single lab","pmids":["40528180"],"is_preprint":false},{"year":2024,"finding":"CGRP-RAMP1 signaling mediates a subset of meningeal macrophage calcium responses to cortical spreading depolarization (CSD): CGRP/RAMP1 signaling mediates the increase (but not the decrease) in CSD-mediated Ca2+ signaling in meningeal macrophages in vivo.","method":"Intravital two-photon calcium imaging in awake Pf4Cre:TIGRE2.0-GCaMP6s reporter mice; CGRP/RAMP1 pharmacological manipulation during CSD","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 / Weak — preprint, intravital imaging with pharmacological manipulation, single lab, mechanistic assignment based on partial CGRP/RAMP1 blockade effect","pmids":["bio_10.1101_2025.10.01.679335"],"is_preprint":true},{"year":2026,"finding":"Sensory neurons inhibit breast cancer cell invadopodia and metastasis via direct CGRP signaling through the CRLR/RAMP1 complex on cancer cells; CGRP induces rapid intracellular cAMP increases and subsequent RhoC activation in cancer cells, suppressing invadopodia assembly and ECM degradation. Loss of RAMP1 function enhances 3D spheroid invasion and in vivo lung metastasis. Invasive breast cancer subtypes show progressively decreased RAMP1/CRLR expression.","method":"Microfluidic device (DACIT) for axon-cancer cell co-culture, CGRP treatment with CRLR/RAMP1 knockdown, intracellular cAMP and RhoC activity assays, 3D spheroid invasion assays, in vivo metastasis experiments, in silico RNAseq and patient sample analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — preprint, but multiple orthogonal methods including novel microfluidic device, signaling assays, in vivo metastasis, and patient data; single lab","pmids":["42079108"],"is_preprint":true},{"year":2005,"finding":"CGRP exerts antiapoptotic effects in H9c2 cardiomyoblasts under oxidative stress specifically through the RAMP1/CRLR complex: CGRP pretreatment inhibits phosphatidylserine externalization, caspase-3 activation, and DNA fragmentation induced by H2O2, increases Bcl-2 and prevents Bax increase; CGRP(8-37) (RAMP1/2 inhibitor) abolishes protection, while adrenomedullin (RAMP2 agonist) does not protect, pinpointing RAMP1/CRLR as the mediating complex.","method":"MTT cell viability assay, annexin V staining, caspase-3 activity, DNA fragmentation, RT-PCR for RAMP isoforms, dot blot for Bcl-2/Bax, pharmacological dissection with CGRP8-37 and ADM in H9c2 cells","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple apoptosis readouts plus pharmacological receptor subtype dissection; single lab","pmids":["16242145"],"is_preprint":false},{"year":2013,"finding":"RAMP1 overexpression in MG-63 osteoblast-like cells promotes CRLR surface localization and enhances CGRP-mediated osteogenic differentiation, including increased collagen I expression, alkaline phosphatase activity, and mineral deposition (Alizarin Red staining), indicating RAMP1 quantity directly modulates CGRP receptor surface expression and downstream osteogenic signaling.","method":"Stable RAMP1 transfection in MG-63 cells, Western blot and RT-PCR for RAMP1/CRLR expression, immunofluorescence for CRLR distribution, ALP activity assay, Alizarin Red mineralization staining","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — stable overexpression with multiple functional readouts; single lab","pmids":["22949393"],"is_preprint":false},{"year":2024,"finding":"CGRP-RAMP1 signaling activates p38 MAPK in osteoblasts during mechanical stimulation; low-magnitude compressive force upregulates RAMP1 expression and phosphorylates p38, and CGRP treatment similarly upregulates RAMP1 and p38 phosphorylation with increased osteogenic markers RUNX2 and osteocalcin, suggesting RAMP1 acts upstream of p38 in osteogenic differentiation.","method":"Osteoblast compressive force model, CGRP treatment, Western blot for RAMP1, RUNX2, osteocalcin, p38, and p-p38; Alizarin Red staining","journal":"Journal of orthopaedic surgery and research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — correlative Western blot data with CGRP treatment, no genetic KO or direct pathway perturbation; single lab","pmids":["38825686"],"is_preprint":false},{"year":2025,"finding":"In vitiligo, oxidative stress impairs Treg expansion via dysregulated CGRP-RAMP1-Gαi3 signaling: RAMP1+ Tregs are increased in active vitiligo; H2O2 and CGRP upregulate Gαi3 while suppressing p-CREB and PCNA in Treg-like cells, impairing proliferation. CGRP injection exacerbates depigmentation in a mouse vitiligo model.","method":"Flow cytometry of CLA+ Tregs from patients, in vitro CGRP/H2O2 stimulation of MT-2 cells with Western blot for Gαi3, p-CREB, PCNA; intradermal CGRP injection in mouse vitiligo model","journal":"Free radical biology & medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — in vitro signaling data plus in vivo phenotype but mechanistic pathway not rigorously dissected with RAMP1-specific perturbation; single lab","pmids":["41453541"],"is_preprint":false}],"current_model":"RAMP1 is a single-pass transmembrane accessory protein that heterodimerizes with CLR (CRLR) to form a functional CGRP receptor at the cell surface—a process that requires RAMP1 for proper CLR trafficking, establishes a defined extracellular ligand-binding interface (involving RAMP1 residues Trp74, Trp84, Tyr66, His97 and CLR Met42), and enables agonist-driven Gs/cAMP signaling and β-arrestin–dynamin-dependent clathrin-mediated endocytosis of the ternary CLR/RAMP1/β-arrestin complex; beyond CGRP receptor assembly, RAMP1 signaling regulates neuroimmune communication (modulating macrophage phenotype, T-cell responses, lymphangiogenesis, and gut barrier protection via goblet cells), supports liver regeneration and fibrosis through YAP/TAZ and TGFβ1/Smad2 pathways, and is subject to post-translational regulation by C-mannosylation at Trp56 (enhancing protein stability and cell migration) and transcriptional regulation by NKX3.1 and estrogen signaling."},"narrative":{"mechanistic_narrative":"RAMP1 is a single-pass transmembrane accessory protein that confers ligand selectivity onto the calcitonin receptor-like receptor (CLR/CRLR): coexpression of RAMP1 converts CLR from an adrenomedullin receptor into a functional CGRP receptor by generating high-affinity CGRP binding sites and CGRP-stimulated cAMP signaling, while competing with RAMP2 for CLR association [PMID:10342881]. RAMP1 is required for CLR to reach the cell surface—expressed alone it is retained in the ER/Golgi as a disulfide-linked homodimer, but with CLR it forms a 1:1 heterodimer that traffics to the plasma membrane and engages G proteins and β-arrestin upon CGRP stimulation, driving dynamin- and β-arrestin–dependent clathrin-mediated endocytosis of a stable CLR/RAMP1/β-arrestin ternary complex [PMID:11535606, PMID:17503773]. The RAMP1 extracellular domain adopts a three-helix, disulfide-stabilized fold [PMID:16632510] in which discrete residues partition into a CLR-recognition surface governing receptor trafficking (Tyr66, His97, helix 1) and a CGRP-recognition surface tuning agonist potency (Leu69, Thr73, residues 86–94) [PMID:19072332, PMID:20017504]; residues at the CLR/RAMP1 extracellular interface, notably RAMP1 Trp74 and Trp84, form the binding site for clinically used non-peptide antagonists (BIBN4096BS, telcagepant/MK-0974) [PMID:19914210, PMID:20188075]. Receptor abundance is set by RAMP1 levels, and post-translational C-mannosylation at Trp56 enhances RAMP1 stability and cell migration without affecting CLR trafficking [PMID:35942636, PMID:22949393]. Beyond receptor assembly, RAMP1 transduces CGRP-driven neuroimmune and tissue-repair programs: it shapes macrophage and T-cell phenotypes and lymphangiogenesis [PMID:31911634, PMID:29078910, PMID:39504351], protects the gut barrier through goblet-cell mucus secretion [PMID:36243004], and governs liver biology bidirectionally via YAP/TAZ and TGFβ1/Smad2 signaling—supporting regeneration and limiting ischemia-reperfusion injury while promoting fibrosis [PMID:32329113, PMID:39103072, PMID:38638379]. RAMP1 expression is transcriptionally repressed by NKX3.1 and regulated by estrogen, linking it to prostate cancer MAPK signaling and to cyclical hormonal control of the CGRP system [PMID:23867798, PMID:40528180].","teleology":[{"year":1999,"claim":"Established the founding function of RAMP1: it is the molecular switch that determines CLR ligand identity, answering why CLR can act as either an adrenomedullin or a CGRP receptor.","evidence":"Radioligand binding and CRE-luciferase cAMP assays with CRLR/RAMP1/RAMP2 combinations in COS-7 and UMR-106 cells","pmids":["10342881"],"confidence":"High","gaps":["Did not resolve the structural basis of selectivity","Did not address trafficking or signaling beyond cAMP"]},{"year":2001,"claim":"Showed RAMP1 is an obligate chaperone for CLR surface delivery and defined the agonist-induced endocytic fate of the receptor, explaining how the heterodimer is assembled and internalized.","evidence":"Subcellular fractionation, co-IP, disulfide analysis, and internalization assays with dominant-negative dynamin/β-arrestin in transfected cells","pmids":["11535606"],"confidence":"High","gaps":["Stoichiometry of the signaling complex not structurally resolved","Did not map ligand-contact residues"]},{"year":2007,"claim":"Demonstrated in living cells that CLR/RAMP1 heterodimerization is selective and that a trafficking-deficient RAMP1 mutant still binds CLR, distinguishing complex conformation from heterodimerization per se.","evidence":"BRET titration, radioligand binding, cAMP assays, and mutagenesis in living cells","pmids":["17503773"],"confidence":"High","gaps":["Conformational defect not structurally defined","Homodimer functional role unresolved"]},{"year":2006,"claim":"Provided the first structural framework for the RAMP1 extracellular domain and assigned its disulfide bonds, enabling residue-level interpretation of CLR and ligand contacts.","evidence":"Ab initio molecular modeling with site-directed mutagenesis to map disulfide bonds","pmids":["16632510"],"confidence":"Medium","gaps":["No experimental crystal structure","Modeled interface predictions required functional validation"]},{"year":2010,"claim":"Mapped RAMP1 surface into separable CLR-recognition and CGRP-recognition determinants, explaining how a single accessory protein controls both trafficking and pharmacology.","evidence":"Alanine scanning and RAMP1-RAMP3 chimera analysis with cell-surface ELISA and cAMP assays in COS-7 cells","pmids":["19072332","20017504"],"confidence":"Medium","gaps":["Contributions inferred functionally, not from a co-structure","Did not resolve allosteric coupling between sites"]},{"year":2010,"claim":"Localized the binding site of clinical non-peptide CGRP antagonists to the CLR/RAMP1 extracellular interface, identifying RAMP1 Trp74/Trp84 as key contacts and providing the pharmacological rationale for the receptor as a migraine drug target.","evidence":"Site-directed/alanine mutagenesis of RAMP1 and CLR with quantitative radioligand binding for BIBN4096BS, telcagepant, and MK-0974","pmids":["19914210","20188075"],"confidence":"High","gaps":["Antagonist poses inferred from affinity shifts rather than direct structure","Position-74 contributions to AM vs CGRP pharmacology only partly resolved (21402116)"]},{"year":2018,"claim":"Defined the transmembrane CGRP-binding contacts of the activated receptor, extending the ligand interface from the RAMP1/CLR ectodomain into the CLR TM bundle.","evidence":"Genetic code expansion/unnatural amino acid mutagenesis with photoaffinity cross-linking and CGRP analogue validation","pmids":["30004692"],"confidence":"High","gaps":["RAMP1 contribution to TM-bundle contacts not directly addressed","Full activated-state structure not solved here"]},{"year":2013,"claim":"Linked RAMP1 abundance to disease via transcriptional control, showing NKX3.1 directly represses RAMP1 and that RAMP1 supports prostate cancer proliferation through MAPK signaling.","evidence":"ChIP-seq, Nkx3.1 KO mice, RAMP1 shRNA knockdown with in vitro/in vivo tumorigenicity and pathway readouts","pmids":["23867798"],"confidence":"Medium","gaps":["Whether MAPK effects are CGRP-ligand-dependent not established","Mechanism connecting RAMP1 to MEK1/ERK not fully defined"]},{"year":2014,"claim":"Moved RAMP1 from receptor biochemistry into in vivo physiology, showing CGRP/RAMP1 signaling drives airway inflammation through non-smooth-muscle cells.","evidence":"RAMP1 KO mice and smooth-muscle-specific CLR mice in an ovalbumin asthma model with airway resistance and inflammatory readouts","pmids":["25010197"],"confidence":"Medium","gaps":["Specific endothelial/immune cell type not pinpointed","Downstream signaling in those cells not defined"]},{"year":2019,"claim":"Established RAMP1 as a broad regulator of immune and metabolic physiology, controlling macrophage polarization, T-cell-driven lymphangiogenesis, amylin anorectic signaling, and stress hematopoiesis.","evidence":"RAMP1 (and RAMP3) KO mice across sepsis, lymphedema/lymphangiogenesis, feeding, and bone marrow transplantation models with cell-depletion and in vitro stimulation","pmids":["22656887","31911634","29078910","31881259","30674976","30462657"],"confidence":"Medium","gaps":["Cell-autonomous vs systemic CGRP sources often unresolved","Downstream signaling effectors largely correlative across these models"]},{"year":2022,"claim":"Identified C-mannosylation at Trp56 as a post-translational determinant of RAMP1 stability and migration, separable from its CLR-trafficking function.","evidence":"Mass spectrometry site identification with C-mannosylation-deficient mutants in stability and migration assays","pmids":["35942636"],"confidence":"Medium","gaps":["Migration mechanism downstream of C-mannosylation unknown","Physiological context of the modification not tested in vivo"]},{"year":2022,"claim":"Demonstrated direct neuro-epithelial signaling, with nociceptor CGRP acting on goblet-cell RAMP1 to drive mucus secretion and protect against colitis.","evidence":"Epithelial-specific RAMP1 KO, nociceptor ablation, chemogenetic activation, and DSS colitis with CGRP rescue","pmids":["36243004"],"confidence":"High","gaps":["Intracellular pathway from RAMP1 to mucin exocytosis not defined","Human relevance based on expression rather than function"]},{"year":2024,"claim":"Defined RAMP1 as a context-dependent hub in liver injury, supporting regeneration and limiting ischemia-reperfusion damage while promoting fibrosis, all converging on YAP/TAZ and TGFβ1/Smad2 signaling.","evidence":"RAMP1 KO mice in hepatectomy, CCl4, ischemia-reperfusion, and fibrosis models with ERK/YAP/Smad2 readouts and CGRP/inhibitor rescue in slices, primary hepatocytes, and LX-2 cells","pmids":["32329113","39103072","38638379"],"confidence":"High","gaps":["How a single receptor produces opposing pro/anti-fibrotic outcomes is unresolved","Direct coupling of RAMP1/CLR to YAP regulation not biochemically defined"]},{"year":2024,"claim":"Extended the CGRP-RAMP1 axis into immune-cancer and neuroimmune crosstalk, shaping commensal-specific T cells, endometriosis macrophages, and breast cancer invasion via cAMP/RhoC.","evidence":"Conditional RAMP1 KO, intravital imaging, RAMP1 KO macrophage co-cultures, pharmacological CGRP blockade, and microfluidic axon-cancer co-culture (some preprint)","pmids":["38451947","39504351","42079108","39196292"],"confidence":"High","gaps":["Cell-type-specific signaling pathways incompletely mapped","Breast cancer and meningeal macrophage findings are preprint-stage"]},{"year":2025,"claim":"Connected RAMP1 to hormonal control of the CGRP system, showing estrogen upregulates Ramp1 and that RAMP1 is required for cyclical regulation of CGRP-system genes in the trigeminal ganglion.","evidence":"RT-qPCR across the estrous cycle in WT and Ramp1 KO mice with exogenous hormone administration and sex-stratified analysis","pmids":["40528180"],"confidence":"Medium","gaps":["Direct estrogen-responsive elements in RAMP1 not mapped","Functional consequence for headache pathophysiology not established"]},{"year":null,"claim":"How RAMP1 mechanistically directs opposing physiological outcomes (protective vs pathogenic) in different tissues, and how its ectodomain conformation couples to divergent intracellular effectors beyond cAMP, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No experimental high-resolution structure of the human CLR/RAMP1 complex in the timeline","Tissue-specific effector selection (Gs/cAMP vs ERK vs YAP/Smad2) not mechanistically explained","Whether non-receptor RAMP1 functions exist independent of CLR is untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,4,30]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,1,28]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,4]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,2,30]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[1]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,16,21]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[12,14,18,19,20,24]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,2]}],"complexes":["CGRP receptor (CLR/RAMP1 heterodimer)","CLR/RAMP1/β-arrestin ternary complex"],"partners":["CALCRL","ARRB1","RAMP2","RAMP3","NKX3.1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O60894","full_name":"Receptor 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Required for the transport of CALCRL to the plasma membrane (PubMed:9620797). Together with CALCRL, form the receptor complex for the calcitonin gene-related peptides CGRP1/CALCA and CGRP2/CALCB (PubMed:33602864, PubMed:9620797). Together with CALCR, form the AMYR1 receptor complex for amylin/IAPP and CGRP1/CALCA (PubMed:35324283, PubMed:38603770)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/O60894/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RAMP1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RAMP1","total_profiled":1310},"omim":[{"mim_id":"608682","title":"ADRENOMEDULLIN 2; ADM2","url":"https://www.omim.org/entry/608682"},{"mim_id":"606121","title":"CGRP RECEPTOR COMPONENT; CRCP","url":"https://www.omim.org/entry/606121"},{"mim_id":"605155","title":"RECEPTOR ACTIVITY-MODIFYING PROTEIN 3; RAMP3","url":"https://www.omim.org/entry/605155"},{"mim_id":"605154","title":"RECEPTOR ACTIVITY-MODIFYING PROTEIN 2; RAMP2","url":"https://www.omim.org/entry/605154"},{"mim_id":"605153","title":"RECEPTOR ACTIVITY-MODIFYING PROTEIN 1; RAMP1","url":"https://www.omim.org/entry/605153"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nuclear membrane","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"blood vessel","ntpm":533.5},{"tissue":"endometrium 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cancers.","date":"2025","source":"BMJ oncology","url":"https://pubmed.ncbi.nlm.nih.gov/41158750","citation_count":3,"is_preprint":false},{"pmid":"38825686","id":"PMC_38825686","title":"Involvement of RAMP1/p38MAPK signaling pathway in osteoblast differentiation in response to mechanical stimulation: a preliminary study.","date":"2024","source":"Journal of orthopaedic surgery and research","url":"https://pubmed.ncbi.nlm.nih.gov/38825686","citation_count":3,"is_preprint":false},{"pmid":"38638379","id":"PMC_38638379","title":"RAMP1 Protects Hepatocytes against Ischemia-reperfusion Injury by Inhibiting the ERK/YAP Pathway.","date":"2024","source":"Journal of clinical and translational hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/38638379","citation_count":2,"is_preprint":false},{"pmid":"41453541","id":"PMC_41453541","title":"Oxidative stress impairs the expansion of regulatory T cells in active vitiligo via dysregulated CGRP-RAMP1-Gαi3 signaling.","date":"2025","source":"Free radical 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biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/41649670","citation_count":0,"is_preprint":false},{"pmid":"42079108","id":"PMC_42079108","title":"Sensory neurons inhibit invadopodia and metastasis via direct CGRP-RAMP1-cAMP signaling to cancer cells.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/42079108","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.01.679335","title":"Intravital calcium imaging of meningeal macrophages reveals niche-specific dynamics and aberrant responses to brain hyperexcitability","date":"2025-10-02","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.01.679335","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.13.659569","title":"Determinants of Improved CGRP Peptide Binding Kinetics Revealed by Enhanced Molecular Simulations","date":"2025-06-17","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.13.659569","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.10.09.617487","title":"Amylin receptor subunit interactions are modulated by agonists and determine signaling","date":"2024-10-09","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.09.617487","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.10.25.620369","title":"Loss of Calcitonin Gene Related Receptor component protein (RCP) in nervous system can bias “gepant” 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responses.\",\n      \"method\": \"Radioligand binding assays ([125I]hαCGRP, [125I]rADM) and CRE-luciferase cAMP reporter assays in transiently transfected COS-7 and UMR-106 cells with combinations of rCRLR, hRAMP1, and hRAMP2\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with binding and functional (cAMP) assays, multiple receptor combinations tested, replicated across two cell lines\",\n      \"pmids\": [\"10342881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"When expressed alone, RAMP1 is retained intracellularly in the ER and Golgi as a disulfide-linked homodimer; coexpression with CRLR redirects RAMP1 to the cell surface as a 1:1 heterodimer (without intermolecular disulfide bonds) and promotes intramolecular disulfide bond formation within RAMP1. CGRP activation leads to CRLR phosphorylation and dynamin- and β-arrestin-dependent endocytosis of a stable CRLR/RAMP1/β-arrestin ternary complex via clathrin-coated pits.\",\n      \"method\": \"Subcellular fractionation, co-immunoprecipitation, disulfide bond analysis, CGRP-stimulated internalization assays with dominant-negative dynamin and β-arrestin mutants in transfected cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (biochemical fractionation, co-IP, dominant-negative constructs, trafficking assays) in a single focused study\",\n      \"pmids\": [\"11535606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CRLR and RAMP1 associate selectively to form heterodimers at the cell surface, as shown by BRET titration; both proteins can also form homodimers. RAMP1 is required for CRLR to engage G proteins and β-arrestin upon CGRP stimulation; a trafficking-deficient RAMP1 mutant still heterodimerizes with CRLR, indicating deficient surface targeting results from altered complex conformation rather than loss of heterodimerization.\",\n      \"method\": \"Bioluminescence resonance energy transfer (BRET) titration in living cells, radioligand binding, cAMP production assay, mutagenesis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — BRET in living cells combined with binding and functional assays and mutagenesis, multiple orthogonal methods in one study\",\n      \"pmids\": [\"17503773\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The extracellular domain of RAMP1 adopts a three-helix structure with disulfide bonds at Cys27–Cys82, Cys40–Cys72, and Cys57–Cys104 (determined by site-directed mutagenesis); modeling identified Phe93, Tyr100, and Phe101 as a putative CLR-binding interface and Trp74/Phe92 as potential ligand interaction sites.\",\n      \"method\": \"Ab initio molecular modeling of RAMP1 extracellular domain, site-directed mutagenesis to map disulfide bonds\",\n      \"journal\": \"Biophysical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — disulfide bond assignments validated by mutagenesis, but structural model is computational with no crystal structure; single lab\",\n      \"pmids\": [\"16632510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Alanine mutagenesis of RAMP1 identified Tyr66 and His97 as critical for CLR trafficking to the cell surface (CLR recognition site), while Leu69 and Thr73 in helix 2 contribute to CGRP potency (CGRP recognition site); Met48 in helix 1 had only a modest effect on CLR surface expression.\",\n      \"method\": \"Alanine scanning mutagenesis of RAMP1 coexpressed with CLR in COS-7 cells; cell-surface ELISA and cAMP assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic alanine mutagenesis with functional readouts (trafficking and cAMP), multiple residues tested, single lab but comprehensive\",\n      \"pmids\": [\"19072332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Non-peptide CGRP receptor antagonists BIBN4096BS and MK-0974 interact with the CGRP receptor via Met42 of CLR and Trp74 of RAMP1; Trp74Lys mutation reduces BIBN4096BS affinity >300-fold and similarly reduces MK-0974 affinity, and Met42Ala reduces BIBN4096BS affinity 48-fold and MK-0974 affinity ~900-fold, placing the antagonist binding site at the CLR/RAMP1 extracellular domain interface.\",\n      \"method\": \"Site-directed mutagenesis of CLR residues 23–63 and RAMP1 Trp74, radioligand binding and functional assays in transfected cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis with quantitative binding affinities for two independent antagonists, confirms and extends prior Trp74 finding, single lab but rigorous\",\n      \"pmids\": [\"19914210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Tryptophan-84 of RAMP1 is critical for both high-affinity non-peptide antagonist (telcagepant, BIBN4096BS) binding and agonist (CGRP and CGRP8-37) potency; Arg67 is important for telcagepant but not compound 3 (BIBN4096BS analog) binding, indicating distinct contact points for different antagonists within the RAMP1 N-terminal domain.\",\n      \"method\": \"Alanine replacement mutagenesis of RAMP1 residues, radioligand binding and cAMP assays in cells coexpressing CLR\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis with quantitative binding and functional readouts for both agonists and antagonists, single lab\",\n      \"pmids\": [\"20188075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RAMP1-RAMP3 chimera analysis showed that helix 1 of RAMP1 and residues 62–69 in helix 2 are required for CLR trafficking; residues 86–89 of RAMP1 at the helix 2–3 junction are important for hαCGRP and adrenomedullin potency but not hβCGRP potency, suggesting a peptide-binding role; residues 90–94 have dual roles in CLR recognition and CGRP binding.\",\n      \"method\": \"RAMP1–RAMP3 chimera expression with CLR in COS-7 cells; cell-surface ELISA and cAMP assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — chimeric protein approach with multiple segment exchanges and functional readouts, single lab\",\n      \"pmids\": [\"20017504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Position 74 of RAMP1 (Trp74) vs RAMP3 (Glu74) differentially affects adrenomedullin potency at the respective CLR complexes; geometry and charge at this residue contribute to differential AM pharmacology at AM2 and CGRP receptors, while CGRP and AM2/intermedin potency are largely unaffected by substitutions at this position in RAMP1.\",\n      \"method\": \"Site-directed mutagenesis of RAMP1 and RAMP3 at position 74 with multiple amino acid substitutions, coexpressed with CLR in COS-7 cells; cAMP assays and ELISA for surface expression\",\n      \"journal\": \"Peptides\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis across both RAMPs with multiple substitutions and functional readouts, single lab\",\n      \"pmids\": [\"21402116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Photoaffinity cross-linking using unnatural amino acid mutagenesis in CLR ECL2 identified key contacts for CGRP within the transmembrane bundle: I284 in ECL2 and L291 at the top of TM5 are primary contacts; M223 in TM3 and F349 in TM6 are minor contacts. Molecular modeling indicates CGRP sits at the top of the TM bundle with Thr6 contacting H295 (important for activation) and Leu12/Ala13 contacting I284.\",\n      \"method\": \"Genetic code expansion and unnatural amino acid (p-azido-Phe) mutagenesis in CLR ECL2, photoaffinity cross-linking with fluorescein-CGRP analog, functional validation with [Leu16Ala]-CGRP analogue\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — photoaffinity cross-linking plus unnatural amino acid mutagenesis plus functional peptide analogue validation; multiple orthogonal methods in one study\",\n      \"pmids\": [\"30004692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NKX3.1 directly represses RAMP1 expression in prostate cells; multiple NKX3.1 binding sites were identified in the RAMP1 locus by ChIP-seq. RAMP1 knockdown reduces prostate cancer cell proliferation and tumorigenicity, associated with reduced MEK1 expression and decreased phospho-ERK1/2 levels, placing RAMP1 upstream of the MAPK signaling pathway in prostate cancer.\",\n      \"method\": \"ChIP-seq for NKX3.1 binding at RAMP1 locus, Nkx3.1 knockout mice analysis, shRNA knockdown of RAMP1 in prostate cancer cell lines with in vitro and in vivo tumorigenicity assays, gene expression profiling\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq plus KO mice plus functional knockdown with pathway readouts, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"23867798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RAMP1 deficiency in mice reduces antigen-induced airway hyperresponsiveness and lung inflammation in an ovalbumin asthma model; loss of CLR from smooth muscle cells specifically does NOT alter airway resistance, indicating CGRP/RAMP1 signaling drives asthma pathology via endothelial and inflammatory cells rather than smooth muscle cells.\",\n      \"method\": \"RAMP1 knockout mice (exon 3 excision) and smooth-muscle-specific CLR heterozygous mice subjected to ovalbumin sensitization/challenge; airway resistance (methacholine challenge) and inflammatory cell measurements\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined phenotypic readout, cell-type-specific CLR deletion identifies non-smooth muscle mechanism; single lab\",\n      \"pmids\": [\"25010197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RAMP1 deficiency improves bacterial clearance in the early phase of polymicrobial septic peritonitis by reducing IL-10-mediated immunosuppression; CGRP directly increases IL-10 production in macrophages via RAMP1 signaling in response to LPS, and RAMP1 deficiency increases neutrophil recruitment and defensin-α1 release.\",\n      \"method\": \"RAMP1 KO mice in cecal ligation and puncture model; cytokine measurements in plasma and peritoneal lavage, in vitro CGRP stimulation of macrophages, neutrophil and defensin assays\",\n      \"journal\": \"Immunobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus in vitro macrophage CGRP stimulation with cytokine readouts; single lab, two complementary methods\",\n      \"pmids\": [\"22656887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP1 mediates amylin's anorectic signaling: RAMP1/3 double KO mice are insensitive to both amylin and salmon calcitonin (sCT) food intake suppression; RAMP1 single KO mice respond to amylin but not sCT, while RAMP3 KO mice respond to sCT but not amylin. RAMP1 is also required for normal fat utilization and meal pattern regulation.\",\n      \"method\": \"RAMP1, RAMP3, and RAMP1/3 global KO mice; acute intraperitoneal amylin/sCT injection food intake assays, metabolic measurements, meal pattern analysis\",\n      \"journal\": \"Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined pharmacological and feeding phenotype, multiple KO lines compared; single lab\",\n      \"pmids\": [\"31881259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RAMP1 signaling in CD4+ T cells promotes inflammation-associated lymphangiogenesis by driving VEGF-C and VEGF-D expression; RAMP1-deficient mice show suppressed LPS-induced lymphangiogenesis, reduced CD4+ cell numbers, and a shift toward pro-inflammatory (M1) macrophage phenotype with increased CD11b+ cells.\",\n      \"method\": \"RAMP1 KO mice with LPS-induced peritoneal inflammation model; FITC-dextran lymphatic flow assay, immunostaining for CD4+/CD11b+ cells, VEGF-C/D expression analysis, CD4+ cell depletion experiments\",\n      \"journal\": \"Laboratory investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus cell depletion plus functional lymphatic flow assay; single lab\",\n      \"pmids\": [\"31911634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"RAMP1 signaling promotes lymphangiogenesis during secondary lymphedema by supporting VEGF-C/VEGFR3 expression and reparative macrophage polarization; RAMP1-deficient mice show sustained tail lymphedema, suppressed lymphangiogenesis, dilated/dysfunctional lymphatics, and elevated M1 macrophage gene expression (TNF-α, IL-1β) with decreased M2 markers (IL-10).\",\n      \"method\": \"RAMP1 KO mice in surgical tail lymphedema model; tail diameter measurements, VEGF-C/VEGFR3 expression, macrophage marker analysis\",\n      \"journal\": \"The Journal of surgical research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with functional lymphatic readouts and macrophage phenotyping; single lab\",\n      \"pmids\": [\"29078910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RAMP1 deficiency severely impairs liver regeneration after partial hepatectomy and carbon tetrachloride injury; mechanistically, RAMP1 loss leads to hyperphosphorylation of YAP at Ser127 and Ser397, reducing YAP/TAZ protein levels and expression of YAP-controlled cell cycle regulators. In vitro, CGRP treatment of liver slice cultures and primary human hepatocytes increases YAP protein and reduces YAP phosphorylation.\",\n      \"method\": \"RAMP1 KO mice subjected to 70% partial hepatectomy and CCl4 model; liver mass recovery, hepatocyte proliferation (Ki67), YAP/TAZ protein levels and phosphorylation (Western blot), ex vivo CGRP treatment of liver slices and primary hepatocytes\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO in two injury models plus in vitro mechanistic validation in multiple systems (mouse and human tissue); multiple orthogonal methods\",\n      \"pmids\": [\"32329113\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RAMP1 is C-mannosylated at Trp56 (identified by mass spectrometry); C-mannosylation enhances RAMP1 protein stability and promotes cell migration activity but does not affect RAMP1 plasma membrane trafficking or its function in transporting CRLR to the cell surface.\",\n      \"method\": \"Mass spectrometry to identify C-mannosylation site, C-mannosylation-deficient RAMP1 mutants, protein stability assays, cell migration assays\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — MS identification of modification site plus functional mutagenesis for stability and migration; single lab, two orthogonal methods\",\n      \"pmids\": [\"35942636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RAMP1 is expressed on intestinal goblet cells in mice and humans; nociceptor (Nav1.8+CGRP+) signaling via the CGRP-RAMP1 pathway induces rapid goblet cell emptying and mucus secretion. Loss of epithelial Ramp1 increases susceptibility to colitis; CGRP administration protects nociceptor-ablated mice against colitis.\",\n      \"method\": \"RAMP1 cell-specific KO (epithelial), nociceptor ablation, chemogenetic nociceptor activation, capsaicin treatment, mouse colitis model (DSS), CGRP administration rescue experiments; histology, mucus thickness measurement\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic cell-type-specific KO, nociceptor ablation and chemogenetic activation, pharmacological rescue, multiple complementary approaches in one study\",\n      \"pmids\": [\"36243004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The CGRP-RAMP1 axis in skin mediates direct neuroimmune communication: commensal colonization upregulates RAMP1 on CD8+ T lymphocytes; CGRP-RAMP1 signaling in commensal-specific T cells constrains Type 17 responses and moderates the activation status of microbiota-reactive lymphocytes, shaping the overall skin epithelial activation state and wound healing responses.\",\n      \"method\": \"Intravital imaging of T cell proximity to nerve fibers, flow cytometry of RAMP1+ T cells after commensal colonization, RAMP1 conditional KO in T cells, wound healing assays, commensal-specific T cell transfer experiments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — intravital imaging plus conditional genetic KO plus functional immunological readouts; multiple orthogonal methods\",\n      \"pmids\": [\"38451947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CGRP/RAMP1 signaling in macrophages drives endometriosis lesion growth and pain: CGRP stimulation changes macrophage phenotype toward a pro-endometriosis state with impaired efferocytosis and support of endometrial cell growth in a RAMP1-dependent manner. FDA-approved CGRP-RAMP1 blockers reduce mechanical hyperalgesia, spontaneous pain, and lesion size in mice.\",\n      \"method\": \"Nonsurgical mouse endometriosis model, nociceptor ablation, in vitro CGRP-stimulated macrophage co-culture with endometrial cells (RAMP1 KO macrophages as controls), efferocytosis assays, pharmacological CGRP blockade\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple in vitro and in vivo approaches, genetic KO macrophages, pharmacological rescue, and human tissue samples; strong mechanistic evidence\",\n      \"pmids\": [\"39504351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CGRP-RAMP1 signaling promotes liver fibrosis via TGFβ1/Smad2 and YAP pathways: RAMP1 deficiency attenuates fibrogenesis and hepatic stellate cell (HSC) activity. In LX-2 HSC cells, CGRP induces TGFβ1 production and downstream Smad2 signaling, increases α-SMA expression and collagen synthesis, and promotes YAP activation and nuclear translocation downstream of TGFβ1/Smad2.\",\n      \"method\": \"RAMP1 KO mice in liver fibrosis model; in vitro CGRP stimulation of LX-2 cells; Western blot for Smad2 phosphorylation, α-SMA, collagen, YAP nuclear translocation\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo KO model plus in vitro mechanistic cell signaling with multiple pathway readouts; single lab\",\n      \"pmids\": [\"39103072\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RAMP1 protects against hepatic ischemia-reperfusion injury by inhibiting ERK/MAPK activation and YAP phosphorylation; RAMP1 KO mice show increased apoptosis and liver damage associated with elevated ERK and YAP phosphorylation, and ERK or YAP inhibitors reduce apoptosis in RAMP1 KO livers.\",\n      \"method\": \"RAMP1 KO mice in 70% liver ischemia-reperfusion model; histology, serum transaminase, ERK/YAP phosphorylation (Western blot), pharmacological rescue with SCH772984 (ERK inhibitor) and PY-60 (YAP inhibitor)\",\n      \"journal\": \"Journal of clinical and translational hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with pharmacological rescue experiments validating specific pathway; single lab\",\n      \"pmids\": [\"38638379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In dental pulp, CGRP released from sensory nerves binds RAMP1 on dental pulp stem cells (DPSCs) to promote collective cell migration to injury sites; sensory denervation impairs DPSC recruitment and pulp repair. Single-cell RNA-seq identified the CGRP-Ramp1 axis as the communication mechanism; BIBN4096 (CGRP receptor antagonist) blocks the pro-migratory effect.\",\n      \"method\": \"Sensory denervation model, indirect co-culture of DPSCs with trigeminal neurons, CGRP recombinant protein and BIBN4096 antagonist treatment, single-cell RNA-seq, immunohistochemistry, migration assays\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo denervation plus in vitro co-culture with pharmacological antagonism plus scRNA-seq; single lab\",\n      \"pmids\": [\"39196292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RAMP1 expressed on Kupffer cells (KCs) is a critical regulator of immune-mediated hepatitis: RAMP1 KO mice show severe ConA-induced liver injury with increased inflammatory cytokines; KC depletion ameliorates damage; co-culture of KCs with splenic T cells increases cytokine expression from both cell types in a RAMP1-dependent manner. Adoptive transfer of RAMP1-deficient splenic T cells causes a modest increase in liver injury.\",\n      \"method\": \"RAMP1 KO mice in ConA hepatitis model; KC depletion, splenectomy, T cell depletion, adoptive transfer of splenic T cells, KC/T cell co-culture with cytokine measurements\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus cell depletion plus adoptive transfer plus co-culture; multiple approaches, single lab\",\n      \"pmids\": [\"30462657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CGRP/CRLR/RAMP1 signaling in hematopoietic progenitor cells is required for stress-induced hematopoiesis: Ramp1-deficient bone marrow cells have decreased repopulation capacity and increased ROS production and apoptosis under proliferative stress, while steady-state hematopoiesis is maintained. Continuous CGRP exposure reduces immature hematopoietic and differentiated myeloid cells in vivo.\",\n      \"method\": \"Ramp1 KO mice, BM transplantation/repopulation assays, ROS and apoptosis measurements in transplanted BM cells, continuous CGRP infusion in vivo\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with BM transplantation assay and ROS/apoptosis readouts plus CGRP infusion experiment; single lab\",\n      \"pmids\": [\"30674976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Ramp1 expression in the trigeminal ganglion (TG) varies across the estrous cycle (peaking in proestrus) in inverse correlation with Calca (CGRPα); Ramp1 expression correlates with Esr2 (ERβ), and estrogen treatment upregulates Ramp1. In Ramp1 KO mice, the cyclical variation of Calca, Ramp2, and Ramp3 seen in WT is absent, and basal Calca is elevated in males, establishing RAMP1 as essential for hormonal regulation of the CGRP system in the TG.\",\n      \"method\": \"RT-qPCR across estrous cycle stages in WT and Ramp1 KO mice, exogenous estrogen/progesterone administration, sex-stratified analysis\",\n      \"journal\": \"The journal of headache and pain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus hormone manipulation with quantitative gene expression across multiple timepoints and both sexes; single lab\",\n      \"pmids\": [\"40528180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CGRP-RAMP1 signaling mediates a subset of meningeal macrophage calcium responses to cortical spreading depolarization (CSD): CGRP/RAMP1 signaling mediates the increase (but not the decrease) in CSD-mediated Ca2+ signaling in meningeal macrophages in vivo.\",\n      \"method\": \"Intravital two-photon calcium imaging in awake Pf4Cre:TIGRE2.0-GCaMP6s reporter mice; CGRP/RAMP1 pharmacological manipulation during CSD\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 / Weak — preprint, intravital imaging with pharmacological manipulation, single lab, mechanistic assignment based on partial CGRP/RAMP1 blockade effect\",\n      \"pmids\": [\"bio_10.1101_2025.10.01.679335\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Sensory neurons inhibit breast cancer cell invadopodia and metastasis via direct CGRP signaling through the CRLR/RAMP1 complex on cancer cells; CGRP induces rapid intracellular cAMP increases and subsequent RhoC activation in cancer cells, suppressing invadopodia assembly and ECM degradation. Loss of RAMP1 function enhances 3D spheroid invasion and in vivo lung metastasis. Invasive breast cancer subtypes show progressively decreased RAMP1/CRLR expression.\",\n      \"method\": \"Microfluidic device (DACIT) for axon-cancer cell co-culture, CGRP treatment with CRLR/RAMP1 knockdown, intracellular cAMP and RhoC activity assays, 3D spheroid invasion assays, in vivo metastasis experiments, in silico RNAseq and patient sample analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — preprint, but multiple orthogonal methods including novel microfluidic device, signaling assays, in vivo metastasis, and patient data; single lab\",\n      \"pmids\": [\"42079108\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CGRP exerts antiapoptotic effects in H9c2 cardiomyoblasts under oxidative stress specifically through the RAMP1/CRLR complex: CGRP pretreatment inhibits phosphatidylserine externalization, caspase-3 activation, and DNA fragmentation induced by H2O2, increases Bcl-2 and prevents Bax increase; CGRP(8-37) (RAMP1/2 inhibitor) abolishes protection, while adrenomedullin (RAMP2 agonist) does not protect, pinpointing RAMP1/CRLR as the mediating complex.\",\n      \"method\": \"MTT cell viability assay, annexin V staining, caspase-3 activity, DNA fragmentation, RT-PCR for RAMP isoforms, dot blot for Bcl-2/Bax, pharmacological dissection with CGRP8-37 and ADM in H9c2 cells\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple apoptosis readouts plus pharmacological receptor subtype dissection; single lab\",\n      \"pmids\": [\"16242145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RAMP1 overexpression in MG-63 osteoblast-like cells promotes CRLR surface localization and enhances CGRP-mediated osteogenic differentiation, including increased collagen I expression, alkaline phosphatase activity, and mineral deposition (Alizarin Red staining), indicating RAMP1 quantity directly modulates CGRP receptor surface expression and downstream osteogenic signaling.\",\n      \"method\": \"Stable RAMP1 transfection in MG-63 cells, Western blot and RT-PCR for RAMP1/CRLR expression, immunofluorescence for CRLR distribution, ALP activity assay, Alizarin Red mineralization staining\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — stable overexpression with multiple functional readouts; single lab\",\n      \"pmids\": [\"22949393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CGRP-RAMP1 signaling activates p38 MAPK in osteoblasts during mechanical stimulation; low-magnitude compressive force upregulates RAMP1 expression and phosphorylates p38, and CGRP treatment similarly upregulates RAMP1 and p38 phosphorylation with increased osteogenic markers RUNX2 and osteocalcin, suggesting RAMP1 acts upstream of p38 in osteogenic differentiation.\",\n      \"method\": \"Osteoblast compressive force model, CGRP treatment, Western blot for RAMP1, RUNX2, osteocalcin, p38, and p-p38; Alizarin Red staining\",\n      \"journal\": \"Journal of orthopaedic surgery and research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — correlative Western blot data with CGRP treatment, no genetic KO or direct pathway perturbation; single lab\",\n      \"pmids\": [\"38825686\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In vitiligo, oxidative stress impairs Treg expansion via dysregulated CGRP-RAMP1-Gαi3 signaling: RAMP1+ Tregs are increased in active vitiligo; H2O2 and CGRP upregulate Gαi3 while suppressing p-CREB and PCNA in Treg-like cells, impairing proliferation. CGRP injection exacerbates depigmentation in a mouse vitiligo model.\",\n      \"method\": \"Flow cytometry of CLA+ Tregs from patients, in vitro CGRP/H2O2 stimulation of MT-2 cells with Western blot for Gαi3, p-CREB, PCNA; intradermal CGRP injection in mouse vitiligo model\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — in vitro signaling data plus in vivo phenotype but mechanistic pathway not rigorously dissected with RAMP1-specific perturbation; single lab\",\n      \"pmids\": [\"41453541\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RAMP1 is a single-pass transmembrane accessory protein that heterodimerizes with CLR (CRLR) to form a functional CGRP receptor at the cell surface—a process that requires RAMP1 for proper CLR trafficking, establishes a defined extracellular ligand-binding interface (involving RAMP1 residues Trp74, Trp84, Tyr66, His97 and CLR Met42), and enables agonist-driven Gs/cAMP signaling and β-arrestin–dynamin-dependent clathrin-mediated endocytosis of the ternary CLR/RAMP1/β-arrestin complex; beyond CGRP receptor assembly, RAMP1 signaling regulates neuroimmune communication (modulating macrophage phenotype, T-cell responses, lymphangiogenesis, and gut barrier protection via goblet cells), supports liver regeneration and fibrosis through YAP/TAZ and TGFβ1/Smad2 pathways, and is subject to post-translational regulation by C-mannosylation at Trp56 (enhancing protein stability and cell migration) and transcriptional regulation by NKX3.1 and estrogen signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RAMP1 is a single-pass transmembrane accessory protein that confers ligand selectivity onto the calcitonin receptor-like receptor (CLR/CRLR): coexpression of RAMP1 converts CLR from an adrenomedullin receptor into a functional CGRP receptor by generating high-affinity CGRP binding sites and CGRP-stimulated cAMP signaling, while competing with RAMP2 for CLR association [#0]. RAMP1 is required for CLR to reach the cell surface—expressed alone it is retained in the ER/Golgi as a disulfide-linked homodimer, but with CLR it forms a 1:1 heterodimer that traffics to the plasma membrane and engages G proteins and β-arrestin upon CGRP stimulation, driving dynamin- and β-arrestin–dependent clathrin-mediated endocytosis of a stable CLR/RAMP1/β-arrestin ternary complex [#1, #2]. The RAMP1 extracellular domain adopts a three-helix, disulfide-stabilized fold [#3] in which discrete residues partition into a CLR-recognition surface governing receptor trafficking (Tyr66, His97, helix 1) and a CGRP-recognition surface tuning agonist potency (Leu69, Thr73, residues 86–94) [#4, #7]; residues at the CLR/RAMP1 extracellular interface, notably RAMP1 Trp74 and Trp84, form the binding site for clinically used non-peptide antagonists (BIBN4096BS, telcagepant/MK-0974) [#5, #6]. Receptor abundance is set by RAMP1 levels, and post-translational C-mannosylation at Trp56 enhances RAMP1 stability and cell migration without affecting CLR trafficking [#17, #30]. Beyond receptor assembly, RAMP1 transduces CGRP-driven neuroimmune and tissue-repair programs: it shapes macrophage and T-cell phenotypes and lymphangiogenesis [#14, #15, #20], protects the gut barrier through goblet-cell mucus secretion [#18], and governs liver biology bidirectionally via YAP/TAZ and TGFβ1/Smad2 signaling—supporting regeneration and limiting ischemia-reperfusion injury while promoting fibrosis [#16, #21, #22]. RAMP1 expression is transcriptionally repressed by NKX3.1 and regulated by estrogen, linking it to prostate cancer MAPK signaling and to cyclical hormonal control of the CGRP system [#10, #26].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established the founding function of RAMP1: it is the molecular switch that determines CLR ligand identity, answering why CLR can act as either an adrenomedullin or a CGRP receptor.\",\n      \"evidence\": \"Radioligand binding and CRE-luciferase cAMP assays with CRLR/RAMP1/RAMP2 combinations in COS-7 and UMR-106 cells\",\n      \"pmids\": [\"10342881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of selectivity\", \"Did not address trafficking or signaling beyond cAMP\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed RAMP1 is an obligate chaperone for CLR surface delivery and defined the agonist-induced endocytic fate of the receptor, explaining how the heterodimer is assembled and internalized.\",\n      \"evidence\": \"Subcellular fractionation, co-IP, disulfide analysis, and internalization assays with dominant-negative dynamin/β-arrestin in transfected cells\",\n      \"pmids\": [\"11535606\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the signaling complex not structurally resolved\", \"Did not map ligand-contact residues\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated in living cells that CLR/RAMP1 heterodimerization is selective and that a trafficking-deficient RAMP1 mutant still binds CLR, distinguishing complex conformation from heterodimerization per se.\",\n      \"evidence\": \"BRET titration, radioligand binding, cAMP assays, and mutagenesis in living cells\",\n      \"pmids\": [\"17503773\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational defect not structurally defined\", \"Homodimer functional role unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Provided the first structural framework for the RAMP1 extracellular domain and assigned its disulfide bonds, enabling residue-level interpretation of CLR and ligand contacts.\",\n      \"evidence\": \"Ab initio molecular modeling with site-directed mutagenesis to map disulfide bonds\",\n      \"pmids\": [\"16632510\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimental crystal structure\", \"Modeled interface predictions required functional validation\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapped RAMP1 surface into separable CLR-recognition and CGRP-recognition determinants, explaining how a single accessory protein controls both trafficking and pharmacology.\",\n      \"evidence\": \"Alanine scanning and RAMP1-RAMP3 chimera analysis with cell-surface ELISA and cAMP assays in COS-7 cells\",\n      \"pmids\": [\"19072332\", \"20017504\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Contributions inferred functionally, not from a co-structure\", \"Did not resolve allosteric coupling between sites\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Localized the binding site of clinical non-peptide CGRP antagonists to the CLR/RAMP1 extracellular interface, identifying RAMP1 Trp74/Trp84 as key contacts and providing the pharmacological rationale for the receptor as a migraine drug target.\",\n      \"evidence\": \"Site-directed/alanine mutagenesis of RAMP1 and CLR with quantitative radioligand binding for BIBN4096BS, telcagepant, and MK-0974\",\n      \"pmids\": [\"19914210\", \"20188075\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Antagonist poses inferred from affinity shifts rather than direct structure\", \"Position-74 contributions to AM vs CGRP pharmacology only partly resolved (21402116)\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined the transmembrane CGRP-binding contacts of the activated receptor, extending the ligand interface from the RAMP1/CLR ectodomain into the CLR TM bundle.\",\n      \"evidence\": \"Genetic code expansion/unnatural amino acid mutagenesis with photoaffinity cross-linking and CGRP analogue validation\",\n      \"pmids\": [\"30004692\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RAMP1 contribution to TM-bundle contacts not directly addressed\", \"Full activated-state structure not solved here\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Linked RAMP1 abundance to disease via transcriptional control, showing NKX3.1 directly represses RAMP1 and that RAMP1 supports prostate cancer proliferation through MAPK signaling.\",\n      \"evidence\": \"ChIP-seq, Nkx3.1 KO mice, RAMP1 shRNA knockdown with in vitro/in vivo tumorigenicity and pathway readouts\",\n      \"pmids\": [\"23867798\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MAPK effects are CGRP-ligand-dependent not established\", \"Mechanism connecting RAMP1 to MEK1/ERK not fully defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Moved RAMP1 from receptor biochemistry into in vivo physiology, showing CGRP/RAMP1 signaling drives airway inflammation through non-smooth-muscle cells.\",\n      \"evidence\": \"RAMP1 KO mice and smooth-muscle-specific CLR mice in an ovalbumin asthma model with airway resistance and inflammatory readouts\",\n      \"pmids\": [\"25010197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific endothelial/immune cell type not pinpointed\", \"Downstream signaling in those cells not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established RAMP1 as a broad regulator of immune and metabolic physiology, controlling macrophage polarization, T-cell-driven lymphangiogenesis, amylin anorectic signaling, and stress hematopoiesis.\",\n      \"evidence\": \"RAMP1 (and RAMP3) KO mice across sepsis, lymphedema/lymphangiogenesis, feeding, and bone marrow transplantation models with cell-depletion and in vitro stimulation\",\n      \"pmids\": [\"22656887\", \"31911634\", \"29078910\", \"31881259\", \"30674976\", \"30462657\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-autonomous vs systemic CGRP sources often unresolved\", \"Downstream signaling effectors largely correlative across these models\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified C-mannosylation at Trp56 as a post-translational determinant of RAMP1 stability and migration, separable from its CLR-trafficking function.\",\n      \"evidence\": \"Mass spectrometry site identification with C-mannosylation-deficient mutants in stability and migration assays\",\n      \"pmids\": [\"35942636\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Migration mechanism downstream of C-mannosylation unknown\", \"Physiological context of the modification not tested in vivo\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated direct neuro-epithelial signaling, with nociceptor CGRP acting on goblet-cell RAMP1 to drive mucus secretion and protect against colitis.\",\n      \"evidence\": \"Epithelial-specific RAMP1 KO, nociceptor ablation, chemogenetic activation, and DSS colitis with CGRP rescue\",\n      \"pmids\": [\"36243004\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Intracellular pathway from RAMP1 to mucin exocytosis not defined\", \"Human relevance based on expression rather than function\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined RAMP1 as a context-dependent hub in liver injury, supporting regeneration and limiting ischemia-reperfusion damage while promoting fibrosis, all converging on YAP/TAZ and TGFβ1/Smad2 signaling.\",\n      \"evidence\": \"RAMP1 KO mice in hepatectomy, CCl4, ischemia-reperfusion, and fibrosis models with ERK/YAP/Smad2 readouts and CGRP/inhibitor rescue in slices, primary hepatocytes, and LX-2 cells\",\n      \"pmids\": [\"32329113\", \"39103072\", \"38638379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single receptor produces opposing pro/anti-fibrotic outcomes is unresolved\", \"Direct coupling of RAMP1/CLR to YAP regulation not biochemically defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the CGRP-RAMP1 axis into immune-cancer and neuroimmune crosstalk, shaping commensal-specific T cells, endometriosis macrophages, and breast cancer invasion via cAMP/RhoC.\",\n      \"evidence\": \"Conditional RAMP1 KO, intravital imaging, RAMP1 KO macrophage co-cultures, pharmacological CGRP blockade, and microfluidic axon-cancer co-culture (some preprint)\",\n      \"pmids\": [\"38451947\", \"39504351\", \"42079108\", \"39196292\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-type-specific signaling pathways incompletely mapped\", \"Breast cancer and meningeal macrophage findings are preprint-stage\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Connected RAMP1 to hormonal control of the CGRP system, showing estrogen upregulates Ramp1 and that RAMP1 is required for cyclical regulation of CGRP-system genes in the trigeminal ganglion.\",\n      \"evidence\": \"RT-qPCR across the estrous cycle in WT and Ramp1 KO mice with exogenous hormone administration and sex-stratified analysis\",\n      \"pmids\": [\"40528180\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct estrogen-responsive elements in RAMP1 not mapped\", \"Functional consequence for headache pathophysiology not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RAMP1 mechanistically directs opposing physiological outcomes (protective vs pathogenic) in different tissues, and how its ectodomain conformation couples to divergent intracellular effectors beyond cAMP, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No experimental high-resolution structure of the human CLR/RAMP1 complex in the timeline\", \"Tissue-specific effector selection (Gs/cAMP vs ERK vs YAP/Smad2) not mechanistically explained\", \"Whether non-receptor RAMP1 functions exist independent of CLR is untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 4, 30]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 1, 28]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 2, 30]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 16, 21]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [12, 14, 18, 19, 20, 24]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 2]}\n    ],\n    \"complexes\": [\"CGRP receptor (CLR/RAMP1 heterodimer)\", \"CLR/RAMP1/β-arrestin ternary complex\"],\n    \"partners\": [\"CALCRL\", \"ARRB1\", \"RAMP2\", \"RAMP3\", \"NKX3.1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}